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Sample records for leu-phe knockdown resistance

  1. Evidence of increasing Leu-Phe knockdown resistance mutation in Anopheles gambiae from Niger following a nationwide long-lasting insecticide-treated nets implementation

    PubMed Central

    Czeher, Cyrille; Labbo, Rabiou; Arzika, Ibrahim; Duchemin, Jean-Bernard

    2008-01-01

    Background At the end of 2005, a nationwide long-lasting insecticide-treated net (LLIN) distribution targeting the most vulnerable populations was implemented throughout Niger. A large number of studies in Africa have reported the existence of anopheline populations resistant to various insecticides, partly due to knockdown resistance (kdr) mutations, but few operational wide-scale control programmes were coupled with the monitoring of such mutations. The distribution of the kdr-west (kdr-w) Leu-Phe mutation was studied in Anopheles gambiae s.l. populations from Niger and temporal variations were monitored following the nationwide LLIN implementation. Methods Mosquitoes were collected from 14 localities during the wet seasons of 2005, 2006 and 2007 with additional sampling in the capital city, Niamey. After morphological identification of Anopheles gambiae s.l. specimens, DNA extracts were used for the determination of species and molecular forms of the Anopheles gambiae complex and for the detection of the kdr-w mutation. Results Around 1,500 specimens collected in the three consecutive years were analysed. All Anopheles arabiensis specimens analysed were homozygous susceptible, whereas the few Anopheles gambiae S forms exhibited a high overall kdr-w frequency. The M form samples exhibited a low overall kdr-w frequency before the LLIN distribution, that increased significantly in the two wet season collections following the LLIN distribution. Higher kdr frequencies were repeatedly noticed within host-seeking females compared to resting ones in indoor collections. In addition, preliminary results in M form urban populations from Niamey showed far higher kdr frequencies than in all of the rural sites studied. Discussion This study describes the first case of kdr mutation in Anopheles gambiae populations from Niger. It is suspected that the LLIN have caused the important temporal increase of kdr-w mutation observed during this study. While the kdr mutation is still

  2. Development of potent antagonists for formyl peptide receptor 1 based on Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH.

    PubMed

    Hayashi, Ryo; Kitajima, Toshiki; Mizuguchi, Hikaru; Fujimoto, Miki; Yamaguchi, Aya; Koga, Shuichiro; Koga, Yuya; Osada, Satoshi; Kodama, Hiroaki

    2014-08-01

    While stimulation of formyl peptide receptors (FPRs) on the surface of human neutrophils induces several immune responses, under conditions of continuous activation of the receptor by agonists such as formyl-Met-Leu-Phe-OH (fMLP), neutrophil-dependent tissue damage ensues. Thus, FPR antagonists could be anticipated as drugs for FPR-related disease. In this study, Boc-Phe-D-Leu-Phe-D-Leu-Phe-OH (Boc-FlFlF), one of several FPR subtype selective antagonists, was chosen and the positions at the Phe residues were optimized. We found that substitution with unnatural amino acids resulted in an improvement of two orders of magnitude. The most potent antagonist indicated FPR subtype selectivity at 1 μM. In addition to finding a potent antagonist, the structure-activity trends observed in this study should be valuable in designing a new type of FPR subtype selective antagonist. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Selective involvement of kappa opioid and phencyclidine receptors in the analgesic and motor effects of dynorphin-A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro.

    PubMed

    Shukla, V K; Bansinath, M; Dumont, M; Lemaire, S

    1992-09-18

    Dynorphin A-(1-13)-Tyr-Leu-Phe-Asn-Gly-Pro (Dyn Ia; 1-8 nmol) injected intracerebroventricularly in the mouse produces two independent behavioral effects: (1) a norbinaltorphimine (kappa opioid antagonist)-reversible analgesia in the acetic acid-induced writhing test and (2) motor dysfunction characterized by wild running, pop-corn jumping, hindlimb jerking and barrel rolling and antagonized by the irreversible phencyclidine (PCP) and sigma (sigma) receptor antagonist, metaphit and the non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists, dextromethorphan and ketamine. The specific involvement of the PCP receptor in the motor effects of Dyn Ia is supported by the direct competitive interaction of the peptide with the binding of [3H]MK-801 (Ki: 0.63 microM) and [3H]TCP (Ki: 4.6 microM) to mouse brain membrane preparations.

  4. In vitro studies of human monocyte migration across endothelium in response to leukotriene B4 and f-Met-Leu-Phe.

    PubMed Central

    Migliorisi, G.; Folkes, E.; Pawlowski, N.; Cramer, E. B.

    1987-01-01

    Relatively little is known about monocyte emigration from the vasculature or about the factors that regulate this process. In this study, a human in vitro model of a blood vessel wall was used for examination of monocyte transendothelial migration. Umbilical vein endothelial cells were grown to confluency on amnion connective tissue, and human monocytes were stimulated to cross the monolayer in response to the chemoattractants leukotriene B4 or f-Met-Leu-Phe. The pattern and time course of monocyte migration were similar for the two chemotactic factors. In both cases, approximately 40-50% of the adherent monocytes extended single or multiple pseudopods into the apical endothelial surface. This indenting behavior was also observed in the absence of chemotactic factors. It was not affected by the medium (M199 or Gey's) or method of monocyte isolation. Neutrophils also displayed this behavior, but only about half as many neutrophils as monocytes indented the endothelial surface. The integrity of the endothelium remained intact as the monocytes traversed the monolayer. When the monocytes reached the basal surface of the endothelium, they frequently wedged themselves between the basal surface of the endothelium and its basal lamina. The monocytes then invaded the basal lamina and accumulated in the connective tissue. In response to both f-Met-Leu-Phe and leukotriene B4, monocyte migration across the endothelium began as early as 10 minutes. The average rate of accumulation in the connective tissue peaked at 30 minutes; and by 60 minutes, 25-35% of the monocytes had traversed the monolayer. Approximately two to three times as many monocytes traversed the endothelium under conditions of chemotaxis as under conditions of chemokinesis or random migration. These studies provide the basis for understanding the process of monocyte migration out of the bloodstream and lay the foundation for the study of their differentiation into macrophages in the connective tissue. Images

  5. Characterization of P-Rex1 for its role in fMet-Leu-Phe-induced superoxide production in reconstituted COS(phox) cells.

    PubMed

    Nie, Baoming; Cheng, Ni; Dinauer, Mary C; Ye, Richard D

    2010-05-01

    P-Rex1 (phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1) is a Rac-specific guanine nucleotide exchange factor activated by Gbetagamma subunits and by PtdIns((3,4,5))P(3). Recent studies indicate that P-Rex1 plays an important role in signaling downstream of neutrophil chemoattractant receptors. Here we report that heterologous expression of P-Rex1, but not Vav1, reconstitutes formyl peptide receptor 1 (FPR1)-mediated NADPH oxidase activation in the transgenic COS(phox) cells expressing gp91(phox), p22(phox), p67(phox) and p47(phox). A successful reconstitution requires the expression of a full-length P-Rex1 with intact DH and PH domains, and is accompanied by P-Rex1 membrane localization as well as Rac1 activation. P-Rex1-dependent superoxide generation in the reconstituted COS(phox) cells was further enhanced by expression of the novel PKC isoform PKCdelta and by overexpression of Akt. Heterologous expression of P-Rex1 in COS(phox) cells potentiated fMet-Leu-Phe-induced Akt phosphorylation, whereas expression of a constitutively active form of Akt enhanced Rac1 activation. In contrast, a dominant negative Akt mutant reduced the fMet-Leu-Phe stimulated superoxide generation as well as Rac1 activation. These results demonstrate that in COS(phox) cells, P-Rex1 is a critical component for FPR1-mediated signaling leading to NADPH oxidase activation, and there is a crosstalk between the P-Rex1-Rac pathway and Akt in superoxide generation.

  6. Acute and subacute toxicity of chemotactic conjugates between monoclonal antibody and fMet-Leu-Phe in humans: a phase I clinical trial.

    PubMed

    Obrist, R; Schmidli, J; Müller, R; Gallati, H; Obrecht, J P

    1991-01-01

    Conjugates of the chemotactic peptide fMet-Leu-Phe (fMLP) to IgG retain chemotactic and antigen recognition function in vitro and enhance intra-tumour macrophage numbers in a guinea pig model. We report a study approved by the ethics committee on the acute toxicity of fMLP conjugates in ten consenting cancer patients with metastasizing melanoma and colon cancer. They were given increasing single doses (1-2500 micrograms) IgG-fMLP made with the anti-melanoma monoclonal antibody (mAb) 9.2.27. Clinical examinations and blood cell counts, urinalysis, electrolytes, and liver and kidney function tests before and after the infusion and weekly thereafter revealed no relevant toxicities. One patient had a herpes zooster exacerbation on day 1, which was judged to be coincidental. Peak post-infusion conjugate serum concentrations fell to unmeasurable levels within a few days. In no case was a human humoral anti-(mouse Ig) immune response detected.

  7. Altered postreceptor signal transduction of formyl-Met-Leu-Phe receptors in polymorphonuclear leukocytes of patients with non-insulin-dependent diabetes mellitus.

    PubMed

    Fóris, G; Paragh, G; Dezsõ, B; Keresztes, T; Balogh, Z; Szabó, J

    1998-01-01

    The signal transduction of the formyl-Met-Leu-Phe (FMLP) receptor in polymorphonuclear leukocytes (PMNLs) from patients with non-insulin-dependent diabetes mellitus (NIDDM) was compared to that of PMNLs obtained from healthy volunteers. According to our previous studies in this group of patients neither the decrease in insulin binding capacity nor the enhanced insulin-degrading enzyme activity was involved. In control PMNLs, 10 nM FMLP induced a pertussis toxin-sensitive increase in phosphatidyl inositol (PI) cleavage and a subsequent Ca2+ signaling from the intracellular pools. On the other hand, the FMLP-induced protein kinase C (PKC) activation and translocation into the membrane could not be detected in these cells via the measurement of 32P incorporation into histone. In contrast, in PMNLs of this special group of patients suffering from NIDDM the FMLP stimulus produced a significantly low increase in PI cleavage and Ca2+ signaling from the intracellular pools. Moreover, in resting PMNLs of these patients with NIDDM, not only the [Ca2+]i but also the membrane-bound PKC activity was found to be significantly increased. In addition, PKC translocation into the cell membrane of diabetic PMNLs could be further triggered with FMLP as judged by the measurement of 32P incorporation into histone. Based on these results, it appears that the signaling of FMLP receptors in PMNLs of some NIDDM patients may have an alternative pathway through Ca2+ influx from extracellular medium, arachidonic acid cascade, and PKC activation.

  8. Distribution of the members of Anopheles gambiae and pyrethroid knock-down resistance gene (kdr) in Guinea-Bissau, West Africa.

    PubMed

    Dabiré, K R; Diabaté, A; Agostinho, F; Alves, F; Manga, L; Faye, O; Baldet, T

    2008-04-01

    An entomological survey conducted in 2002 in Guinea Bissau aimed i) to study the distribution of the members of Anopheles gambiae Giles complex (Diptera: Culicidae) throughout four ecological areas extended from mangrove to savannah ii) to evaluate the insecticide susceptibility status of these malaria vectors exposed to permethrin 0.75% and DDT4%, and finally iii) to investigate the occurrence and the spread of the Leu-Phe knock down resistance (kdr) gene associated with pyrethroid and DDT resistance within these vector populations. Adult female mosquitoes issued from indoor morning collections were tested using WHO procedures, test kits and impregnated papers to assess their insecticide susceptibility status. Tested specimens were identified by PCR assays and characterized for the kdr gene. Malaria vectors were mainly dominated elsewhere by An. gambiae s.s. (both S and M molecular forms) living in sympatry with low proportion of An. melas in the littoral. An. gambiae s.s. tested populations were fully susceptible both to permethrin 0.75% and to DDT 4% irrespective to their location and ecotypes. The Leu-Phe kdr mutation was detected at low frequency only in two sites respectively urban (Bissau) and Guinea-savannah (Gabu) areas. It occurred only in the S molecular form in Gabu (at the frequency of 0.14) and both in the S and M molecular forms in Bissau at the frequency of 0.06 and 0.02 respectively. These results suggested that the populations of An. gambiae s.s., the most frequent malaria vector in Guinea Bissau, still remain cross-susceptible to pyrethroids and DDT This susceptibility status and the frequency of resistance mechanism such as the kdr mutation must be monitored in the future particularly in the urban and savannah areas with continuous and intensive use of insecticides.

  9. Performance of Eriopis connexa (Coleoptera: Coccinellidae) resistant to lambda-cyhalothrin after extended recovery from knockdown.

    PubMed

    Santos, D S; Rodrigues, A R S; Torres, J B; Lira, R

    2016-12-01

    A population of the predatory lady beetle Eriopis connexa (Germar) (Coleoptera: Coccinellidae) was recorded as resistant to lambda-cyhalothrin. Adults exposed to this insecticide have recovered from knockdown after 72 h. Thus, the performance of resistant (R) and susceptible (S) populations of E. connexa not exposed to insecticide (R0 and S0) and R adults recovering from knockdown 24, 48, and 72 h after exposure (R24, R48, and R72) was studied. In addition, the fertility life table parameters were calculated for one generation considering the progenies from R0, S0, and R24 populations. The recovery rate from knockdown was 69.4% for R-adults, and greater recovery rate was observed within 48 h following lambda-cyhalothrin exposure. The S-females produced about 50% more eggs and lived longer, when compared with R-females irrespective of the recovery periods after knockdown. The R-females produced similar number of eggs and exhibited similar longevity across all treatments (R0, R24, R48, and R72). Progenies produced by R- and S-populations did not exhibit consistent differences in development and survival. The fertility life table parameters showed higher intrinsic rate of population growth (rm) and lower mean generation time (T) for R0- and R24-females, when compared with those for S0-females. Thus, the time interval needed to recover from knockdown is not related to the adaptive cost of resistance in E. connexa.

  10. Role of HSF activation for resistance to heat, cold and high-temperature knock-down.

    PubMed

    Nielsen, Morten Muhlig; Overgaard, Johannes; Sørensen, Jesper Givskov; Holmstrup, Martin; Justesen, Just; Loeschcke, Volker

    2005-12-01

    Regulation of heat shock proteins (Hsps) by the heat shock factor (HSF) and the importance of these proteins for resistance to heat stress is well documented. Less characterized is the importance of Hsps for cold stress resistance although Hsp70 is known to be induced following long-term cold exposure in Drosophila melanogaster. In this study, a temperature-sensitive HSF mutant line was used to investigate the role of HSF activation following heat hardening, rapid cold hardening (RCH) and long-term cold acclimation (LTCA) on heat and cold resistance, and this was correlated with Hsp70 expression. In addition, the effect of HSF activation on high-temperature knock-down resistance was evaluated. We found a significantly decreased HSF activation in the mutant line as compared to a corresponding control line following heat hardening, and this was correlated with decreased heat resistance of the mutant line. However, we did not find this difference in HSF activity to be important for resistance to cold stress or high-temperature knock-down. The findings indicate that induction of stress genes regulated by HSF, such as Hsps, although occurring following LTCA, are not of major importance for cold stress resistance and neither for RCH nor high-temperature knock-down resistance in D. melanogaster.

  11. Knockdown of nucleophosmin by RNA interference reverses multidrug resistance in resistant leukemic HL-60 cells.

    PubMed

    Lin, Minhui; Hu, Jianda; Liu, Tingbo; Li, Jing; Chen, Buyuan; Chen, Xinji

    2013-09-01

    Nucleophosmin, a multifunctional nucleolar phosphoprotein, is involved in many cellular activities. However, the role of NPM in drug-resistance of leukemia has not yet been explored. We designed and selected one shRNA targeting on NPM gene transduction into HL-60 and HL-60/ADR cell lines (an adriamycin resistant cell line) by lentivirus. Cell proliferation, apoptosis and differentiation were assessed. The expressions of the related genes and proteins were detected by real-time quantitative RT-PCR and Western blotting. The results showed obvious down-regulation of NPM mRNA and protein levels after NPM RNAi. NPM-targeted RNAi also resulted in many cellular changes, such as, suppressing cell proliferation and inducing cell differentiation. Down-regulation of NPM gene could arrest the cell cycle progression, an increase in the proportion of G0/G1 phase in knockdown groups. NPM gene silencing could also induce pro-apoptotic genes and proteins expression, and inhibit anti-apoptotic genes/proteins expression. Furthermore, IC50 of two chemotherapeutic agents (adriamycin and ADR; daunorubicin and DNR) to HL-60 and HL-60/ADR cells decreased, especially more remarkable on HL-60/ADR cells. IC50 of ADR on HL-60/ADR cells was reduced from 12.544 ± 0.851 μmol/L (before NPM RNAi) to 6.331 ± 0.522 μmol/L (after NPM RNAi), IC50 of DNR was reduced from 2.152 ± 0.143 μmol/L (before NPM RNAi) to 1.116 ± 0.093 μmol/L (after NPM RNAi). The relative reversal rate of HL-60/ADR cells on ADR was 50.2%, and on DNR was 48.9%. In conclusion, our results demonstrated that shRNA expression vectors could effectively reduce NPM expression and restore the drug sensitivity of resistant leukemic cells to conventional chemotherapeutic agents.

  12. Response of Two Heat Shock Genes to Selection for Knockdown Heat Resistance in Drosophila Melanogaster

    PubMed Central

    McColl, G.; Hoffmann, A. A.; McKechnie, S. W.

    1996-01-01

    To identify genes involved in stress resistance and heat hardening, replicate lines of Drosophila melanogaster were selected for increased resistance to knockdown by a 39° heat stress. Two selective regimes were used, one with and one without prior hardening. Mean knockdown times were increased from ~5 min to >20 min after 18 generations. Initial realized heritabilities were as high as 10% for lines selected without hardening, and crosses between lines indicated simple additive gene effects for the selected phenotypes. To survey allelic variation and correlated selection responses in two candidate stress genes, hsr-omega and hsp68, we applied denaturing gradient gel electrophoresis to amplified DNA sequences from small regions of these genes. After eight generations of selection, allele frequencies at both loci showed correlated responses for selection following hardening, but not without hardening. The hardening process itself was associated with a hsp68 frequency change in the opposite direction to that associated with selection that followed hardening. These stress loci are closely linked on chromosome III, and the hardening selection established a disequilibrium, suggesting an epistatic effect on resistance. The data indicate that molecular variation in both hsr-omega and hsp68 contribute to natural heritable variation for hardened heat resistance. PMID:8844150

  13. Molecular Ecology of Pyrethroid Knockdown Resistance in Culex pipiens pallens Mosquitoes

    PubMed Central

    Zhang, Donghui; Shi, Linna; Zhou, Guofa; Gong, Maoqing; Zhou, Huayun; Sun, Yan; Ma, Lei; He, Ji; Hong, Shanchao; Zhou, Dan; Xiong, Chunrong; Chen, Chen; Zou, Ping; Zhu, Changliang; Yan, Guiyun

    2010-01-01

    Pyrethroid insecticides have been extensively used in China and worldwide for public health pest control. Accurate resistance monitoring is essential to guide the rational use of insecticides and resistance management. Here we examined the nucleotide diversity of the para-sodium channel gene, which confers knockdown resistance (kdr) in Culex pipiens pallens mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations. We developed and validated allele-specific PCR and the real-time TaqMan methods for resistance diagnosis. The real-time TaqMan method is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity. Significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014S mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance monitoring in natural Cx. pipiens pallens populations in the East China region. The laboratory selection experiment found that L1014F mutation frequency, but not L1014S mutation, responded to deltamethrin selection, suggesting that the L1014F mutation is the key mutation conferring resistance to deltamethrin. High L1014F mutation frequency detected in six populations of Cx. pipens pallens suggests high prevalence of pyrethroid resistance in Eastern China, calling for further surveys to map the resistance in China and for investigating alternative mosquito control strategies. PMID:20657783

  14. Diversity of knockdown resistance alleles in a single house fly population facilitates adaptation to pyrethroid insecticides.

    PubMed

    Kasai, S; Sun, H; Scott, J G

    2017-02-01

    Insecticide use exerts a tremendous selection force on house fly populations, but the frequencies of the initial resistance mutations may not reach high levels if they have a significant fitness cost in the absence of insecticides. However, with the continued use of the same (or similar) insecticides, it is expected that new mutations (conferring equal or greater resistance, but less of a fitness cost) will evolve. Pyrethroid insecticides target the insect voltage sensitive sodium channel (VSSC) and have been widely used for control of house flies at animal production facilities for more than three decades. There are three Vssc mutations known that cause resistance to pyrethroids in house flies: knockdown resistance (kdr, L1014F), kdr-his (L1014H) and super-kdr (M918T + L1014F). Whether or not there are any new mutations in house fly populations has not been examined for decades. We collected house flies from a dairy in Kansas (USA) and selected this population for three generations. We discovered multiple new Vssc alleles, including two that give very high levels of resistance to most pyrethroids. The importance of these findings to understanding the evolution of insecticide resistance, designing appropriate resistance monitoring and management schemes, and the future of pyrethroids for house fly control are discussed. © 2016 The Royal Entomological Society.

  15. Knockdown Resistance Mutations in Aedes aegypti (Diptera: Culicidae) From Puerto Rico.

    PubMed

    Ponce-García, Gustavo; Del Río-Galvan, Samantha; Barrera, Roberto; Saavedra-Rodriguez, Karla; Villanueva-Segura, Karina; Felix, Gilberto; Amador, Manuel; Flores, Adriana E

    2016-11-01

    Permethrin resistance is widespread in Aedes aegypti (L.), the main dengue, zika, and chikungunya virus vector in Latin America and the Caribbean. A common mechanism of resistance to pyrethroids-knockdown resistance (kdr)-is conferred through mutations in the insect's voltage-dependent sodium channel. In this mosquito, around 10 replacement substitutions in the voltage-gated sodium channel gene (vgsc) have been reported in pyrethroid-resistant strains. Two of these mutations, named Ile1,016 and Cys1,534, are widespread in mosquito populations from Latin America and the Caribbean. This study assessed the levels of permethrin resistance and the frequency of two kdr mutations in eight Ae. aegypti populations collected in Puerto Rico in 2013. Permethrin resistance factors ranged from 33-214-fold relative to the New Orleans reference strain. The frequency of kdr mutation Ile1,016 ranged from 0.65 to fixation (1.0), and for Cys1,534 frequencies varied from 0.8 to fixation. Alarmingly, two populations-Carolina and Caguas-reached fixation at both loci. Our results suggest that permethrin effectiveness for Ae. aegypti control is compromised in these collections from Puerto Rico. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  16. Determination, mechanism and monitoring of knockdown resistance in permethrin-resistant human head lice, Pediculus humanus capitis

    PubMed Central

    Clark, J. Marshall

    2009-01-01

    Permethrin resistance has been reported worldwide and clinical failures to commercial pediculicides containing permethrin have likewise occurred. Permethrin resistance in head lice populations from the U.S. is widespread but is not yet uniform and the level of resistance is relatively low (~4–8 fold). Permethrin-resistant lice are cross-resistant to pyrethrins, PBO-synergized pyrethrins and to DDT. Nix®, when applied to human hair tufts following manufacture’s instructions, did not provide 100% control when assessed by the hair tuft bioassay in conjunction with the in vitro rearing system. Resistance to permethrin is due to knockdown resistance (kdr), which is the result of three point mutations within the α-subunit gene of the voltage-gated sodium channel that causes amino acid substitutions, leading to nerve insensitivity. A three-tiered resistance monitoring system has been established based on molecular resistance detection techniques. Quantitative sequencing (QS) has been developed to predict the kdr allele frequency in head lice at a population level. The speed, simplicity and accuracy of QS made it an ideal candidate for a routine primary resistance monitoring tool to screen a large number of louse populations as an alternative to conventional bioassay. As a secondary monitoring method, real-time PASA (rtPASA) has been devised for a more precise determination of low resistance allele frequencies. To obtain more detailed information on resistance allele zygosity, as well as allele frequency, serial invasive signal amplification reaction (SISAR) has been developed as an individual genotyping method. Our approach of using three tiers of molecular resistance detection should facilitate large-scale routine resistance monitoring of permethrin resistance in head lice using field-collected samples. PMID:20161186

  17. Molecular detection of knockdown resistance (kdr) in Blattella germanica (Blattodea: Blattellidae) from northwestern Iran.

    PubMed

    Gholizadeh, S; Nouroozi, B; Ladonni, H

    2014-09-01

    Pyrethroid insecticides are highly insecticidal compounds that are widely used against the German cockroach, a significant household insect pest. In several insect species, there is a point mutation in the para-type sodium channel gene associated with knockdown resistance (kdr). In the current study, genomic DNA was analyzed in the region where the kdr and super-kdr (an enhanced form of pyrethroid resistance) mutations reside in Blatella germanica (L., 1767) (Blattodea: Blattellidae) collected from Iran. Studies on the extracted DNA from hand-captured German cockroach specimens were conducted by polymerase chain reaction and sequencing to detect related mutations. The kdr mutation, substitution of G for C (L1014F), which results in amino acid replacement (leucine with phenylalanine), was detected in all 18 sequenced specimens from three different locations. However, the super-kdr mutation (M918T), which is detected in super-kdr house flies, was not found in the sequences of the current study. The high ratio of the kdr mutation in a field population of B. germanica in Urmia confirms that the individuals are homozygous. These data should be helpful in designing and implementing a control program and resistance management.

  18. Monooxygenase Levels and Knockdown Resistance (kdr) Allele Frequencies in Anopheles gambiae and Anopheles arabiensis in Kenya

    PubMed Central

    Chen, Hong; Githeko, Andrew K; Githure, John I; Mutunga, James; Zhou, Guofa; Yan, Guiyun

    2013-01-01

    Pyrethroid-treated bed nets and indoor spray are important components of malaria control strategies in Kenya. Information on resistance to pyrethroid insecticides in Anopheles gambiae and An. arabiensis populations is essential to the selection of appropriate insecticides and the management of insecticide resistance. Monooxygenase activity and knockdown resistance (kdr) allele frequency are biochemical and molecular indicators of mosquito resistance to pyrethroids. This study determined baseline information on monooxygenase activity and kdr allele frequency in anopheline mosquitoes in the western region, the Great Rift Valley-central province region, and the coastal region of Kenya. A total of 1990 field-collected individuals, representing 12 An. gambiae and 22 An. arabiensis populations was analyzed. We found significant among-population variation in monooxygenase activity in An. gambiae and An. arabiensis and substantial variability among individuals within populations. Nine out of 12 An. gambiae populations exhibited significantly higher average monooxygenase activity than the susceptible Kisumu reference strain. The kdr alleles (L1014S) were detected in three An. gambiae populations, and one An. arabiensis population in western Kenya, but not in the Rift Valley-central region and the coastal Kenya region. All genotypes with the kdr alleles were heterozygous, and the conservative estimation of kdr allele frequency was below 1% in these four populations. Information on monooxygenase activity and kdr allele frequency reported in this study provided baseline data for monitoring insecticide resistance changes in Kenya during the era when large-scale insecticide-treated bednet and indoor residual spray campaigns were being implemented. PMID:18402140

  19. Knockdown of LYRM1 rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

    PubMed

    Zhang, Min; Qin, Zhen-Ying; Dai, Yong-mei; Wang, Yu-Mei; Zhu, Guan-zhong; Zhao, Ya-Ping; Ji, Chen-Bo; Zhu, Jin-Gai; Shi, Chun-Mei; Qiu, Jie; Cao, Xin-Guo; Guo, Xi-Rong

    2014-09-01

    LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.

  20. Knockdown Resistance Allele Frequencies in North American Head Louse (Anoplura: Pediculidae) Populations

    PubMed Central

    Yoon, Kyong Sup; Previte, Domenic J.; Hodgdon, Hilliary E.; Poole, Bryan C.; Kwon, Deok Ho; El-Ghar, Gamal E. Abo; Lee, Si Hyeock; Clark, J. Marshall

    2014-01-01

    The study examines the extent and frequency of a knockdown-type resistance allele (kdr type) in North American populations of human head lice. Lice were collected from 32 locations in Canada and the United States. DNA was extracted from individual lice and used to determine their zygosity using the serial invasive signal amplification technique to detect the kdr-type T917I (TI) mutation, which is most responsible for nerve insensitivity that results in the kdr phenotype and permethrin resistance. Previously sampled sites were resampled to determine if the frequency of the TI mutation was changing. The TI frequency was also reevaluated using a quantitative sequencing method on pooled DNA samples from selected sites to validate this population genotyping method. Genotyping substantiated that TI occurs at high levels in North American lice (88.4%). Overall, the TI frequency in U.S. lice was 84.4% from 1999 to 2009, increased to 99.6% from 2007 to 2009, and was 97.1% in Canadian lice in 2008. Genotyping results using the serial invasive signal amplification reaction (99.54%) and quantitative sequencing (99.45%) techniques were highly correlated. Thus, the frequencies of TI in North American head louse populations were found to be uniformly high, which may be due to the high selection pressure from the intensive and widespread use of the pyrethrins- or pyrethroid-based pediculicides over many years, and is likely a main cause of increased pediculosis and failure of pyrethrins- or permethrin-based products in Canada and the United States. Alternative approaches to treatment of head lice infestations are critically needed. PMID:24724296

  1. Knockdown of the Inhibitor of Apoptosis BRUCE Sensitizes Resistant Breast Cancer Cells to Chemotherapeutic Agents.

    PubMed

    Garrison, Jason B; Ge, Chunmin; Che, Lixiao; Pullum, Derek A; Peng, Guang; Khan, Sohaib; Ben-Jonathan, Nira; Wang, Jiang; Du, Chunying

    2015-04-01

    Management of patients with breast cancer often fails because of inherent or acquired resistance to chemotherapy. BRUCE (BIR repeat containing ubiquitin-conjugating enzyme) is a member of the inhibitor of apoptosis protein (IAP) family. It has various cellular functions including suppression of apoptosis and promotion of cytokinesis. Furthermore, it pays a critical role in promotion of DNA damage repair and preservation of genome stability, a new function recently reported by our group. Although BRUCE is expressed in breast cancer cell lines, its expression in human primary breast tumors and its contribution to chemoresistance in breast cancers has not been explored. Chemotherapeutic drugs are used in the treatment of breast cancer patients. However, they are not effective to all patients and patients often develop resistance. Consequently we explored if BRUCE protein level, as judged by immunohistochemistry (IHC), is higher in primary breast tumors than normal breast tissue. We also examined if depletion of BRUCE, using a lentiviral shRNA approach, enhances cell sensitivity to multiple chemotherapeutic agents, including cisplatin, an agent that induces DNA damage by generating DNA cross-links, and taxol, a microtubule stabilizer and mitotic inhibitor. The reason for including these two chemotherapeutic agents in this study is that they hit two essential cellular processes of DNA repair and cytokinesis in which BRUCE plays critical roles. IHC analysis of BRUCE revealed significantly higher levels of BRUCE in primary breast tumors than normal breast tissue. Knockdown of BRUCE protein expression by lentiviral shRNA resulted in increased sensitivity to cisplatin in the resistant breast cancer MDB-MD-231 cell line. Moreover, depletion of BRUCE in this cell line achieved a more profound level of cell killing when coupled to low doses of cisplatin and taxol combined, rather than either drug used alone. Our data suggest that elevated protein levels of BRUCE in breast

  2. PCR-based assay to survey for knockdown resistance to pyrethroid acaricides in human scabies mites (Sarcoptes scabiei var hominis).

    PubMed

    Pasay, Cielo; Walton, Shelley; Fischer, Katja; Holt, Deborah; McCarthy, James

    2006-04-01

    Permethrin, in the form of a topical cream, is being increasingly used for community-based programs to control endemic scabies. The development of resistance has reduced the use of pyrethroids for the control of many arthropods of economic and health importance. The best recognized form of pyrethroid resistance, known as knockdown resistance or kdr, has been linked to specific mutations in the target of these agents, the para-homologous voltage-sensitive sodium channel gene (Vssc). To develop tools to study resistance to pyrethroid acaricides, we cloned 3711 and 6151 bp, respectively, of cDNA and genomic fragments of the Vssc gene from scabies mite, Sarcoptes scabiei. The sequence encompasses the major polymorphic amino acid residues associated with pyrethroid resistance. A polymerase chain reaction-based strategy has been developed that enables genotyping individual scabies mites. This will facilitate early detection and monitoring of pyrethroid resistance in scabies mite populations under drug selection pressure.

  3. CGI-58 knockdown sequesters diacylglycerols in lipid droplets/ER-preventing diacylglycerol-mediated hepatic insulin resistance

    PubMed Central

    Cantley, Jennifer L.; Yoshimura, Toru; Camporez, Joao Paulo G.; Zhang, Dongyan; Jornayvaz, Francois R.; Kumashiro, Naoki; Guebre-Egziabher, Fitsum; Jurczak, Michael J.; Kahn, Mario; Guigni, Blas A.; Serr, Julie; Hankin, Joseph; Murphy, Robert C.; Cline, Gary W.; Bhanot, Sanjay; Manchem, Vara Prasad; Brown, J. Mark; Samuel, Varman T.; Shulman, Gerald I.

    2013-01-01

    Comparative gene identification 58 (CGI-58) is a lipid droplet-associated protein that promotes the hydrolysis of triglyceride by activating adipose triglyceride lipase. Loss-of-function mutations in CGI-58 in humans lead to Chanarin–Dorfman syndrome, a condition in which triglyceride accumulates in various tissues, including the skin, liver, muscle, and intestines. Therefore, without adequate CGI-58 expression, lipids are stored rather than used for fuel, signaling intermediates, and membrane biosynthesis. CGI-58 knockdown in mice using antisense oligonucleotide (ASO) treatment also leads to severe hepatic steatosis as well as increased hepatocellular diacylglycerol (DAG) content, a well-documented trigger of insulin resistance. Surprisingly, CGI-58 knockdown mice remain insulin-sensitive, seemingly dissociating DAG from the development of insulin resistance. Therefore, we sought to determine the mechanism responsible for this paradox. Hyperinsulinemic-euglycemic clamp studies reveal that the maintenance of insulin sensitivity with CGI-58 ASO treatment could entirely be attributed to protection from lipid-induced hepatic insulin resistance, despite the apparent lipotoxic conditions. Analysis of the cellular compartmentation of DAG revealed that DAG increased in the membrane fraction of high fat-fed mice, leading to PKCɛ activation and hepatic insulin resistance. However, DAG increased in lipid droplets or lipid-associated endoplasmic reticulum rather than the membrane of CGI-58 ASO-treated mice, and thus prevented PKCɛ translocation to the plasma membrane and induction of insulin resistance. Taken together, these results explain the disassociation of hepatic steatosis and DAG accumulation from hepatic insulin resistance in CGI-58 ASO-treated mice, and highlight the importance of intracellular compartmentation of DAG in causing lipotoxicity and hepatic insulin resistance. PMID:23302688

  4. Determination of knockdown resistance allele frequencies in global human head louse populations using the serial invasive signal amplification reaction

    PubMed Central

    Hodgdon, Hilliary E.; Yoon, Kyong Sup; Previte, Domenic J.; Kim, Hyo Jeong; Aboelghar, Gamal E.; Lee, Si Hyeock; Clark, J. Marshall

    2010-01-01

    BACKGROUND Pediculosis is the most prevalent parasitic infestation of humans. Resistance to pyrethrin- and pyrethroid-based pediculicides is due to knockdown (kdr)-type point mutations in the voltage-sensitive sodium channel α-subunit gene. Early detection of resistance is crucial for the selection of effective management strategies. RESULTS Kdr allele frequencies of lice from 14 countries were determined using serial invasive signal amplification reaction. Lice collected from Uruguay, UK and Australia had kdr allele frequencies of 100% while lice from Ecuador, Papua New Guinea, South Korea and Thailand had kdr allele frequencies of 0%. The remaining 7 countries investigated, including seven US populations, two Argentina, Brazil, Denmark, Czech Republic, Egypt and Israel, displayed variable kdr allele frequencies, ranging from 11% to 97%. CONCLUSION The newly developed and validated SISAR method is suitable for accurate monitoring of kdr allele frequencies in head lice. Proactive management is needed where kdr-type resistance is not yet saturated. Based on sodium channel insensitivity and its occurrence in louse populations resistant to pyrethrin- and pyrethroid-based pediculicides, the T917I mutation appears a key marker for resistance. Results from the Egyptian population, however, indicate that phenotypic resistance of lice with single or double mutations (M815I and/or L920F) should also be determined. PMID:20564731

  5. Widespread distribution of knockdown resistance mutations in the bed bug, Cimex lectularius (Hemiptera: Cimicidae), populations in the United States.

    PubMed

    Zhu, Fang; Wigginton, John; Romero, Alvaro; Moore, Ali; Ferguson, Kimberly; Palli, Roshan; Potter, Michael F; Haynes, Kenneth F; Palli, Subba R

    2010-04-01

    We previously reported high deltamethrin resistance in bed bugs, Cimex lectularius, collected from multiple areas of the United States (Romero et al., 2007). Recently, two mutations, the Valine to Leucine mutation (V419L) and the Leucine to Isoleucine mutation (L925I) in voltage-gated sodium channel alpha-subunit gene, had been identified to be responsible for knockdown resistance (kdr) to deltamethrin in bed bugs collected from New York (Yoon et al., 2008). The current study was undertaken to investigate the distribution of these two kdr mutations in 110 bed bug populations collected in the United States. Out of the 17 bed bug populations that were assayed for deltamethrin susceptibility, two resistant populations collected in the Cincinnati area and three deltamethrin-susceptible lab colonies showed neither of the two reported mutations (haplotype A). The remaining 12 populations contained L925I or both V419L and L925I mutations in voltage-gated sodium channel alpha-subunit gene (haplotypes B&C). In 93 populations that were not assayed for deltamethrin susceptibility, 12 contained neither of the two mutations (haplotype A) and 81 contained L925I or V419L or both mutations (haplotypes B-D). Thus, 88% of the bed bug populations collected showed target-site mutations. These data suggest that deltamethrin resistance conferred by target-site insensitivity of sodium channel is widely spread in bed bug populations across the United States.

  6. Nuclear proteomics with XRCC3 knockdown to reveal the development of doxorubicin-resistant uterine cancer.

    PubMed

    Chang, Jo-Fan; Lin, Szu-Ting; Hung, Eric; Lu, Yi-Ling; Soon May, Eugenie Wong; Lo, Yi-Wen; Chou, Hsiu-Chuan; Chan, Hong-Lin

    2014-06-01

    The nucleus is a key organelle in mammary cells, which is responsible for several cellular functions including cell proliferation, gene expression, and cell survival. In addition, the nucleus is the primary targets of doxorubicin treatment. In the current study, low-abundance nuclear proteins were enriched for proteomic analysis by using a state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the nuclear protein profiling changes responsible for the development of doxorubicin resistance in human uterine cancer cells. The results of the nuclear proteomic analysis indicated that more than 2100 protein features were resolved from an equal pooled amount of three purified nuclear proteins and 117 differentially expressed spots were identified. Of these 117 identified proteins, 48 belonged to nuclear proteins and a positive correlation was observed between the expression levels of 32 of these nuclear proteins and an increase in drug resistance. According to our review of relevant research, nuclear proteins such as DNA repair protein XRCC3 (XRCC3) have not been reported to play roles in the formation of doxorubicin resistance. Previous studies have used RNA interference and cell viability analysis to evidence the essential roles of XRCC3 on its potency in the formation of doxorubicin resistance. To sum up, our nuclear proteomic approaches enabled us to identify numerous proteins, including XRCC3, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for treating doxorubicin-resistant uterine cancer.

  7. Combination therapy utilizing shRNA knockdown and an optimized resistant transgene for rescue of diseases caused by misfolded proteins.

    PubMed

    Li, Chengwen; Xiao, Pingjie; Gray, Steven James; Weinberg, Marc Scott; Samulski, R Jude

    2011-08-23

    Molecular knockdown of disease proteins and restoration of wild-type activity represent a promising but challenging strategy for the treatment of diseases that result from the accumulation of misfolded proteins (i.e., Huntington disease, amyotrophic lateral sclerosis, and α-1 antitrypsin deficiency). In this study we used alpha-1 antitrypsin (AAT) deficiency with the piZZ mutant phenotype as a model system to evaluate the efficiency of gene-delivery approaches that both silence the piZZ transcript (e.g., shRNA) and restore circulating wild-type AAT expression from resistant codon-optimized AAT (AAT-opt) transgene cassette using adeno-associated virus (AAV) vector delivery. After systemic injection of a self-complimentary AAV serotype 8 (scAAV8) vector encoding shRNA in piZZ transgenic mice, both mutant AAT mRNA in the liver and defected serum protein level were inhibited by 95%, whereas liver pathology, as monitored by dPAS and fibrosis staining, reversed. To restore blood AAT levels in AAV8/shRNA-treated mice, several strategies to restore functional AAT levels were tested, including using AAV AAT-opt transgene cassettes targeted to muscle and liver, or combination vectors carrying piZZ shRNA and AAT-opt transgenes separately, or a single bicistronic AAV vector. With these molecular approaches, we observed over 90% knockdown of mutant AAT with a 13- to 30-fold increase of circulating wild-type AAT protein from the shRNA-resistant AAT-opt cassette. The molecular approaches applied in this study can simultaneously prevent liver pathology and restore blood AAT concentration in AAT deficiencies. Based on these observations, similar gene-therapy strategies could be considered for any diseases caused by accumulation of misfolded proteins.

  8. The L1014F point mutation in the house fly Vssc1 sodium channel confers knockdown resistance to pyrethroids.

    PubMed

    Smith, T J; Lee, S H; Ingles, P J; Knipple, D C; Soderlund, D M

    1997-10-01

    Voltage-sensitive sodium channels encoded by a full-length cDNA corresponding to the Vssc1 gene of the house fly (Musca domestica) were expressed in Xenopus laevis oocytes either alone or in combination with the tipE gene product of Drosophila melanogaster and were characterized by two-electrode voltage clamp. Vssc1 cRNA alone produced very small (50-150 nA) sodium currents, whereas the combination of Vssc1 and tipE cRNAs produced robust (0.5-3 microA), rapidly inactivating sodium currents. The pyrethroid insecticide cismethrin prolonged the sodium current carried by Vssc1/tipE sodium channels during a depolarizing pulse and induced a tail current after repolarization. The Vssc1 cDNA was specifically mutated to substitute phenylalanine for leucine at position 1014 of the inferred amino acid sequence (L1014F), a polymorphism shown previously to be associated with the kdr (knockdown resistance) trait of the house fly. The L1014F substitution reduced the sensitivity of expressed house fly sodium channels to cismethrin at least 10-fold and increased the rate of decay of pyrethroid-induced sodium tail currents. These results demonstrate that the resistance-associated L1014F mutation confers a reduction in the sensitivity of house fly sodium channels to pyrethroids that is sufficient to account for the kdr resistance trait.

  9. Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing.

    PubMed

    Gellatly, Kyle J; Krim, Sarah; Palenchar, Daniel J; Shepherd, Katie; Yoon, Kyong Sup; Rhodes, Christopher J; Lee, Si Hyeock; Marshall Clark, J

    2016-05-01

    Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of the kdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of the kdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (±SD) for all analyzed lice was 98.3 ± 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase of kdr-type mutations in lice over time. © The Authors 2016. Published by Oxford University Press on behalf of Entomological Society of America.

  10. Expansion of the Knockdown Resistance Frequency Map for Human Head Lice (Phthiraptera: Pediculidae) in the United States Using Quantitative Sequencing

    PubMed Central

    Gellatly, Kyle J.; Krim, Sarah; Palenchar, Daniel J.; Shepherd, Katie; Yoon, Kyong Sup; Rhodes, Christopher J.; Lee, Si Hyeock; Marshall Clark, J.

    2016-01-01

    Pediculosis is a prevalent parasitic infestation of humans, which is increasing due, in part, to the selection of lice resistant to either the pyrethrins or pyrethroid insecticides by the knockdown resistance (kdr) mechanism. To determine the extent and magnitude of the kdr-type mutations responsible for this resistance, lice were collected from 138 collection sites in 48 U.S. states from 22 July 2013 to 11 May 2015 and analyzed by quantitative sequencing. Previously published data were used for comparisons of the changes in the frequency of the kdr-type mutations over time. Mean percent resistance allele frequency (mean % RAF) values across the three mutation loci were determined from each collection site. The overall mean % RAF (±SD) for all analyzed lice was 98.3 ± 10%. 132/138 sites (95.6%) had a mean % RAF of 100%, five sites (3.7%) had intermediate values, and only a single site had no mutations (0.0%). Forty-two states (88%) had a mean % RAF of 100%. The frequencies of kdr-type mutations did not differ regardless of the human population size that the lice were collected from, indicating a uniformly high level of resistant alleles. The loss of efficacy of the Nix formulation (Prestige Brand, Tarrytown, NY) from 1998 to 2013 was correlated to the increase in kdr-type mutations. These data provide a plausible reason for the decrease in the effectiveness of permethrin in the Nix formulation, which is the parallel increase of kdr-type mutations in lice over time. PMID:27032417

  11. The cytochrome P450 CYP6P4 is responsible for the high pyrethroid resistance in knockdown resistance-free Anopheles arabiensis

    PubMed Central

    Ibrahim, Sulaiman S.; Riveron, Jacob M.; Stott, Robert; Irving, Helen; Wondji, Charles S.

    2016-01-01

    Pyrethroid insecticides are the front line vector control tools used in bed nets to reduce malaria transmission and its burden. However, resistance in major vectors such as Anopheles arabiensis is posing a serious challenge to the success of malaria control. Herein, we elucidated the molecular and biochemical basis of pyrethroid resistance in a knockdown resistance-free Anopheles arabiensis population from Chad, Central Africa. Using heterologous expression of P450s in Escherichia coli coupled with metabolism assays we established that the over-expressed P450 CYP6P4, located in the major pyrethroid resistance (rp1) quantitative trait locus (QTL), is responsible for resistance to Type I and Type II pyrethroid insecticides, with the exception of deltamethrin, in correlation with field resistance profile. However, CYP6P4 exhibited no metabolic activity towards non-pyrethroid insecticides, including DDT, bendiocarb, propoxur and malathion. Combining fluorescent probes inhibition assays with molecular docking simulation, we established that CYP6P4 can bind deltamethrin but cannot metabolise it. This is possibly due to steric hindrance because of the large vdW radius of bromine atoms of the dihalovinyl group of deltamethrin which docks into the heme catalytic centre. The establishment of CYP6P4 as a partial pyrethroid resistance gene explained the observed field resistance to permethrin, and its inability to metabolise deltamethrin probably explained the high mortality from deltamethrin exposure in the field populations of this Sudano-Sahelian An. arabiensis. These findings describe the heterogeneity in resistance towards insecticides, even from the same class, highlighting the need to thoroughly understand the molecular basis of resistance before implementing resistance management/control tools. PMID:26548743

  12. The Effects of Tenascin C Knockdown on Trabecular Meshwork Outflow Resistance

    PubMed Central

    Keller, Kate E.; Vranka, Janice A.; Haddadin, Ramez I.; Kang, Min-Hyung; Oh, Dong-Jin; Rhee, Douglas J.; Yang, Yong-feng; Sun, Ying Ying; Kelley, Mary J.; Acott, Ted S.

    2013-01-01

    Purpose. Tenascin C (TNC) is a matricellular glycoprotein whose expression in adult tissue is indicative of tissue remodeling. The purpose of the current study was to determine the localization of TNC in trabecular meshwork (TM) tissue and to analyze the effects of TNC on intraocular pressure (IOP). Methods. Human TM frontal sections were immunostained with anti-TNC and imaged by confocal microscopy. TNC mRNA and protein levels were quantitated in anterior segments perfused at physiological and elevated pressure. Short, hairpin RNA (shRNA) silencing lentivirus targeting full-length TNC (shTNC) was applied to anterior segment perfusion organ cultures. The IOPs and central corneal thickness (CCT) of wild-type, TNC−/−, and tenascin X (TNX−/−) knockout mice were measured. Results. TNC was distributed in the juxtacanalicular (JCT) region of adult human TM, predominantly in the basement membrane underlying the inner wall of Schlemm's canal. Application of shTNC lentivirus to human and porcine anterior segments in perfusion culture did not significantly affect outflow rate. Although TNC was upregulated in response to pressure, there was no difference in outflow rate when shTNC-silenced anterior segments were subjected to elevated pressure. Furthermore, IOPs and CCTs were not significantly different between TNC−/− or TNX−/− and wild-type mice. Conclusions. TNC does not appear to contribute directly to outflow resistance. However, TNC immunolocalization in the JCT of adult human eyes suggests that certain areas of the TM are being continuously remodeled with or without an IOP increase. PMID:23882691

  13. Distribution of knock-down resistance mutations in Anopheles gambiae molecular forms in west and west-central Africa

    PubMed Central

    Santolamazza, Federica; Calzetta, Maria; Etang, Josiane; Barrese, Elena; Dia, Ibrahima; Caccone, Adalgisa; Donnelly, Martin J; Petrarca, Vincenzo; Simard, Frederic; Pinto, Joao; della Torre, Alessandra

    2008-01-01

    Background Knock-down resistance (kdr) to DDT and pyrethroids in the major Afrotropical vector species, Anopheles gambiae sensu stricto, is associated with two alternative point mutations at amino acid position 1014 of the voltage-gated sodium channel gene, resulting in either a leucine-phenylalanine (L1014F), or a leucine-serine (L1014S) substitution. In An. gambiae S-form populations, the former mutation appears to be widespread in west Africa and has been recently reported from Uganda, while the latter, originally recorded in Kenya, has been recently found in Gabon, Cameroon and Equatorial Guinea. In M-form populations surveyed to date, only the L1014F mutation has been found, although less widespread and at lower frequencies than in sympatric S-form populations. Methods Anopheles gambiae M- and S-form specimens from 19 sites from 11 west and west-central African countries were identified to molecular form and genotyped at the kdr locus either by Hot Oligonucleotide Ligation Assay (HOLA) or allele-specific PCR (AS-PCR). Results The kdr genotype was determined for about 1,000 An. gambiae specimens. The L1014F allele was found at frequencies ranging from 6% to 100% in all S-form samples (N = 628), with the exception of two samples from Angola, where it was absent, and coexisted with the L1014S allele in samples from Cameroon, Gabon and north-western Angola. The L1014F allele was present in M-form samples (N = 354) from Benin, Nigeria, and Cameroon, where both M- and S-forms were sympatric. Conclusion The results represent the most comprehensive effort to analyse the overall distribution of the L1014F and L1014S mutations in An. gambiae molecular forms, and will serve as baseline data for resistance monitoring. The overall picture shows that the emergence and spread of kdr alleles in An. gambiae is a dynamic process and that there is marked intra- and inter-form heterogeneity in resistance allele frequencies. Further studies are needed to determine: i) the

  14. A simplified high-throughput method for pyrethroid knock-down resistance (kdr) detection in Anopheles gambiae

    PubMed Central

    Lynd, Amy; Ranson, Hilary; McCall, P J; Randle, Nadine P; Black, William C; Walker, Edward D; Donnelly, Martin J

    2005-01-01

    Background A single base pair mutation in the sodium channel confers knock-down resistance to pyrethroids in many insect species. Its occurrence in Anopheles mosquitoes may have important implications for malaria vector control especially considering the current trend for large scale pyrethroid-treated bednet programmes. Screening Anopheles gambiae populations for the kdr mutation has become one of the mainstays of programmes that monitor the development of insecticide resistance. The screening is commonly performed using a multiplex Polymerase Chain Reaction (PCR) which, since it is reliant on a single nucleotide polymorphism, can be unreliable. Here we present a reliable and potentially high throughput method for screening An. gambiae for the kdr mutation. Methods A Hot Ligation Oligonucleotide Assay (HOLA) was developed to detect both the East and West African kdr alleles in the homozygous and heterozygous states, and was optimized for use in low-tech developing world laboratories. Results from the HOLA were compared to results from the multiplex PCR for field and laboratory mosquito specimens to provide verification of the robustness and sensitivity of the technique. Results and Discussion The HOLA assay, developed for detection of the kdr mutation, gives a bright blue colouration for a positive result whilst negative reactions remain colourless. The results are apparent within a few minutes of adding the final substrate and can be scored by eye. Heterozygotes are scored when a sample gives a positive reaction to the susceptible probe and the kdr probe. The technique uses only basic laboratory equipment and skills and can be carried out by anyone familiar with the Enzyme-linked immunosorbent assay (ELISA) technique. A comparison to the multiplex PCR method showed that the HOLA assay was more reliable, and scoring of the plates was less ambiguous. Conclusion The method is capable of detecting both the East and West African kdr alleles in the homozygous and

  15. Detection of a Knockdown Resistance Mutation Associated with Permethrin Resistance in the Body Louse Pediculus humanus corporis by Use of Melting Curve Analysis Genotyping

    PubMed Central

    Drali, Rezak; Benkouiten, Samir; Badiaga, Sékéné; Bitam, Idir

    2012-01-01

    Louse-borne diseases are prevalent in the homeless, and body louse eradication has thus far been unsuccessful in this population. We aim to develop a rapid and robust genotyping method usable in large field-based clinical studies to monitor permethrin resistance in the human body louse Pediculus humanus corporis. We assessed a melting curve analysis genotyping method based on real-time PCR using hybridization probes to detect the M815I-T917I-L920F knockdown resistance (kdr) mutation in the paraorthologous voltage-sensitive sodium channel (VSSC) α subunit gene, which is associated with permethrin resistance. The 908-bp DNA fragment of the VSSC gene, encoding the α subunit of the sodium channel and encompassing the three mutation sites, was PCR sequenced from 65 lice collected from a homeless population. We noted a high prevalence of the 3 indicated mutations in the body lice collected from homeless people (100% for the M815I and L920F mutations and 56.73% for the T917I mutation). These results were confirmed by melting curve analysis genotyping, which had a calculated sensitivity of 100% for the M815I and T917I mutations and of 98% for the L920F mutation. The specificity was 100% for M815I and L920F and 96% for T917I. Melting curve analysis genotyping is a fast, sensitive, and specific tool that is fully compatible with the analysis of a large number of samples in epidemiological surveys, allowing the simultaneous genotyping of 96 samples in just over an hour (75 min). Thus, it is perfectly suited for the epidemiological monitoring of permethrin resistance in human body lice in large-scale clinical studies. PMID:22573588

  16. Knockdown of NYGGF4 (PID1) rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes.

    PubMed

    Shi, Chun-Mei; Wang, Yu-Mei; Zhang, Chun-Mei; Qiu, Jie; Shen, Ya-Hui; Zhu, Jin-Gai; Chen, Lin; Xu, Guang-Feng; Zhao, Ya-Ping; Ji, Chen-Bo; Guo, Xi-Rong

    2012-11-01

    NYGGF4 is a recently identified gene that is involved in obesity-associated insulin resistance. Previous data from this laboratory have demonstrated that NYGGF4 overexpression might contribute to the development of insulin resistance (IR) and to mitochondrial dysfunction. Additionally, NYGGF4 knockdown enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We designed this study to determine whether silencing of NYGGF4 in 3T3-L1 adipocytes could rescue the effect of insulin sensitivity and mitochondrial function induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to ascertain further the mechanism of NYGGF4 involvement in obesity-associated insulin resistance. We found that 3T3-L1 adipocytes, incubated with 5μM FCCP for 12h, had decreased levels of insulin-stimulated glucose uptake and had impaired insulin-stimulated GLUT4 translocation. Silencing also diminished insulin-stimulated tyrosinephosphorylation of IRS-1 and serine phosphorylation of Akt. This phenomenon contrasts with the effect of NYGGF4 knockdown on insulin sensitivity and describes the regulatory function of NYGGF4 in adipocytes insulin sensitivity. We next analyzed the mitochondrial function in NYGGF4-silenced adipocytes incubated with FCCP. NYGGF4 knockdown partly rescued the dissipation of mitochondrial mass, mitochondrial DNA, intracellular ATP synthesis, and intracellular reactive oxygen species (ROS) production occurred following the addition of FCCP, as well as inhibition of mitochondrial transmembrane potential (ΔΨm) in 3T3-L1 adipocytes incubated with FCCP. Collectively, our results suggested that addition of silencing NYGGF4 partly rescued the effect of insulin resistance and mitochondrial dysfunction in NYGGF4 silenced 3T3-L1 adipocytes incubated with FCCP, which might explain the involvement of NYGGF4-induced IR and the development of NYGGF4 in mitochondrial function.

  17. TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

    SciTech Connect

    Zhang, Wanlu; Tang, Zhuqi; Zhu, Xiaohui; Xia, Nana; Zhao, Yun; Wang, Suxin; Cui, Shiwei; Wang, Cuifang

    2015-11-20

    High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway. - Highlights: • TRAF1 accelerated PA-induced IR in HepG2 cells mediated through NF-κB signaling. • Knockdown of TRAF1 alleviated PA-induced IR in HepG2 cells. • Knockdown of TRAF1 alleviated PA-induced lipid accumulation in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced suppression of glucose uptake in HepG2 cells. • Knockdown of TRAF1 reversed PA-induced gluconeogenesis in HepG2 cells.

  18. First Report on Co-Occurrence Knockdown Resistance Mutations and Susceptibility to Beta-Cypermethrin in Anopheles sinensis from Jiangsu Province, China

    PubMed Central

    Tan, Wei-Long; Wang, Zhong-Ming; Li, Chun-Xiao; Chu, Hong-Liang; Xu, Yan; Dong, Yan-De; Wang, Zhong-can; Chen, Dong-Ya; Liu, Hui; Liu, Da-Peng; Liu, Nannan; Sun, Jun; Zhao, Tongyan

    2012-01-01

    The increasing prevalence of insecticide resistance in Anopheles sinensis, a major vector of malaria in Jiangsu province in eastern China, threatens to compromise the successful use of insecticides in malaria control strategies. It is therefore vital to understand the insecticide resistance status of An. sinensis in the region. This study examined the nucleotide diversity of the para-sodium channel and knockdown resistance (kdr) in five field populations of adult An. sinensis mosquitoes collected in Jiangsu province, identifying the L1014F and L1014C substitutions for the first time. Competitive polymerase chain reaction (PCR) amplification of specific allele (cPASA) and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) for resistance diagnosis were developed and validated. Comparing the results with direct sequencing revealed that the PCR-RFLP method was more sensitive and specific whereas the cPASA method was more convenient and suitable. The significant positive correlation between kdr allele frequency and bioassay-based resistance phenotype demonstrates that the frequency of L1014F and L1014C substitutions in the kdr gene provides a useful molecular marker for monitoring beta-cypermethrin resistance in natural populations of An. sinensis. Our results point to the L1014F substitution as the key mutation associated with beta-cypermethrin resistance. The high resistance and mutation frequency detected in the five populations also suggest cross-resistance with other pyrethroids may occur in An. sinensis, highlighting the need for further surveys to map insecticide resistance in China and the adoption of a rational management of insecticide application for resistance management and mosquito vector control. PMID:22272229

  19. RNA interference-mediated knockdown of the hydroxyacid-oxoacid transhydrogenase gene decreases thiamethoxam resistance in adults of the whitefly Bemisia tabaci

    PubMed Central

    Yang, Xin; Xie, Wen; Li, Ru-mei; Zhou, Xiao-mao; Wang, Shao-li; Wu, Qing-jun; Yang, Ni-na; Xia, Ji-xing; Yang, Ze-zong; Guo, Li-tao; Liu, Ya-ting; Zhang, You-jun

    2017-01-01

    Bemisia tabaci has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we investigated whether hydroxyacid-oxoacid transhydrogenase (HOT) is involved in resistance to the neonicotinoid insecticide thiamethoxam in the whitefly. We cloned the full-length gene that encodes HOT in B. tabaci. Its cDNA contains a 1428-bp open reading frame encoding 475 amino acid residues. Then we evaluated the mRNA expression level of HOT in different developmental stages, and found HOT expression was significantly greater in thiamethoxam resistance adults than in thiamethoxam susceptible adults. Subsequently, seven field populations of B. tabaci adults were sampled, the expression of mRNA level of HOT significant positive correlated with thiamethoxam resistance level. At last, we used a modified gene silencing system to knock-down HOT expression in B. tabaci adults. The results showed that the HOT mRNA levels decreased by 57% and thiamethoxam resistance decreased significantly after 2 days of feeding on a diet containing HOT dsRNA. The results indicated that down-regulation of HOT expression decreases thiamethoxam resistance in B. tabaci adults. PMID:28117358

  20. RNA interference-mediated knockdown of the hydroxyacid-oxoacid transhydrogenase gene decreases thiamethoxam resistance in adults of the whitefly Bemisia tabaci.

    PubMed

    Yang, Xin; Xie, Wen; Li, Ru-Mei; Zhou, Xiao-Mao; Wang, Shao-Li; Wu, Qing-Jun; Yang, Ni-Na; Xia, Ji-Xing; Yang, Ze-Zong; Guo, Li-Tao; Liu, Ya-Ting; Zhang, You-Jun

    2017-01-24

    Bemisia tabaci has developed a high level of resistance to thiamethoxam, a second generation neonicotinoid insecticide that has been widely used to control this pest. In this study, we investigated whether hydroxyacid-oxoacid transhydrogenase (HOT) is involved in resistance to the neonicotinoid insecticide thiamethoxam in the whitefly. We cloned the full-length gene that encodes HOT in B. tabaci. Its cDNA contains a 1428-bp open reading frame encoding 475 amino acid residues. Then we evaluated the mRNA expression level of HOT in different developmental stages, and found HOT expression was significantly greater in thiamethoxam resistance adults than in thiamethoxam susceptible adults. Subsequently, seven field populations of B. tabaci adults were sampled, the expression of mRNA level of HOT significant positive correlated with thiamethoxam resistance level. At last, we used a modified gene silencing system to knock-down HOT expression in B. tabaci adults. The results showed that the HOT mRNA levels decreased by 57% and thiamethoxam resistance decreased significantly after 2 days of feeding on a diet containing HOT dsRNA. The results indicated that down-regulation of HOT expression decreases thiamethoxam resistance in B. tabaci adults.

  1. Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

    PubMed

    Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

    2016-05-01

    Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

  2. Dynamics of multiple insecticide resistance in the malaria vector Anopheles gambiae in a rice growing area in South-Western Burkina Faso

    PubMed Central

    Dabiré, Kounbobr Roch; Diabaté, Abdoulaye; Djogbenou, Luc; Ouari, Ali; N'Guessan, Raphaël; Ouédraogo, Jean-Bosco; Hougard, Jean-Marc; Chandre, Fabrice; Baldet, Thierry

    2008-01-01

    Background Insecticide resistance of the main malaria vector, Anopheles gambiae, has been reported in south-western Burkina Faso, West Africa. Cross-resistance to DDT and pyrethroids was conferred by alterations at site of action in the sodium channel, the Leu-Phe kdr mutation; resistance to organophosphates and carbamates resulted from a single point mutation in the oxyanion hole of the acetylcholinesterase enzyme designed as ace-1R. Methods An entomological survey was carried out during the rainy season of 2005 at Vallée du Kou, a rice growing area in south-western Burkina Faso. At the Vallée du Kou, both insecticide resistance mechanisms have been previously described in the M and S molecular forms of An. gambiae. This survey aimed i) to update the temporal dynamics and the circumsporozoite infection rate of the two molecular forms M and S of An. gambiae ii) to update the frequency of the Leu-Phe kdr mutation within these forms and finally iii) to investigate the occurrence of the ace-1R mutation. Mosquitoes collected by indoor residual collection and by human landing catches were counted and morphologically identified. Species and molecular forms of An. gambiae, ace-1R and Leu-Phe kdr mutations were determined using PCR techniques. The presence of the circumsporozoite protein of Plasmodium falciparum was determined using ELISA. Results Anopheles gambiae populations were dominated by the M form. However the S form occurred in relative important proportion towards the end of the rainy season with a maximum peak in October at 51%. Sporozoite rates were similar in both forms. The frequency of the Leu-Phe kdr mutation in the S form reached a fixation level while it is still spreading in the M form. Furthermore, the ace-1R mutation prevailed predominately in the S form and has just started spreading in the M form. The two mutations occurred concomitantly both in M and S populations. Conclusion These results showed that the Vallée du Kou, a rice growing area

  3. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

    PubMed Central

    Myburgh, Renier; Ivic, Sandra; Pepper, Michael S.; Gers-Huber, Gustavo; Li, Duo; Audigé, Annette; Rochat, Mary-Aude; Jaquet, Vincent; Regenass, Stephan; Manz, Markus G.; Salmon, Patrick; Krause, Karl-Heinz

    2015-01-01

    ABSTRACT Gene-engineered CD34+ hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+ T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+ cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+ T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a

  4. Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s.

    PubMed

    Martinez-Torres, D; Chandre, F; Williamson, M S; Darriet, F; Bergé, J B; Devonshire, A L; Guillet, P; Pasteur, N; Pauron, D

    1998-05-01

    Pyrethroid-impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage-gated sodium channel protein recently shown to be associated with mutations of the para-type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR-based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

  5. Molecular evidence for historical presence of knock-down resistance in Anopheles albimanus, a key malaria vector in Latin America.

    PubMed

    Lol, Juan C; Castellanos, María E; Liebman, Kelly A; Lenhart, Audrey; Pennington, Pamela M; Padilla, Norma R

    2013-09-18

    Anopheles albimanus is a key malaria vector in the northern neotropics. Current vector control measures in the region are based on mass distributions of long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) with pyrethroids. Resistance to pyrethroid insecticides can be mediated by increased esterase and/or multi-function oxidase activity and/or mutations in the voltage-gated sodium channel gene. The aim of this work was to characterize the homologous kdr region of the voltage-gated sodium channel gene in An. albimanus and to conduct a preliminary retrospective analysis of field samples collected in the 1990's, coinciding with a time of intense pyrethroid application related to agricultural and public health insect control in the region. Degenerate primers were designed to amplify the homologous kdr region in a pyrethroid-susceptible laboratory strain (Sanarate) of An. albimanus. Subsequently, a more specific primer pair was used to amplify and sequence the region that contains the 1014 codon associated with pyrethroid resistance in other Anopheles spp. (L1014F, L1014S or L1014C). Direct sequencing of the PCR products confirmed the presence of the susceptible kdr allele in the Sanarate strain (L1014) and the presence of homozygous-resistant kdr alleles in field-collected individuals from Mexico (L1014F), Nicaragua (L1014C) and Costa Rica (L1014C). For the first time, the kdr region in An. albimanus is described. Furthermore, molecular evidence suggests the presence of kdr-type resistance in field-collected An. albimanus in Mesoamerica in the 1990s. Further research is needed to conclusively determine an association between the genotypes and resistant phenotypes, and to what extent they may compromise current vector control efforts.

  6. Molecular evidence for historical presence of knock-down resistance in Anopheles albimanus, a key malaria vector in Latin America

    PubMed Central

    2013-01-01

    Background Anopheles albimanus is a key malaria vector in the northern neotropics. Current vector control measures in the region are based on mass distributions of long-lasting insecticidal nets (LLINs) and focal indoor residual spraying (IRS) with pyrethroids. Resistance to pyrethroid insecticides can be mediated by increased esterase and/or multi-function oxidase activity and/or mutations in the voltage-gated sodium channel gene. The aim of this work was to characterize the homologous kdr region of the voltage-gated sodium channel gene in An. albimanus and to conduct a preliminary retrospective analysis of field samples collected in the 1990’s, coinciding with a time of intense pyrethroid application related to agricultural and public health insect control in the region. Methods Degenerate primers were designed to amplify the homologous kdr region in a pyrethroid-susceptible laboratory strain (Sanarate) of An. albimanus. Subsequently, a more specific primer pair was used to amplify and sequence the region that contains the 1014 codon associated with pyrethroid resistance in other Anopheles spp. (L1014F, L1014S or L1014C). Results Direct sequencing of the PCR products confirmed the presence of the susceptible kdr allele in the Sanarate strain (L1014) and the presence of homozygous-resistant kdr alleles in field-collected individuals from Mexico (L1014F), Nicaragua (L1014C) and Costa Rica (L1014C). Conclusions For the first time, the kdr region in An. albimanus is described. Furthermore, molecular evidence suggests the presence of kdr-type resistance in field-collected An. albimanus in Mesoamerica in the 1990s. Further research is needed to conclusively determine an association between the genotypes and resistant phenotypes, and to what extent they may compromise current vector control efforts. PMID:24330978

  7. Knockdown resistance in Anopheles vagus, An. sinensis, An. paraliae and An. peditaeniatus populations of the Mekong region

    PubMed Central

    2010-01-01

    Background In the Mekong region (Vietnam, Cambodia and Laos), a large investigation was conducted to assess the susceptibility of Anopheles species against DDT and pyrethroids. In this study, the resistance status of the potential malaria vectors An. vagus, An. sinensis, An. paraliae and An. peditaeniatus was assessed. Methods Bioassays were performed on field collected unfed female mosquitoes using the standard WHO susceptibility tests. In addition, the DIIS6 region of the para-type sodium channel gene was amplified and sequenced and four allele-specific PCR assays were developed to assess the kdr frequencies. Results In Southern Vietnam all species were DDT and pyrethroid resistant, which might suggest the presence of a kdr resistance mechanism. Sequence-analysis of the DIIS6 region of the para-type sodium channel gene revealed the presence of a L1014S kdr mutation in An. vagus, An. sinensis and An. paraliae. In An. peditaeniatus, a low frequency L1014S kdr mutation was found in combination with a high frequency L1014F kdr mutation. For pyrethroids and DDT, no genotypic differentiation was found between survivors and non-survivors for any of these species. In the two widespread species, An. vagus and An. sinensis, kdr was found only in southern Vietnam and in Cambodia near the Vietnamese border. Conclusions Different levels of resistance were measured in Laos, Cambodia and Vietnam. The kdr mutation in different Anopheles species seems to occur in the same geographical area. These species breed in open agricultural lands where malaria endemicity is low or absent and vector control programs less intensive. It is therefore likely that the selection pressure occurred on the larval stages by insecticides used for agricultural purposes. PMID:20646327

  8. Gut microbiota from metabolic disease-resistant, macrophage-specific RIP140 knockdown mice improves metabolic phenotype and gastrointestinal integrity

    PubMed Central

    Lin, Yi-Wei; Montassier, Emmanuel; Knights, Dan; Wei, Li-Na

    2016-01-01

    While fecal microbiota transplantation (FMT) presents an attractive therapeutic strategy, it remains unclear how to choose the microbiota repertoire that most effectively transfers benefit to recipients. We identified a beneficial taxonomic repertoire in a transgenic mouse model (RIP140mϕKD) which resists the development of high fat diet (HFD)-induced metabolic diseases due to enhanced anti-inflammation engineered by lowering receptor interacting protein (RIP140) expression in macrophage. We confirmed using FMT from HFD-fed RIP140mϕKD to wild type (WT) mice that recipient mice acquired the microbiota repertoire of donor mice. Importantly, FMT from RIP140mϕKD to WT not only effectively transferred the beneficial taxonomic repertoire to WT recipients, but also enabled recipient animals acquiring the anti-inflammatory status of RIP140mϕKD donor animals and avoid HFD-induced insulin resistance, which is associated with significantly improved intestinal integrity. We conclude that FMT can transfer not only microbiota but also the donors’ intestinal innate immune status and improved intestinal integrity. PMID:27929078

  9. Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP Response Element Binding Protein (CREB)

    PubMed Central

    Erion, Derek M.; Ignatova, Irena D.; Yonemitsu, Shin; Nagai, Yoshio; Chatterjee, Paula; Weismann, Dirk; Hsiao, Jennifer J.; Zhang, Dongyan; Iwasaki, Takanori; Stark, Romana; Flannery, Clare; Kahn, Mario; Carmean, Christopher M.; Yu, Xing Xian; Murray, Susan F.; Bhanot, Sanjay; Monia, Brett P.; Cline, Gary W.; Samuel, Varman T.; Shulman, Gerald I.

    2009-01-01

    Summary In patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contributes to fasting and postprandial hyperglycemia. Since CREB is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice and a STZ-treated high-fat fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with NAFLD and T2DM by down regulation of both lipogenic and gluconeogenic gene expression. PMID:19945407

  10. Knockdown transgenic Lrrk Drosophila resists paraquat-induced locomotor impairment and neurodegeneration: A therapeutic strategy for Parkinson's disease.

    PubMed

    Quintero-Espinosa, Diana; Jimenez-Del-Rio, Marlene; Velez-Pardo, Carlos

    2017-02-15

    Leucine-rich repeat kinase 2 (LRRK2) has been linked to familial and sporadic Parkinson's disease. However, it is still unresolved whether LRRK2 in dopaminergic (DAergic) neurons may or may not aggravate the phenotype. We demonstrate that knocking down (KD) the Lrrk gene by RNAi in DAergic neurons untreated or treated with paraquat (PQ) neither affected the number of DAergic clusters, tyrosine hydroxylase (TH) protein levels, lifespan nor locomotor activity when compared to control (i.e. TH/+) flies. KD transgenic Lrrk flies dramatically increased locomotor activity in presence of TH enzyme inhibitor alpha-methyl-para-tyrosine (aMT), whereas no effect on lifespan was observed in both fly lines. Most importantly, KD Lrrk flies had reduced lipid peroxidation (LPO) index alone or in presence of PQ and the antioxidant minocycline (MC, 0.5mM). Taken together, these findings suggest that Lrrk appears unessential for the viability of DAergic neurons in D. melanogaster. Moreover, Lrrk might negatively regulate homeostatic levels of dopamine, thereby dramatically increasing locomotor activity, extending lifespan, and reducing oxidative stress (OS). Our data also indicate that reduced expression of Lrrk in the DAergic neurons of transgenic TH>Lrrk-RNAi/+ flies conferred PQ resistance and absence of neurodegeneration. The present findings support the notion that reduced/suppressed LRRK2 expression might delay or prevent motor symptoms and/or frank Parkinsonism in individuals at risk to suffer autosomal dominant Parkinsonism (AD-P) by blocking OS-induced neurodegenerative processes in the DAergic neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Relationship between the para-homologous sodium channel point mutation (g --> c at nucleotide 2979) and knockdown resistance in the German cockroach using multiplex polymerase chain reaction to discern genotype.

    PubMed

    Valles, Steven M; Perera, Omaththage P; Strong, Charles A

    2003-06-01

    Extensive use of pyrethroid insecticides for urban pest control has led to widespread pyrethroid resistance in the German cockroach. A mutation at nucleotide position 2979 (G to C, causing a leucine to phenylalanine change) in the S6 transmembrane segment of domain II of the para-homologous voltage-gated sodium channel has been previously identified in knockdown-resistant cockroaches and demonstrated by site-directed mutagenesis to reduce channel sensitivity to pyrethroids. In a recent survey, 83% of pyrethroid-resistant German cockroach populations were found to possess this mutation. A German cockroach strain with a low incidence of the L993F mutation was subjected to selection pressure with cypermethrin and subsequently evaluated over several generations for the knockdown resistance phenotype. Correspondingly, we determined the genotype of individual cockroaches of each population at the 2979 position of the para-homologous gene. Genotype was discerned by development of a polymerase chain reaction method that employed a mismatched primer-template set. A direct relationship was observed between mean knockdown time and the presence of the kdr mutation. Furthermore, individuals homozygous for the kdr mutation exhibited a significantly higher mean knockdown time than heterozygotes or wildtype cockroaches. This is the first report demonstrating the progressive expression of the kdr allele in response to insecticide selection pressure.

  12. QTL for the thermotolerance effect of heat hardening, knockdown resistance to heat and chill-coma recovery in an intercontinental set of recombinant inbred lines of Drosophila melanogaster.

    PubMed

    Norry, Fabian M; Scannapieco, Alejandra C; Sambucetti, Pablo; Bertoli, Carlos I; Loeschcke, Volker

    2008-10-01

    The thermotolerance effect of heat hardening (also called short-term acclimation), knockdown resistance to high temperature (KRHT) with and without heat hardening and chill-coma recovery (CCR) are important phenotypes of thermal adaptation in insects and other organisms. Drosophila melanogaster from Denmark and Australia were previously selected for low and high KRHT, respectively. These flies were crossed to construct recombinant inbred lines (RIL). KRHT was higher in heat-hardened than in nonhardened RIL. We quantify the heat-hardening effect (HHE) as the ratio in KRHT between heat-hardened and nonhardened RIL. Composite interval mapping revealed a more complex genetic architecture for KRHT without heat-hardening than for KRHT in heat-hardened insects. Five quantitative trait loci (QTL) were found for KRHT, but only two of them were significant after heat hardening. KRHT and CCR showed trade-off associations for QTL both in the middle of chromosome 2 and the right arm of chromosome 3, which should be the result of either pleiotropy or linkage. The major QTL on chromosome 2 explained 18% and 27-33% of the phenotypic variance in CCR and KRHT in nonhardened flies, respectively, but its KRHT effects decreased by heat hardening. We discuss candidate loci for each QTL. One HHE-QTL was found in the region of small heat-shock protein genes. However, HHE-QTL explained only a small fraction of the phenotypic variance. Most heat-resistance QTL did not colocalize with CCR-QTL. Large-effect QTL for CCR and KRHT without hardening (basal thermotolerance) were consistent across continents, with apparent transgressive segregation for CCR. HHE (inducible thermotolerance) was not regulated by large-effect QTL.

  13. Co-occurrence and distribution of East (L1014S) and West (L1014F) African knock-down resistance in Anopheles gambiae sensu lato population of Tanzania

    PubMed Central

    Kabula, Bilali; Kisinza, William; Tungu, Patrick; Ndege, Chacha; Batengana, Benard; Kollo, Douglas; Malima, Robert; Kafuko, Jessica; Mohamed, Mahdi; Magesa, Stephen

    2014-01-01

    Objective Insecticide resistance molecular markers can provide sensitive indicators of resistance development in Anopheles vector populations. Assaying these makers is of paramount importance in the resistance monitoring programme. We investigated the presence and distribution of knock-down resistance (kdr) mutations in Anopheles gambiae s.l. in Tanzania. Methods Indoor-resting Anopheles mosquitoes were collected from 10 sites and tested for insecticide resistance using the standard WHO protocol. Polymerase chain reaction-based molecular diagnostics were used to genotype mosquitoes and detect kdr mutations. Results The An. gambiae tested were resistance to lambdacyhalothrin in Muheza, Arumeru and Muleba. Out of 350 An. gambiae s.l. genotyped, 35% were An. gambiae s.s. and 65% An. arabiensis. L1014S and L1014F mutations were detected in both An. gambiae s.s. and An. arabiensis. L1014S point mutation was found at the allelic frequency of 4–33%, while L1014F was at the allelic frequency 6–41%. The L1014S mutation was much associated with An. gambiae s.s. (χ2 = 23.41; P < 0.0001) and L1014F associated with An. arabiensis (χ2 = 11.21; P = 0.0008). The occurrence of the L1014S allele was significantly associated with lambdacyhalothrin resistance mosquitoes (Fisher exact P < 0.001). Conclusion The observed co-occurrence of L1014S and L1014F mutations coupled with reports of insecticide resistance in the country suggest that pyrethroid resistance is becoming a widespread phenomenon among our malaria vector populations. The presence of L1014F mutation in this East African mosquito population indicates the spreading of this gene across Africa. The potential operational implications of these findings on malaria control need further exploration. Objectif Les marqueurs moléculaires de la résistance aux insecticides peuvent fournir des indicateurs sensibles du développement de la résistance dans les populations de vecteurs Anopheles. Le test de ces

  14. With no interaction, knockdown of Apollon and MDR1 reverse the multidrug resistance of human chronic myelogenous leukemia K562/ADM cells.

    PubMed

    Chen, Jie-Ru; Jia, Xiu-Hong; Wang, Hong; Yi, Ying-Jie; Li, You-Jie

    2017-05-01

    Chemotherapy is the main treatment method for patients with chronic myeloid leukemia (CML) and has achieved marked results. However, the acquisition of multidrug resistance (MDR) has seriously affected the quality of life and survival rate of patients. The overexpression of the inhibitors of apoptosis proteins (IAPs) and the adenosine triphosphate (ATP)-dependent binding cassette (ABC) transporters are the two main causes of MDR. Apollon and MDR1 are the most important and representative members, respectively, among the IAPs and ABC transporters. In the present study, we investigated the role of Apollon and MDR1 in chemotherapy resistance and their mechanism of interaction. We respectively knocked down the expression of Apollon and MDR1 using short hairpin RNA (shRNA) in adriamycin (ADM) resistant human CML K562 cells and examined the drug sensitivity, the consequences with regard to ADM accumulation and the alterations in the expression of Apollon and MDR1. The expression levels of Apollon and MDR1 mRNA were higher in the K562/ADM cells compared with the parental K562 cells as determined by reverse transcription‑polymerase chain reaction (RT-PCR). The plasmids of Apollon and MDR1 shRNA were respectively stably transfected into K562/ADM cells using Lipofectamine 2000. The transfection efficiency was detected by fluorescence microscopy. Cell Counting Kit-8 (CCK-8) assay revealed that Apollon or MDR1 knockdown significantly increased the chemosensitivity of the K562/ADM cells to ADM. Flow cytometric assay revealed that K562/ADM/shMDR1 cells exhibited a significantly increased intracellular accumulation of ADM, and that changes were not found in the K562/ADM/shApollon cells. Compared with the parental K562/ADM cells, a significantly decreased expression of Apollon mRNA and protein was determined in the K562/ADM/shApollon cells without affecting the expression of MDR1 as determined by RT-PCR and western blotting. Likewise, the expression levels of MDR1 m

  15. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  16. Knockdown of XAB2 enhances all-trans retinoic acid-induced cellular differentiation in all-trans retinoic acid-sensitive and -resistant cancer cells.

    PubMed

    Ohnuma-Ishikawa, Kumiko; Morio, Tomohiro; Yamada, Takayuki; Sugawara, Yuji; Ono, Makoto; Nagasawa, Masayuki; Yasuda, Akio; Morimoto, Chikao; Ohnuma, Kei; Dang, Nam H; Hosoi, Hajime; Verdin, Eric; Mizutani, Shuki

    2007-02-01

    Xeroderma pigmentosum group A (XPA)-binding protein 2 (XAB2) is composed of 855 amino acids, contains 15 tetratricopeptide repeat motifs, and associates with Cockayne syndrome group A and B proteins and RNA polymerase II, as well as XPA. In vitro and in vivo studies showed that XAB2 is involved in pre-mRNA splicing, transcription, and transcription-coupled DNA repair, leading to preimplantation lethality, and is essential for mouse embryogenesis. Retinoids are effective for the treatment of preneoplastic diseases including xeroderma pigmentosum and other dermatologic diseases such as photoaging. We therefore focused on defining the effect of XAB2 on cellular differentiation in the presence of ATRA treatment. In the present study, we showed that overexpression of XAB2 inhibited ATRA-induced cellular differentiation in human rhabdomyosarcoma cell line, and that knockdown of XAB2 by small interfering RNA (siRNA) increased ATRA-sensitive cellular differentiation in the human promyelocytic leukemia cell line HL60 at both physiologic (10(-9)-10(-8) mol/L) and therapeutic (10(-7) mol/L) concentrations of ATRA. Moreover, we found that XAB2 was associated with retinoic acid receptor alpha (RARalpha) and histone deacetylase 3 in the nuclei. Finally, using siRNA against XAB2, we showed that the ATRA-resistant neuroblastoma cell line IMR-32 underwent cellular differentiation induced by ATRA at a therapeutic concentration (10(-6) mol/L). These results strongly suggest that XAB2 is a component of the RAR corepressor complex with an inhibitory effect on ATRA-induced cellular differentiation and that XAB2 plays a role in ATRA-mediated cellular differentiation as an important aspect of cancer therapy.

  17. Circumsporozoite protein rates, blood-feeding pattern and frequency of knockdown resistance mutations in Anopheles spp. in two ecological zones of Mauritania.

    PubMed

    Lekweiry, Khadijetou Mint; Salem, Mohamed Salem Ould Ahmedou; Cotteaux-Lautard, Christelle; Jarjaval, Fanny; Marin-Jauffre, Adeline; Bogreau, Hervé; Basco, Leonardo; Briolant, Sébastien; Boukhary, Ali Ould Mohamed Salem; Brahim, Khyarhoum Ould; Pagès, Frédéric

    2016-05-05

    Mosquitoes belonging to Anopheles gambiae species complex are the main malaria vector in Mauritania but data on their vector capacities, feeding habits and insecticide susceptibility are still scanty. The objectives of this study were to fill this gap. Adult Anopheles spp. mosquitoes were collected using pyrethrum spray catch method from two ecological zones of Mauritania: Nouakchott (Saharan zone) and Hodh Elgharbi region (Sahelian zone). Circumsporozoite proteins (CSP) for P. falciparum, P. vivax VK210 and P. vivax VK247 were detected by enzyme-linked immunosorbent assay (ELISA) from the female anopheline mosquitoes. To confirm CSP-ELISA results, polymerase chain reaction (PCR) was also performed. Blood meal identification was performed in all engorged females by partial sequencing of the mitochondrial cytochrome b gene. Molecular assessments of pyrethroid knockdown resistance (kdr) and insensitive acetylcholinesterase resistance (ace-1) were conducted. In Nouakchott, the only species of Anopheles identified during the survey was Anopheles arabiensis (356 specimens). In Hodh Elgharbi, 1016 specimens of Anopheles were collected, including 578 (56.9%) Anopheles rufipes, 410 (40.35%) An. arabiensis, 20 (1.96%) An. gambiae, 5 (0.5%) An. pharoensis and 3 (0.3 %) An. funestus. Three of 186 female An. arabiensis collected in Nouakchott and tested by ELISA were found positive for Plasmodium vivax VK210, corresponding to a sporozoite rate of 1.6%; however PCR confirmed infection by P. vivax sporozoite in only one of these. In Hodh Elgharbi, no mosquito was found positive for Plasmodium spp. infection. There was a statistically significant difference in the percentage of human blood-fed Anopheles spp. between Nouakchott (58.7%, 47 of 80 blood-engorged An. arabiensis females) and Hodh Elgharbi (11.1%, 2 of 18 blood-engorged mosquitoes). Analysis of the kdr polymorphisms showed 48.2% (70/145) of East African kdr mutation (L1014S) in Nouakchott compared to 10% (4/40) in Hodh

  18. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods.

    PubMed

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-08-13

    Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe - Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly

  19. Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

    PubMed Central

    Bass, Chris; Nikou, Dimitra; Donnelly, Martin J; Williamson, Martin S; Ranson, Hilary; Ball, Amanda; Vontas, John; Field, Linda M

    2007-01-01

    Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot

  20. Susceptibility of Adult Cat Fleas (Siphonaptera: Pulicidae) to Insecticides and Status of Insecticide Resistance Mutations at the Rdl and Knockdown Resistance Loci.

    PubMed

    Rust, Michael K; Vetter, Richard; Denholm, Ian; Blagburn, Byron; Williamson, Martin S; Kopp, Steven; Coleman, Glen; Hostetler, Joe; Davis, Wendell; Mencke, Norbert; Rees, Robert; Foit, Sabrina; Böhm, Claudia; Tetzner, Kathrin

    2015-08-01

    The susceptibility of 12 field-collected isolates and 4 laboratory strains of cat fleas, Ctenocephalides felis was determined by topical application of some of the insecticides used as on-animal therapies to control them. In the tested field-collected flea isolates the LD50 values for fipronil and imidacloprid ranged from 0.09 to 0.35 ng/flea and 0.02 to 0.19 ng/flea, respectively, and were consistent with baseline figures published previously. The extent of variation in response to four pyrethroid insecticides differed between compounds with the LD50 values for deltamethrin ranging from 2.3 to 28.2 ng/flea, etofenprox ranging from 26.7 to 86.7 ng/flea, permethrin ranging from 17.5 to 85.6 ng/flea, and d-phenothrin ranging from 14.5 to 130 ng/flea. A comparison with earlier data for permethrin and deltamethrin implied a level of pyrethroid resistance in all isolates and strains. LD50 values for tetrachlorvinphos ranged from 20.0 to 420.0 ng/flea. The rdl mutation (conferring target-site resistance to cyclodiene insecticides) was present in most field-collected and laboratory strains, but had no discernible effect on responses to fipronil, which acts on the same receptor protein as cyclodienes. The kdr and skdr mutations conferring target-site resistance to pyrethroids but segregated in opposition to one another, precluding the formation of genotypes homozygous for both mutations.

  1. HOXB4 knockdown reverses multidrug resistance of human myelogenous leukemia K562/ADM cells by downregulating P-gp, MRP1 and BCRP expression via PI3K/Akt signaling pathway.

    PubMed

    Wang, Hong; Jia, Xiu-Hong; Chen, Jie-Ru; Yi, Ying-Jie; Wang, Jian-Yong; Li, You-Jie; Xie, Shu-Yang

    2016-12-01

    Multidrug resistance (MDR) plays a pivotal role in human chronic myelogenous leukemia (CML) chemotherapy failure. MDR is mainly associated with the overexpression of drug efflux transporters of the ATP-binding cassette (ABC) proteins. Phosphoinositide 3-kinase (PI3K)/Akt signaling cascade is involved in the MDR phenotype and is correlated with multidrug resistance 1 (MDR1)/P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) expression in many human malignancies. Homeobox (HOX) B4, a member of the HOX gene family, has been reported to be correlated with occurrence, development, poor prognosis and drug resistance of human leukemia. In the present study, HOXB4 expression was analyzed in K562 cell line and its MDR subline K562/ADM. Compared with K562 cells, drug-resistant K562/ADM cells demonstrated evidently higher HOXB4 expression. In addition, we firstly investigated the reversal effect of HOXB4 deletion on K562/ADM cells and the underlying mechanism. The Cell Counting kit-8 (CCK-8) and flow cytometry assays showed that knockdown of HOXB4 enhanced chemosensitivity and decreased drug efflux in K562/ADM cells. Moreover, HOXB4 knockout led to downregulation of P-gp, MRP1 and BCRP expression and PI3K/Akt signaling activity, suggesting that repression of HOXB4 might be a key point to reverse MDR of K562/ADM cells.

  2. Shrimp with knockdown of LvSOCS2, a negative feedback loop regulator of JAK/STAT pathway in Litopenaeus vannamei, exhibit enhanced resistance against WSSV.

    PubMed

    Wang, Sheng; Song, Xuan; Zhang, Zijian; Li, Haoyang; L, Kai; Yin, Bin; He, Jianguo; Li, Chaozheng

    2016-12-01

    JAK/STAT pathway is one of cytokine signaling pathways and mediates diversity immune responses to protect host from viral infection. In this study, LvSOCS2, a member of suppressor of cytokine signaling (SOCS) families, has been cloned and identified from Litopenaeus vannamei. The full length of LvSOCS2 is 1601 bp, including an 1194 bp open reading frame (ORF) coding for a putative protein of 397 amino acids with a calculated molecular weight of ∼42.3 kDa. LvSOCS2 expression was most abundant in gills and could respond to the challenge of LPS, Vibrio parahaemolyticus, Staphhylococcus aureus, Poly (I: C) and white spot syndrome virus (WSSV). There are several STAT binding motifs presented in the proximal promoter region of LvSOCS2 and its expression was induced by LvJAK or LvSTAT protein in a dose dependent manner, suggesting LvSOCS2 could be the transcriptional target gene of JAK/STAT pathway. Moreover, the transcription of DmVir-1, a read out of the activation of JAK/STAT pathway in Drosophila, was promoted by LvJAK but inhibited by LvSOCS2, indicating that LvSOCS2 could be a negative regulator in this pathway and thus can form a negative feedback loop. Our previous study indicated that shrimp JAK/STAT pathway played a positive role against WSSV. In this study, RNAi-mediated knockdown of LvSOCS2 shrimps showed lower susceptibility to WSSV infection and caused lessened virus loads, which further demonstrated that the JAK/STAT pathway could function as an anti-viral immunity in shrimp.

  3. Knockdown and lethal effects of eight commercial nonconventional and two pyrethroid insecticides against moderately permethrin-resistant adult bed bugs, Cimex lectularius (L.) (Hemiptera: Cimicidae)

    USDA-ARS?s Scientific Manuscript database

    The common bed bug, Cimex lectularius (L.) (Hemiptera: Cimicidae) is undergoing a rapid resurgence in the United States during the last decade which has created a notable pest management challenge largely because the pest has developed resistance against DDT, organophosphates, carbamates, and pyreth...

  4. A novel amino acid substitution in the para-sodium channel gene in Rhipicephalus microplus (Acari: Ixodidae) associated with knockdown resistance.

    PubMed

    Aguirre, Marcelino; Flores, Adriana E; Alvarez, Genoveva; Molina, Alberto; Rodriguez, Iram; Ponce, Gustavo

    2010-12-01

    Resistance acquired by the tick Rhipicephalus microplus (Canestrini) to different types of ixodicides in Mexico has had a negative impact on national and local livestock, mainly due to the transmission of diseases such as babesiosis and anaplasmosis, among others. The technique used for the diagnosis of resistance was that in the bioassays noted in the Norma Oficial Mexicana (NOM-006-ZOO-1994). The purpose of this investigation was the determination of resistance to pyrethroids through isoleucine-phenylalanine mutation in the gene KDR, in a population of ticks from Montemorelos, NL, Mexico. Preliminary bioassays demonstrated resistance to cypermethrin and deltamethrin (27.4%) and flumethrin (36.7-34.7%). To identify the mutation, DNA was extracted from 100 mg of larvae (pools), 10 pools were assessed by PCR, in which a pair of primers designed with the program Oligo 2.0 and Amplify 1.2 amplified a 136 bp fragment containing the mutation. The PCR product was subsequently sequenced to confirm the presence of the mutation. A strain susceptible to pyrethroid insecticides (Mora strain) was used as control, but it did not show the mutation. However, the mutation was detected in 4 out of 10 samples of the strain Montemorelos.

  5. Mitochondrial proteomics with siRNA knockdown to reveal ACAT1 and MDH2 in the development of doxorubicin-resistant uterine cancer

    PubMed Central

    Lo, Yi-Wen; Lin, Szu-Ting; Chang, Shing-Jyh; Chan, Chia-Hao; Lyu, Kevin W; Chang, Jo-Fan; May, Eugenie Wong Soon; Lin, Dai-Ying; Chou, Hsiu-Chuan; Chan, Hong-Lin

    2015-01-01

    Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer. PMID:25639359

  6. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA.

    PubMed

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E; Siegfried, Blair D

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA's and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA's. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance.

  7. Knockdown of RNA Interference Pathway Genes in Western Corn Rootworms (Diabrotica virgifera virgifera Le Conte) Demonstrates a Possible Mechanism of Resistance to Lethal dsRNA

    PubMed Central

    Vélez, Ana María; Khajuria, Chitvan; Wang, Haichuan; Narva, Kenneth E.; Siegfried, Blair D.

    2016-01-01

    RNA interference (RNAi) is being developed as a potential tool for insect pest management. Increased understanding of the RNAi pathway in target insect pests will provide information to use this technology effectively and to inform decisions related to resistant management strategies for RNAi based traits. Dicer 2 (Dcr2), an endonuclease responsible for formation of small interfering RNA’s and Argonaute 2 (Ago2), an essential catalytic component of the RNA-induced silencing complex (RISC) have both been associated with the RNAi pathway in a number of different insect species including the western corn rootworm, Diabrotica virgifera virgifera (Coleoptera: Chrysomelidae). We identified both genes from a transcriptome library generated from different tissues and developmental stages of the western corn rootworm, an important target pest for transgenic plants expressing dsRNA targeting essential genes. The expression of these genes was suppressed by more than 90% after injecting gene specific dsRNA into adult rootworms. The injected beetles were then fed vATPase A dsRNA which has previously been demonstrated to cause mortality in western corn rootworm adults. The suppression of both RNAi pathway genes resulted in reduced mortality after subsequent exposure to lethal concentrations of vATPase A dsRNA as well as increased vATPase A expression relative to control treatments. Injections with dsRNA for a non-lethal target sequence (Laccase 2) did not affect mortality or expression caused by vATPase A dsRNA indicating that the results observed with Argo and Dicer dsRNA were not caused by simple competition among different dsRNA’s. These results confirm that both genes play an important role in the RNAi pathway for western corn rootworms and indicate that selection pressures that potentially affect the expression of these genes may provide a basis for future studies to understand potential mechanisms of resistance. PMID:27310918

  8. Rapid and Reversible Tubulin Tyrosination in Human Neutrophils Stimulated by the Chemotacted Peptide, fMet Leu-Phe

    DTIC Science & Technology

    1993-01-01

    microtubule polymer i~eltramo et aL.. followed by protein A-gold incubation and then stained with uranvyl 1987: Beltramo et al.. 1989 I. Tyrosine canl...bulin subunits ( Barra et aL., 1987: Beltramo et al.. noblots of neutrophil extracts and this difference likely 1.987. 1989: Raybin and Flavin, 1977a

  9. Knockdown of long non-coding RNA prostate cancer-associated ncRNA transcript 1 inhibits multidrug resistance and c-Myc-dependent aggressiveness in colorectal cancer Caco-2 and HT-29 cells.

    PubMed

    Qiao, Lei; Liu, Xiangyu; Tang, Yichao; Zhao, Zheng; Zhang, Jilong; Liu, Huayan

    2017-09-07

    The long non-coding RNA (lncRNA) prostate cancer-associated ncRNA transcript 1 (PCAT-1) has been shown to promote prostate cancer cell proliferation through c-Myc and is associated with the poor prognosis of CRC patients. In the current study, it was hypothesized that the effect of PCAT-1 on the aggressiveness of CRC cells was dependent on the function of c-Myc. Human CRC cell lines Caco-2 and HT-29 were transfected with specific PCAT-1 shRNAs, and cell migration, invasiveness, and resistance to 5-fluorouracil were measured. To elucidate the role of c-Myc in PCAT-1 function, c-Myc was overexpressed in PCAT-1-silenced CRC cells and the effect of c-Myc overexpression on the aggressiveness of PCAT-1-silenced cells was detected. The results showed that knockdown of PCAT-1 in CRC cells suppressed cell motility and invasiveness, and sensitized the cells to 5-fluorouracil, as evidenced by the reduced viability and induced apoptosis in PCAT-1-silenced cells compared to the parental cells in response to 5-fluorouracil treatment. The expression of c-Myc in PCAT-1-silenced CRC cells was down-regulated, and forced expression of c-Myc partially restored the invasiveness in PCAT-1-silenced cells. In summary, the findings outlined in the current study suggest that PCAT-1 regulates the invasiveness and drug resistance in CRC cells and that PCAT-1 may promote CRC cell invasion by modulating the expression of c-Myc.

  10. Temporal frequency of knockdown resistance mutations, F1534C and V1016G, in Aedes aegypti in Chiang Mai city, Thailand and the impact of the mutations on the efficiency of thermal fogging spray with pyrethroids.

    PubMed

    Plernsub, Suriya; Saingamsook, Jassada; Yanola, Jintana; Lumjuan, Nongkran; Tippawangkosol, Pongsri; Walton, Catherine; Somboon, Pradya

    2016-10-01

    In Thailand, control of dengue outbreaks is currently attained by the use of space sprays, particularly thermal fogging using pyrethroids, with the aim of killing infected Aedes mosquito vectors in epidemic areas. However, the principal dengue vector, Aedes aegypti, is resistant to pyrethroids conferred mainly by mutations in the voltage-gated sodium channel gene, F1534C and V1016G, termed knockdown resistance (kdr). The objectives of this study were to determine the temporal frequencies of F1534C and V1016G in Ae. aegypti populations in relation to pyrethroid resistance in Chiang Mai city, and to evaluate the impact of the mutations on the efficacy of thermal fogging with the pyrethroid deltamethrin. Larvae and pupae were collected from several areas around Chiang Mai city during 2011-2015 and reared to adulthood for bioassays for deltamethrin susceptibility. These revealed no trend of increasing deltamethrin resistance during the study period (mortality 58.0-69.5%, average 62.8%). This corresponded to no overall change in the frequencies of the C1534 allele (0.55-0.66, average 0.62) and G1016 allele (0.34-0.45, average 0.38), determined using allele specific amplification. Only three genotypes of kdr mutations were detected: C1534 homozygous (VV/CC); G1016/C1534 double heterozygous (VG/FC); and G1016 homozygous (GG/FF) indicating that the F1534C and V1016G mutations occurred on separate haplotypic backgrounds and a lack of recombination between them to date. The F1 progeny females were used to evaluate the efficacy of thermal fogging spray with Damthrin-SP(®) (deltamethrin+S-bioallethrin+piperonyl butoxide) using a caged mosquito bioassay. The thermal fogging spray killed 100% and 61.3% of caged mosquito bioassay placed indoors and outdoors, respectively. The outdoor spray had greater killing effect on C1534 homozygous and had partially effect on double heterozygous mosquitoes, but did not kill any G1016 homozygous mutants living outdoors. As this selection

  11. Turkey knockdown in successive flocks.

    PubMed

    Evans, R D; Edson, R K; Watkins, K L; Robertson, J L; Meldrum, J B; Novilla, M N

    2000-01-01

    Turkey knockdown was diagnosed in three of five flocks of hen turkeys on a single farm within a 12-mo period. The age of birds in the flocks affected ranged from 6 wk 2 days to 7 wk 4 days. The attack rate ranged from 0.02% to 0.30% with a case fatality rate in affected birds ranging from 0 to 74%. The diagnosis was made on the basis of clinical signs and histopathologic lesions associated with knockdown. The feed in all flocks contained bacitracin methylene disalicylate and monensin (Coban). Affected birds were recumbent, demonstrated paresis, and were unable to vocalize. Postmortem examination revealed few significant lesions although pallor of the adductor muscles and petechiation in adductor and gastrocnemius muscles were noted. Birds that had been recumbent for extended periods were severely dehydrated. Consistent microscopic lesions included degeneration, necrosis, and regeneration of adductor, gastrocnemius, and abdominal muscles. No lesion in cardiac tissue was noted. Results of our investigation indicated that changes in water consumption, vitamin E status, and brooder to finisher movement correlated with the occurrence of knockdown. Turkey knockdown was defined in 1993 as any condition identified in a turkey flock that has affected the neuromuscular system to a degree that a turkey is unable to walk or stand. This definition was later modified to...neuromuscular or skeletal systems to a degree that a turkey is unable to walk or stand properly. Knockdown may be associated with numerous feed, management, or disease factors alone or in combination. Dosage of monensin, feed restriction/gorging, water restriction, heat stress, copper, mycotoxins, sodium chloride in feed, and sulfa drugs have all been suggested as contributing factors; however, laboratory studies to duplicate this have not been successful. This report presents observations from a single farm at which three of five hen flocks in a single year experienced knockdown. When a flock was reported as

  12. PCTAIRE1-knockdown sensitizes cancer cells to TNF family cytokines.

    PubMed

    Yanagi, Teruki; Shi, Ranxin; Aza-Blanc, Pedro; Reed, John C; Matsuzawa, Shu-ichi

    2015-01-01

    While PCTAIRE1/PCTK1/Cdk16 is overexpressed in malignant cells and is crucial in tumorigenesis, its function in apoptosis remains unclear. Here we investigated the role of PCTAIRE1 in apoptosis, especially in the extrinsic cell death pathway. Gene-knockdown of PCTAIRE1 sensitized prostate cancer PPC1 and Du145 cells, and breast cancer MDA-MB-468 cells to TNF-family cytokines, including TNF-related apoptosis-inducing ligand (TRAIL). Meanwhile, PCTAIRE1-knockdown did not sensitize non-malignant cells, including diploid fibroblasts IMR-90 and the immortalized prostate epithelial cell line 267B1. PCTAIRE1-knockdown did not up-regulate death receptor expression on the cell surface or affect caspase-8, FADD and FLIP expression levels. PCTAIRE1-knockdown did promote caspase-8 cleavage and RIPK1 degradation, while RIPK1 mRNA knockdown sensitized PPC1 cells to TNF-family cytokines. Furthermore, the kinase inhibitor SNS-032, which inhibits PCTAIRE1 kinase activity, sensitized PPC1 cells to TRAIL-induced apoptosis. Together these results suggest that PCTAIRE1 contributes to the resistance of cancer cell lines to apoptosis induced by TNF-family cytokines, which implies that PCTAIRE1 inhibitors could have synergistic effects with TNF-family cytokines for cytodestruction of cancer cells.

  13. Glucocorticoid receptor knockdown decreases the antioxidant protection of B16 melanoma cells: an endocrine system-related mechanism that compromises metastatic cell resistance to vascular endothelium-induced tumor cytotoxicity.

    PubMed

    Obrador, Elena; Valles, Soraya L; Benlloch, María; Sirerol, J Antoni; Pellicer, José A; Alcácer, Javier; Coronado, Javier Alcácer-F; Estrela, José M

    2014-01-01

    We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in metastatic cells in vivo independent of the tumor location (liver, lung, or subcutaneous). The decrease in γ-GCS activity was associated with lower intracellular GSH levels. Nrf2- and p53-dependent down-regulation of γ-GCS was associated with a decrease in the activities of superoxide dismutase 1 and 2, catalase, glutathione peroxidase, and glutathione reductase, but not of the O2--generating NADPH oxidase. The GCR knockdown-induced decrease in antioxidant protection caused a drastic decrease in the survival of metastatic cells during their interaction with endothelial cells, both in vitro and in vivo; only 10% of cancer cells attached to the endothelium survived compared to 90% survival observed in the controls. This very low rate of metastatic cell survival was partially increased (up to 52%) in vivo by inoculating B16-F10 cells preloaded with GSH ester, which enters the cell and delivers free GSH. Taken together, our results indicate that glucocorticoid signaling influences the survival of metastatic cells during their interaction with the vascular endothelium.

  14. Glucocorticoid Receptor Knockdown Decreases the Antioxidant Protection of B16 Melanoma Cells: An Endocrine System-Related Mechanism that Compromises Metastatic Cell Resistance to Vascular Endothelium-Induced Tumor Cytotoxicity

    PubMed Central

    Obrador, Elena; Valles, Soraya L.; Benlloch, María; Sirerol, J. Antoni; Pellicer, José A.; Alcácer, Javier; Coronado, Javier Alcácer-F.; Estrela, José M.

    2014-01-01

    We previously reported an interorgan system in which stress-related hormones (corticosterone and noradrenaline), interleukin-6, and glutathione (GSH) coordinately regulate metastatic growth of highly aggressive B16-F10 melanoma cells. Corticosterone, at levels measured in tumor-bearing mice, also induces apoptotic cell death in metastatic cells with low GSH content. In the present study we explored the potential role of glucocorticoids in the regulation of metastatic cell death/survival during the early stages of organ invasion. Glucocorticoid receptor (GCR) knockdown decreased the expression and activity of γ-glutamylcysteine synthetase (γ-GCS), the rate-limiting step in GSH synthesis, in metastatic cells in vivo independent of the tumor location (liver, lung, or subcutaneous). The decrease in γ-GCS activity was associated with lower intracellular GSH levels. Nrf2- and p53-dependent down-regulation of γ-GCS was associated with a decrease in the activities of superoxide dismutase 1 and 2, catalase, glutathione peroxidase, and glutathione reductase, but not of the O2−-generating NADPH oxidase. The GCR knockdown-induced decrease in antioxidant protection caused a drastic decrease in the survival of metastatic cells during their interaction with endothelial cells, both in vitro and in vivo; only 10% of cancer cells attached to the endothelium survived compared to 90% survival observed in the controls. This very low rate of metastatic cell survival was partially increased (up to 52%) in vivo by inoculating B16-F10 cells preloaded with GSH ester, which enters the cell and delivers free GSH. Taken together, our results indicate that glucocorticoid signaling influences the survival of metastatic cells during their interaction with the vascular endothelium. PMID:24802641

  15. Association between Three Mutations, F1565C, V1023G and S996P, in the Voltage-Sensitive Sodium Channel Gene and Knockdown Resistance in Aedes aegypti from Yogyakarta, Indonesia

    PubMed Central

    Rochmijati Wuliandari, Juli; Lee, Siu Fai; White, Vanessa Linley; Tantowijoyo, Warsito; Hoffmann, Ary Anthony; Endersby-Harshman, Nancy Margaret

    2015-01-01

    Mutations in the voltage-sensitive sodium channel gene (Vssc) have been identified in Aedes aegypti and some have been associated with pyrethroid insecticide resistance. Whether these mutations cause resistance, alone or in combination with other alleles, remains unclear, but must be understood if mutations are to become markers for resistance monitoring. We describe High Resolution Melt (HRM) genotyping assays for assessing mutations found in Ae. aegypti in Indonesia (F1565C, V1023G, S996P) and use them to test for associations with pyrethroid resistance in mosquitoes from Yogyakarta, a city where insecticide use is widespread. Such knowledge is important because Yogyakarta is a target area for releases of Wolbachia-infected mosquitoes with virus-blocking traits for dengue suppression. We identify three alleles across Yogyakarta putatively linked to resistance in previous research. By comparing resistant and susceptible mosquitoes from bioassays, we show that the 1023G allele is associated with resistance to type I and type II pyrethroids. In contrast, F1565C homozygotes were rare and there was only a weak association between individuals heterozygous for the mutation and resistance to a type I pyrethroid. As the heterozygote is expected to be incompletely recessive, it is likely that this association was due to a different resistance mechanism being present. A resistance advantage conferred to V1023G homozygotes through addition of the S996P allele in the homozygous form was suggested for the Type II pyrethroid, deltamethrin. Screening of V1023G and S996P should assist resistance monitoring in Ae. aegypti from Yogyakarta, and these mutations should be maintained in Wolbachia strains destined for release in this city to ensure that these virus-blocking strains of mosquitoes are not disadvantaged, relative to resident populations. PMID:26463408

  16. ARID1A gene knockdown promotes neuroblastoma migration and invasion.

    PubMed

    Li, C; Xu, Z; Zhao, Z; An, Q; Wang, L; Yu, Y; Piao, D

    2017-03-03

    Neuroblastoma is the most common extracranial solid tumor in childhood which often acquires drug resistance and becomes aggressive phenotypes. The high-risk patients suffer from high mortality due to the limitation of the treatment strategies. ARID1A (AT-rich interactive domain-containing protein 1A), a subunit of SWI/SNF complexes, is considered as a tumor suppressor in many cancers. The aim of the present study was to investigate the effect of ARID1A on migration and invasion in neuroblastoma cells. The shRNA targeting ARID1A was designed and delivered into SK-N-SH cells to knock down ARID1A expression. Knockdown of ARID1A by shRNA significantly increased the viability and invasion ability, and caused G1 arrest inhibition and DNA synthesis increase in SK-N-SH cells. Moreover, Knockdown of ARID1A increased the activity and expression of matrix metalloproteinase (MMP)-2 and -9 in SK-N-SH cells. Furthermore, ARID1A knockdown caused diminished expression of E-cadherin, enhanced expression of N-cadherin and β-catenin nuclear translocation in SK-N-SH cells. These results suggest that loss of ARID1A may associate with the promotion of invasion and metastasis of neuroblastoma. Our findings indicate ARID1A is a tumor suppressor in neuroblastoma.

  17. Annexin A3 Knockdown Suppresses Lung Adenocarcinoma

    PubMed Central

    Liu, Qing-Qing; Zhang, Yue-Hua; Qiu, Jing-Hua

    2016-01-01

    Our previous study identified an elevated abundance of annexin A3 (Anxa3) as a novel prognostic biomarker of lung adenocarcinoma (LADC) through quantitative proteomics analysis. However, the biological functions of Anxa3 in LADC are not fully clear. In this study, in vitro and in vivo assays were performed to investigate the effects of Anxa3 downregulation on the growth, migration, invasion, metastasis, and signaling pathway activation of LADC cells. After Anxa3 downregulation, the growth of A549 and LTEP-a2 LADC cells was slowed and they showed decreased migration and invasion in vitro. Anxa3 knockdown significantly inhibited tumor formation by A549 cells in vivo; while many metastases were formed by control A549 cells, there were obvious reductions in the numbers of lung, liver, and brain metastases formed by Anxa3 knockdown in A549 cells. Furthermore, Anxa3 knockdown significantly decreased MMP-2 and N-cadherin expression and increased E-cadherin expression both in cell lines in vitro and in tumor nodules examined during in vivo tumorigenesis assays. Interestingly, Anxa3 downregulation reduced the phosphorylated levels of MEK and ERK. In summary, Anxa3 knockdown inhibited the growth, migration, invasion, and metastasis of LADC, decreased the activation of the MEK/ERK signaling pathway, and modulated the expression of MMP-2, E-cadherin, and N-cadherin. PMID:27995049

  18. The effect of Msh2 knockdown on methylating agent induced toxicity in DNA glycosylase deficient cells.

    PubMed

    Cooley, N; Elder, R H; Povey, A C

    2010-01-31

    The DNA structure recognition protein MSH2 is an important protein in DNA mismatch repair due to its role in initiating the repair process. To examine the potential interactions between mismatch repair and base excision repair (BER) we have examined the effect of MSH2 knockdown on 6-thioguanine (6-TG), temozolomide (TMZ) and methylmethane sulphonate (MMS) induced toxicity in BER proficient and deficient cell lines. An shRNA expression vector containing Msh2 target sequences was designed and used to transfect mouse embryonic fibroblasts lacking either alkylpurine DNA N-glycosylase (Mpg) or endonuclease III homologue (Nth1). Significant knockdown of Msh2 gene expression was achieved with three different target sequences, with the highest level being shown by Msh2(283). Clonal selection resulted in differing levels of knockdown in Mpg(-/-) cells: (69.0+/-12.1% from 5 cell clones). Transfection of the Msh2(283) sequence in Mpg+/+, Nth1+/+ and Nth1(-/-) cells resulted in average knockdowns of 45.1+/-40.5% (3 clones), 58.0+/-21.4% (5 clones) and 74.9+/-14.8% (3 clones), respectively. Msh2 knockdown resulted in increased resistance to 6-TG in BER (MPG and NTH1) proficient and deficient cell lines with similar levels of knockdown (84+/-4%) but increased resistance to TMZ only in Mpg+/+ and Nth1(-/-) cell lines and not Mpg(-/-) or Nth1+/+ cells as assessed by an MTT assay. Msh2 knockdown had no effect on sensitivity to MMS induced toxicity. In a clonogenic assay, Msh2 silenced Mpg+/+, Mpg(-/-), Nth1+/+ and Nth1(-/-) cells were more resistant to TMZ. These results confirm previous studies showing that MSH2 is a key protein in influencing 6-TG and O(6)-methylguanine induced toxicity but also suggest that the effect of this protein depends upon the presence of other proteins in different DNA repair pathways. 2009 Elsevier Ireland Ltd. All rights reserved.

  19. Knockout mouse production assisted by Blm knockdown

    PubMed Central

    FUKUDA, Mikiko; INOUE, Mayuko; MURAMATSU, Daisuke; MIYACHI, Hitoshi; SHINKAI, Yoichi

    2015-01-01

    Production of knockout mice using targeted embryonic stem cells (ESCs) is a powerful approach for investigating the function of specific genes in vivo. Although the protocol for gene targeting via homologous recombination (HR) in ESCs is already well established, the targeting efficiency varies at different target loci and is sometimes too low. It is known that knockdown of the Bloom syndrome gene, BLM, enhances HR-mediated gene targeting efficiencies in various cell lines. However, it has not yet been investigated whether this approach in ESCs is applicable for successful knockout mouse production. Therefore, we attempted to answer this question. Consistent with previous reports, Blm knockdown enhanced gene targeting efficiencies for three gene loci that we examined by 2.3–4.1-fold. Furthermore, the targeted ESC clones generated good chimeras and were successful in germline transmission. These data suggest that Blm knockdown provides a general benefit for efficient ESC-based and HR-mediated knockout mouse production. PMID:26598326

  20. Genotype effect on lifespan following vitellogenin knockdown.

    PubMed

    Ihle, Kate E; Fondrk, M Kim; Page, Robert E; Amdam, Gro V

    2015-01-01

    Honey bee workers display remarkable flexibility in the aging process. This plasticity is closely tied to behavioral maturation. Workers who initiate foraging behavior at earlier ages have shorter lifespans, and much of the variation in total lifespan can be explained by differences in pre-foraging lifespan. Vitellogenin (Vg), a yolk precursor protein, influences worker lifespan both as a regulator of behavioral maturation and through anti-oxidant and immune functions. Experimental reduction of Vg mRNA, and thus Vg protein levels, in wild-type bees results in precocious foraging behavior, decreased lifespan, and increased susceptibility to oxidative damage. We sought to separate the effects of Vg on lifespan due to behavioral maturation from those due to immune and antioxidant function using two selected strains of honey bees that differ in their phenotypic responsiveness to Vg gene knockdown. Surprisingly, we found that lifespans lengthen in the strain described as behaviorally and hormonally insensitive to Vg reduction. We then performed targeted gene expression analyses on genes hypothesized to mediate aging and lifespan: the insulin-like peptides (Ilp1 and 2) and manganese superoxide dismutase (mnSOD). The two honey bee Ilps are the most upstream components in the insulin-signaling pathway, which influences lifespan in Drosophila melanogaster and other organisms, while manganese superoxide dismutase encodes an enzyme with antioxidant functions in animals. We found expression differences in the llps in fat body related to behavior (llp1 and 2) and genetic background (Ilp2), but did not find strain by treatment effects. Expression of mnSOD was also affected by behavior and genetic background. Additionally, we observed a differential response to Vg knockdown in fat body expression of mnSOD, suggesting that antioxidant pathways may partially explain the strain-specific lifespan responses to Vg knockdown. Copyright © 2014 Elsevier Inc. All rights reserved.

  1. Preparation of cell-lines for conditional knockdown of gene expression and measurement of the knockdown effects on E4orf4-induced cell death.

    PubMed

    Brestovitsky, Anna; Sharf, Rakefet; Kleinberger, Tamar

    2012-10-21

    Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways(1,2). However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time(3-5). Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein(6). Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured(7-9). The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal.

  2. Preparation of Cell-lines for Conditional Knockdown of Gene Expression and Measurement of the Knockdown Effects on E4orf4-Induced Cell Death

    PubMed Central

    Brestovitsky, Anna; Sharf, Rakefet; Kleinberger, Tamar

    2012-01-01

    Functional inactivation of gene expression in mammalian cells is crucial for the study of the contribution of a protein of interest to various pathways1,2. However, conditional knockdown of gene expression is required in cases when constitutive knockdown is not tolerated by cells for a long period of time3-5. Here we describe a protocol for preparation of cell lines allowing conditional knockdown of subunits of the ACF chromatin remodeling factor. These cell lines facilitate the determination of the contribution of ACF to induction of cell death by the adenovirus E4orf4 protein6. Sequences encoding short hairpin RNAs for the Acf1 and SNF2h subunits of the ACF chromatin remodeling factor were cloned next to a doxycycline-inducible promoter in a plasmid also containing a gene for the neomycin resistance gene. Neomycin-resistant cell clones were selected in the presence of G418 and isolated. The resulting cell lines were induced by doxycycline treatment, and once Acf1 or SNF2h expression levels were reduced, the cells were transfected with a plasmid encoding E4orf4 or an empty vector. To confirm the specific effect of the shRNA constructs, Acf1 or SNF2h protein levels were restored to WT levels by cotransfection with a plasmid expressing Acf1 or SNF2h which were rendered resistant to the shRNA by introduction of silent mutations. The ability of E4orf4 to induce cell death in the various samples was determined by a DAPI assay, in which the frequency of appearance of nuclei with apoptotic morphologies in the transfected cell population was measured7-9. The protocol described here can be utilized for determination of the functional contribution of various proteins to induction of cell death by their protein partners in cases when constitutive knockdown may be cell lethal. PMID:23117279

  3. Knockdown of MLO genes reduces susceptibility to powdery mildew in grapevine

    PubMed Central

    Pessina, Stefano; Lenzi, Luisa; Perazzolli, Michele; Campa, Manuela; Dalla Costa, Lorenza; Urso, Simona; Valè, Giampiero; Salamini, Francesco; Velasco, Riccardo; Malnoy, Mickael

    2016-01-01

    Erysiphe necator is the causal agent of powdery mildew (PM), one of the most destructive diseases of grapevine. PM is controlled by sulfur-based and synthetic fungicides, which every year are dispersed into the environment. This is why PM-resistant varieties should become a priority for sustainable grapevine and wine production. PM resistance can be achieved in other crops by knocking out susceptibility S-genes, such as those residing at genetic loci known as MLO (Mildew Locus O). All MLO S-genes of dicots belong to the phylogenetic clade V, including grapevine genes VvMLO7, 11 and 13, which are upregulated during PM infection, and VvMLO6, which is not upregulated. Before adopting a gene-editing approach to knockout candidate S-genes, the evidence that loss of function of MLO genes can reduce PM susceptibility is necessary. This paper reports the knockdown through RNA interference of VvMLO6, 7, 11 and 13. The knockdown of VvMLO6, 11 and 13 did not decrease PM severity, whereas the knockdown of VvMLO7 in combination with VvMLO6 and VvMLO11 reduced PM severity up to 77%. The knockdown of VvMLO7 and VvMLO6 seemed to be important for PM resistance, whereas a role for VvMLO11 does not seem likely. Cell wall appositions (papillae) were present in both resistant and susceptible lines in response to PM attack. Thirteen genes involved in defense were less upregulated in infected mlo plants, highlighting the early mlo-dependent disruption of PM invasion. PMID:27390621

  4. Knockdown of NAPA using short-hairpin RNA sensitizes cancer cells to cisplatin: implications to overcome chemoresistance.

    PubMed

    Wu, Zchong-Zcho; Chao, Chuck C-K

    2010-09-15

    Cisplatin is a widely used anti-cancer drug which targets DNA in replicating cells. In the present study, we found that NAPA--a protein found in the endoplasmic reticulum (ER) and implicated in protein trafficking--protects cells against cisplatin. Accordingly, knockdown of NAPA using lentivirus-encoded shRNA (shNAPA) induced ER stress similar to cisplatin treatment in HEK293 cells. A low dose of cisplatin also elicited a mild ER stress response associated with the accumulation of the protective proteins BiP and NAPA. Remarkably, knockdown of NAPA induced apoptosis and enhanced cisplatin-induced cytotoxicity/apoptosis, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of NAPA increased resistance to cisplatin by reducing cisplatin-induced ER stress and apoptosis. The modulatory effects of shNAPA required the tumor suppressor p53 since the effects of NAPA knockdown were reduced by the p53 inhibitor PFT-alpha and in H1299 cells which are p53-null. A partial reversal of cisplatin resistance was also observed in cisplatin-resistant HeLa cells following knockdown of NAPA. Our results also indicated that calpain is required for ER-mediated apoptosis. Importantly, combined cisplatin/shNAPA treatment suppressed tumor growth in vivo in xenograph experiments performed in nude mice. Taken together, these observations suggest that NAPA represents a target of cisplatin, and that knockdown of NAPA may improve cisplatin-based cancer therapy.

  5. Dihydroceramide Desaturase Knockdown Impacts Sphingolipids and Apoptosis after Photodamage in Human Head and Neck Squamous Carcinoma Cells

    PubMed Central

    BREEN, PAUL; JOSEPH, NICHOLAS; THOMPSON, KYLE; KRAVEKA, JACQUELINE M.; GUDZ, TATYANA I.; LI, LI; RAHMANIYAN, MEHRDAD; BIELAWSKI, JACEK; PIERCE, JASON S.; VAN BUREN, ERIC; BHATTI, GAURAV; SEPAROVIC, DUSKA

    2014-01-01

    Background Dihydroceramide desaturase 1 (DES) is the enzyme responsible for converting dihydroceramide into ceramide in the de novo sphingolipid biosynthesis pathway. Dihydroceramide can inhibit ceramide channel formation to interfere with apoptosis. We have shown that following ceramide synthase knockdown, photodynamic therapy (PDT), a cancer treatment modality, is associated with decreased levels of ceramides and dihydroceramides in cells that are resistant to apoptosis. Aim Here we investigated the effect of DES knockdown on the sphingolipid profile and apoptosis in human head and neck squamous carcinoma cells after PDT with the silicon phthalocyanine Pc 4. Materials and Methods Following siRNA transfection and PDT treatment, quantitative real-time polymerase chain reaction for quantification of DES mRNA, immunoblotting for protein expression, mass spectrometry for sphingolipid analysis, spectrofluorometry for caspase 3-like (DEVDase) activity, flow cytometry for apoptosis detection, and trypan blue assay for cell viability evaluation, were performed. Results Down-regulation of DES led to a substantial increase in levels of dihydroceramides without affecting ceramide levels. PDT-induced accumulation of individual dihydroceramides and global ceramides was increased by DES knockdown. Concomitantly, mitochondrial depolarization, DEVDase activation, late-apoptosis and cell death were attenuated by DES knockdown. Early apoptosis, however, was enhanced. Conclusion Our findings support the following: (i) dihydroceramide reduces pro-apoptotic effects of ceramide; (ii) cells adapt to DES knockdown to become more sensitive to ceramide and early-apoptosis; (iii) DES is a potential molecular target for regulating apoptotic resistance to PDT. PMID:23267130

  6. RNF7 knockdown inhibits prostate cancer tumorigenesis by inactivation of ERK1/2 pathway

    PubMed Central

    Xiao, Yangjiong; Jiang, Yan; Song, Hongmei; Liang, Tao; Li, Yonghui; Yan, Dongliang; Fu, Qiang; Li, Zuowei

    2017-01-01

    Development of castration resistance is a key contributor to mortality in patients with prostate cancer. High expression of RING finger protein 7 (RNF7) in cancer cells is known to play a key role in tumor progression. However, the role of RNF7 in prostate cancer progression is not well elucidated. In this study, we silenced RNF7 by shRNA interference in two castration resistant prostate cancer (CRPC) cell lines, DU145 and PC3. RNF7 knockdown attenuated proliferation and enhanced sensitivity of prostate cancer cells to cisplatin treatment. Invasive property of DU145 and PC3 cells was also attenuated by RNF7 silencing. The underlying mechanisms appear to be associated with accumulation of tumor suppressive proteins p21, p27 and NOXA, while inactivation of ERK1/2 by RNF7 knockdown. We demonstrated that RNF7 knockdown induced growth suppression of prostate cancer cells and inactivated ERK1/2 pathway, which suggested RNF7 might be a potential novel therapeutic target for CRPC. PMID:28252001

  7. Enhanced toxic cloud knockdown spray system for decontamination applications

    SciTech Connect

    Betty, Rita G.; Tucker, Mark D.; Brockmann, John E.; Lucero, Daniel A.; Levin, Bruce L.; Leonard, Jonathan

    2011-09-06

    Methods and systems for knockdown and neutralization of toxic clouds of aerosolized chemical or biological warfare (CBW) agents and toxic industrial chemicals using a non-toxic, non-corrosive aqueous decontamination formulation.

  8. Knockdown of mitogen-activated protein kinase (MAPK) signalling in the midgut of Anopheles stephensi mosquitoes using antisense morpholinos.

    PubMed

    Pietri, J E; Cheung, K W; Luckhart, S

    2014-10-01

    Arthropod-borne infectious diseases are responsible for nearly 1.5 million deaths annually across the globe, with malaria responsible for >50% of these deaths. Recent efforts to enhance malaria control have focused on developing genetically modified Anopheles mosquitoes that are resistant to malaria parasite infection by manipulating proteins that are essential to the immune response. Although this approach has shown promise, the lack of efficient genetic tools in the mosquito makes it difficult to investigate innate immunity using reverse genetics. Current gene knockdown strategies based on small interfering RNA are typically labourious, inefficient, and require extensive training. In the present study, we describe the use of morpholino antisense oligomers to knockdown MEK-ERK signalling in the midgut of Anopheles stephensi through a simple feeding protocol. Anti-MEK morpholino provided in a saline meal was readily ingested by female mosquitoes with minimal toxicity and resulted in knockdown of total MEK protein levels 3-4 days after morpholino feeding. Further, anti-MEK morpholino feeding attenuated inducible phosphorylation of the downstream kinase ERK and, as predicted by previous work, reduced parasite burden in mosquitoes infected with Plasmodium falciparum. To our knowledge, this is the first example of morpholino use for target protein knockdown via feeding in an insect vector. Our results suggest this method is not only efficient for studies of individual proteins, but also for studies of phenotypic control by complex cell signalling networks. As such, our protocol is an effective alternative to current methods for gene knockdown in arthropods.

  9. Knockdown of Tlr4 in the Arcuate Nucleus Improves Obesity Related Metabolic Disorders.

    PubMed

    Zhao, Yongli; Li, Guohua; Li, Ying; Wang, Yuchuan; Liu, Zhengjuan

    2017-08-07

    High-fat diet-induced hypothalamic metabolic inflammation is emerging as a cause for the development of obesity. It is acknowledged that Toll-like receptor4 (TLR4) signaling plays a crucial role in triggering of the hypothalamic metabolic inflammation during the course of diet-induced obesity. Whether hypothalamic arcuate nucleus (ARC)-restricted TLR4 knockdown improves obesity-related metabolic disorders remains unexplored. In this study, we used TLR4 shRNA lentiviral particles to suppress the TLR4 expression in the hypothalamic ARC of diet-induced obese rat model by stereotaxic injection. Our results demonstrate that ARC-restricted TLR4 knockdown protects obese rats from diet-induced weight gain and energy intake, from diet-induced impaired glucose homeostasis and peripheral insulin resistance, and from high-fat diet-induced hepatic steatosis and adipocyte hypertrophy. Thus, we define ARC-restricted TLR4 knockdown as a potential strategy to combat metabolic disorders associated with obesity.

  10. Neuroligin-1 Knockdown Suppresses Seizure Activity by Regulating Neuronal Hyperexcitability.

    PubMed

    Fang, Min; Wei, Jin-Lai; Tang, Bo; Liu, Jing; Chen, Ling; Tang, Zhao-Hua; Luo, Jing; Chen, Guo-Jun; Wang, Xue-Feng

    2016-01-01

    Abnormally synchronized synaptic transmission in the brain leads to epilepsy. Neuroligin-1 (NL1) is a synaptic cell adhesion molecule localized at excitatory synapses. NL1 modulates synaptic transmission and determines the properties of neuronal networks in the mammalian central nervous system. We showed that the expression of NL1 and its binding partner neurexin-1β was increased in temporal lobe epileptic foci in patients and lithium-pilocarpine-treated epileptic rats. We investigated electrophysiological and behavioral changes in epileptic rats after lentivirally mediated NL1 knockdown in the hippocampus to determine whether NL1 suppression prevented seizures and, if so, to explore the probable underlying mechanisms. Our behavioral studies revealed that NL1 knockdown in epileptic rats reduced seizure severity and increased seizure latency. Whole-cell patch-clamp recordings of CA1 pyramidal neurons in hippocampal slices from NL1 knockdown epileptic rats revealed a decrease in spontaneous action potential frequency and a decrease in miniature excitatory postsynaptic current (mEPSC) frequency but not amplitude. The amplitude of N-methyl-D-aspartate receptor (NMDAR)-dependent EPSCs was also selectively decreased. Notably, NL1 knockdown reduced total NMDAR1 expression and the surface/total ratio in the hippocampus of epileptic rats. Taken together, these data indicate that NL1 knockdown in epileptic rats may reduce the frequency and severity of seizures and suppress neuronal hyperexcitability via changes in postsynaptic NMDARs.

  11. Androglobin knockdown inhibits growth of glioma cell lines

    PubMed Central

    Huang, Bo; Lu, Yi-Sheng; Li, Xia; Zhu, Zhi-Chuan; Li, Kui; Liu, Ji-Wei; Zheng, Jing; Hu, Ze-Lan

    2014-01-01

    Globin family was famous for oxygen supply function of its members such as hemoglobin and myoglobin. With the progress of research, several members of this protein family have been proven to play roles in tumors including glioma. Androglobin (ADGB) is a recently identified member of globin family with very few studies about its function. In the present study, we show that ADGB plays an oncogene role in glioma. Lentiviral vector mediated ADGB knockdown inhibited the proliferation of glioma cell lines determined by MTT assay and colony formation assay. ADGB knockdown also increased the apoptosis of glioma cell line U251 assessed by flow cytometry. In addition, western blot showed that ADGB knockdown altered levels of several proteins related to proliferation, survival or apoptosis in U251 cells. These findings suggest ADGB is involved in the progression of glioma in vitro. PMID:24966926

  12. Establishment of conditional vectors for hairpin siRNA knockdowns

    PubMed Central

    Matsukura, Shiro; Jones, Peter A.; Takai, Daiya

    2003-01-01

    Small interference RNA (siRNA) is an emerging methodology in reverse genetics. Here we report the development of a new tetracycline-inducible vector-based siRNA system, which uses a tetracycline-responsive derivative of the U6 promoter and the tetracycline repressor for conditional in vivo transcription of short hairpin RNA. This method prevents potential lethality immediately after transfection of a vector when the targeted gene is indispensable, or the phenotype of the knockdown is lethal or results in a growth abnormality. We show that the controlled knockdown of DNA methyltransferase 1 (DNMT1) in human cancer resulted in growth arrest. Removal of the inducer, doxycycline, from treated cells led to re-expression of the targeted gene. Thus the method allows for a highly controlled approach to gene knockdown. PMID:12888529

  13. The effect of alpha-synuclein knockdown on MPP+ toxicity in models of human neurons.

    PubMed

    Fountaine, Timothy M; Venda, Lara Lourenco; Warrick, Nicholas; Christian, Helen C; Brundin, Patrik; Channon, Keith M; Wade-Martins, Richard

    2008-12-01

    The protein alpha-synuclein is central to the pathophysiology of Parkinson's disease (PD) but its role in the development of neurodegeneration remains unclear. alpha-Synuclein-knockout mice develop without gross abnormality and are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial inhibitor widely used to model parkinsonism. Here we show that differentiated human dopaminergic neuron-like cells also have increased resistance to 1-methyl-4-phenylpyridine (MPP+), the active metabolite of MPTP, when alpha-synuclein is knocked down using RNA interference. In attempting to understand how this occurred we found that lowering alpha-synuclein levels caused changes to intracellular vesicles, dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2), each of which is known to be an important component of the early events leading to MPP+ toxicity. Knockdown of alpha-synuclein reduced the availability of DAT on the neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of VMAT2 molecules per vesicle by 2.8-fold. However, these changes were not associated with any reduction in MPP+ -induced superoxide production, suggesting that alpha-synuclein knockdown may have other downstream effects which are important. We then showed that alpha-synuclein knockdown prevented MPP+ -induced activation of nitric oxide synthase (NOS). Activation of NOS is an essential step in MPTP toxicity and increasing evidence points to nitrosative stress as being important in neurodegeneration. Overall, these results show that as well as having a number of effects on cellular events upstream of mitochondrial dysfunction alpha-synuclein affects pathways downstream of superoxide production, possibly involving regulation of NOS activity.

  14. Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos.

    PubMed

    Lemeer, Simone; Jopling, Chris; Gouw, Joost; Mohammed, Shabaz; Heck, Albert J R; Slijper, Monique; den Hertog, Jeroen

    2008-11-01

    The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.

  15. Knockdown of NAT12/NAA30 reduces tumorigenic features of glioblastoma-initiating cells.

    PubMed

    Mughal, Awais A; Grieg, Zanina; Skjellegrind, Håvard; Fayzullin, Artem; Lamkhannat, Mustapha; Joel, Mrinal; Ahmed, M Shakil; Murrell, Wayne; Vik-Mo, Einar O; Langmoen, Iver A; Stangeland, Biljana

    2015-08-21

    Glioblastoma (GBM) is the most common primary brain malignancy and confers a dismal prognosis. GBMs harbor glioblastoma-initiating cells (GICs) that drive tumorigenesis and contribute to therapeutic resistance and tumor recurrence. Consequently, there is a strong rationale to target this cell population in order to develop new molecular therapies against GBM. Accumulating evidence indicates that Nα-terminal acetyltransferases (NATs), that are dysregulated in numerous human cancers, can serve as therapeutic targets. Microarrays were used to study the expression of several NATs including NAT12/NAA30 in clinical samples and stem cell cultures. The expression of NAT12/NAA30 was analyzed using qPCR, immunolabeling and western blot. We conducted shRNA-mediated knockdown of NAT12/NAA30 gene in GICs and studied the effects on cell viability, sphere-formation and hypoxia sensitivity. Intracranial transplantation to SCID mice enabled us to investigate the effects of NAT12/NAA30 depletion in vivo. Using microarrays we identified genes and biochemical pathways whose expression was altered upon NAT12/NAA30 down-regulation. While decreased expression of the distal 3'UTR of NAT12/NAA30 was generally observed in GICs and GBMs, this gene was strongly up-regulated at the protein level in GBM and GICs. The increased protein levels were not caused by increased levels of the steady state mRNA but rather by other mechanisms. Also, shorter 3'UTR of NAT12/NAA30 correlated with poor survival in glioma patients. As well, we observed previously not described nuclear localization of this typically cytoplasmic protein. When compared to non-silencing controls, cells featuring NAT12/NAA30 knockdown exhibited reduced cell viability, sphere-forming ability, and mitochondrial hypoxia tolerance. Intracranial transplantation showed that knockdown of NAT12/NAA30 resulted in prolonged animal survival. Microarray analysis of the knockdown cultures showed reduced levels of HIF1α and altered expression

  16. Mortality and knockdown effects of imidacloprid and methomyl in house fly (Musca domestica L., Diptera: Muscidae) populations.

    PubMed

    Memmi, Burcu Koçak

    2010-06-01

    In this study, the knockdown and mortality effects of imidacloprid and methomyl were investigated. The residual surface applications were carried out to determine the knockdown effects (KDt50 and KDt95) and mortality (LD50 and LD95) induced by each insecticide. For mortality comparisons, the susceptible house fly (Musca domestica L., Diptera: Muscidae) of a WHO population and three natural field-collected M. domestica populations from Turkey were used. In conclusion, it was found that the resistance to imidacloprid and methomyl was significantly higher in the field populations when compared to the susceptible population from WHO. The results showed that applicators and pest management decision-makers should control and conduct an integrated pest management strategy by including biological agents to prevent the development of high levels of resistance in the field populations.

  17. Insecticidal potency of RNAi-based catalase knockdown in Rhynchophorus ferrugineus (Oliver) (Coleoptera: Curculionidae).

    PubMed

    Al-Ayedh, Hassan; Rizwan-Ul-Haq, Muhammad; Hussain, Abid; Aljabr, Ahmed M

    2016-11-01

    Palm trees around the world are prone to notorious Rhynchophorus ferrugineus, which causes heavy losses of palm plantations. In Middle Eastern countries, this pest is a major threat to date palm orchards. Conventional pest control measures with the major share of synthetic insecticides have resulted in insect resistance and environmental issues. Therefore, in order to explore better alternatives, the RNAi approach was employed to knock down the catalase gene in fifth and tenth larval instars with different dsRNA application methods, and their insecticidal potency was studied. dsRNA of 444 bp was prepared to knock down catalase in R. ferrugineus. Out of the three dsRNA application methods, dsRNA injection into larvae was the most effective, followed by dsRNA application by artificial feeding. Both methods resulted in significant catalase knockdown in various tissues, especially the midgut. As a result, the highest growth inhibition of 123.49 and 103.47% and larval mortality of 80 and 40% were observed in fifth-instar larvae, whereas larval growth inhibition remained at 86.83 and 69.08% with larval mortality at 30 and 10% in tenth-instar larvae after dsRNA injection and artificial diet treatment. The topical application method was the least efficient, with the lowest larval growth inhibition of 57.23 and 45.61% and 0% mortality in fifth- and tenth-instar larvae. Generally, better results were noted at the high dsRNA dose of 5 µL. Catalase enzyme is found in most insect body tissues, and thus its dsRNA can cause broad-scale gene knockdown within the insect body, depending upon the application method. Significant larval mortality and growth inhibition after catalase knockdown in R. ferrugineus confirms its insecticidal potency and suggests a bright future for RNAi-based bioinsecticides in pest control. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  18. Adseverin knockdown inhibits osteoclastogenesis in RAW264.7 cells

    PubMed Central

    QI, WENTING; GAO, YAN; TIAN, JUN; JIANG, HONGWEI

    2014-01-01

    Osteoclastogenesis is a complex process that is highly dependent on the dynamic regulation of the actin cytoskeleton. Adseverin (Ads), a member of the gelsolin superfamily of actin-binding proteins, regulates actin remodeling by severing and capping actin filaments. The objective of the present study was to characterize the role of Ads during osteoclastogenesis by assessing Ads expression and using a knockdown strategy. Immunoblot analyses were used to examine Ads expression during osteoclastogenesis. A stable Ads knockdown macrophage cell line was generated using a retroviral shRNA construct. Osteoclast differentiation was morphologically examined via cell staining with osteoclast specific markers and light microscopy. The results showed that Ads expression was significantly increased in response to receptor activator of nuclear factor-κB ligand during osteoclastogenesis, and Ads was highly expressed in mature osteoclasts. Ads-knockdown macrophages showed major osteoclastogenesis defects, most likely caused by a pre-osteoclast fusion defect. These results indicate that Ads deficiency in monocytes inhibits osteoclastogenesis. Thus, in future studies it could be noteworthy to investigate the function of Ads in bone marrow monocytes during osteoclastogenesis. PMID:25339151

  19. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells

    SciTech Connect

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    Research highlights: {yields} Exogenous VEGF decreases MRP4 expression in a dose-dependent manner. {yields} MRP4 knockdown leads to enhanced cell migration. {yields} MRP4 knockdown suppresses caspase-3-mediated cell apoptosis. {yields} MRP4 knockdown produces cell assembly and cell aggregation. -- Abstract: The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  20. Interaction of Hb Grey Lynn (Vientiane) [α91(FG3)Leu>Phe (α1)] with Hb E [β26(B8) Glu>Lys] and α(+)-thalassemia: Molecular and Hematological Analysis.

    PubMed

    Singha, Kritsada; Fucharoen, Goonnapa; Fucharoen, Supan

    2015-01-01

    Hemoglobin (Hb) Grey Lynn is a Hb variant caused by a mutation at codon 91 of α1-globin gene whereas Hb E is a common β-globin chain variant among Southeast Asian population. We report two hitherto undescribed conditions of Hb Grey Lynn found in Thai individuals. The study was done on two unrelated Thai subjects. Hematological parameters were recorded and Hb analysis was carried out using automated Hb analyzers. Mutations were identified by DNA analysis. Hematological features of the patients were compared with those of various forms of Hb Grey Lynn documented previously. Hb and DNA analyses identified a heterozygous Hb Grey Lynn in one patient and a double heterozygous Hb Grey Lynn and Hb E with α(+)-thalassemia in another. Interaction of α(Grey Lynn) with β(E) chains leads to the formation of a new Hb variant, namely the Hb Grey Lynn E (α(GL)2β(E)2), detectable by liquid chromatography (10.3%) but masked by Hb E on capillary electrophoresis. Interaction of these multiple globin gene defects could lead to complex hemoglobinopathies requiring combined analysis with multiple Hb analyzers followed by DNA testing to provide accurate diagnosis of the cases.

  1. Manipulating the in vivo immune response by targeted gene knockdown.

    PubMed

    Lieberman, Judy

    2015-08-01

    Aptamers, nucleic acids selected for high affinity binding to proteins, can be used to activate or antagonize immune mediators or receptors in a location and cell-type specific manner and to enhance antigen presentation. They can also be linked to other molecules (other aptamers, siRNAs or miRNAs, proteins, toxins) to produce multifunctional compounds for targeted immune modulation in vivo. Aptamer-siRNA chimeras (AsiCs) that induce efficient cell-specific knockdown in immune cells in vitro and in vivo can be used as an immunological research tool or potentially as an immunomodulating therapeutic.

  2. Reversible gene knockdown in mice using a tight, inducible shRNA expression system

    PubMed Central

    Seibler, Jost; Kleinridders, Andre; Küter-Luks, Birgit; Niehaves, Sandra; Brüning, Jens C.; Schwenk, Frieder

    2007-01-01

    RNA interference through expression of short hairpin (sh)RNAs provides an efficient approach for gene function analysis in mouse genetics. Techniques allowing to control time and degree of gene silencing in vivo, however, are still lacking. Here we provide a generally applicable system for the temporal control of ubiquitous shRNA expression in mice. Depending on the dose of the inductor doxycycline, the knockdown efficiency reaches up to 90%. To demonstrate the feasibility of our tool, a mouse model of reversible insulin resistance was generated by expression of an insulin receptor (Insr)-specific shRNA. Upon induction, mice develop severe hyperglycemia within seven days. The onset and progression of the disease correlates with the concentration of doxycycline, and the phenotype returns to baseline shortly after withdrawal of the inductor. On a broad basis, this approach will enable new insights into gene function and molecular disease mechanisms. PMID:17376804

  3. Knockdown of CXCL14 disrupts neurovascular patterning during ocular development.

    PubMed

    Ojeda, Ana F; Munjaal, Ravi P; Lwigale, Peter Y

    2017-03-01

    The C-X-C motif ligand 14 (CXCL14) is a recently discovered chemokine that is highly conserved in vertebrates and expressed in various embryonic and adult tissues. CXCL14 signaling has been implicated to function as an antiangiogenic and anticancer agent in adults. However, its function during development is unknown. We previously identified novel expression of CXCL14 mRNA in various ocular tissues during development. Here, we show that CXCL14 protein is expressed in the anterior eye at a critical time during neurovascular development and in the retina during neurogenesis. We report that RCAS-mediated knockdown of CXCL14 causes severe neural defects in the eye including precocious and excessive innervation of the cornea and iris. Absence of CXCL14 results in the malformation of the neural retina and misprojection of the retinal ganglion neurons. The ocular neural defects may be due to loss of CXCL12 modulation since recombinant CXCL14 diminishes CXCL12-induced axon growth in vitro. Furthermore, we show that knockdown of CXCL14 causes neovascularization of the cornea. Altogether, our results show for the first time that CXCL14 plays a critical role in modulating neurogenesis and inhibiting ectopic vascularization of the cornea during ocular development. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Knockdown of eIF4E suppresses cell proliferation, invasion and enhances cisplatin cytotoxicity in human ovarian cancer cells.

    PubMed

    Wan, Jing; Shi, Fang; Xu, Zhanzhan; Zhao, Min

    2015-12-01

    Eukaryotic initiation factor 4E (eIF4E) plays an important role in cap-dependent translation. The overexpression of eIF4E gene has been found in a variety of human malignancies. In this study, we attempted to identify the potential effects of eIF4E and explore the possibility of eIF4E as a therapeutic target for the treatment of human ovarian cancer. First the activation of eIF4E protein was detected with m7-GTP cap binding assays in ovarian cancer and control cells. Next, the eIF4E-shRNA expression plasmids were used to specifically inhibit eIF4E activity in ovarian cancer cells line A2780 and C200. The effects of knockdown eIF4E gene on cell proliferation, migration and invasion were investigated in vitro. Moreover, the changes of cell cycle and apoptosis of ovarian cancer cells were detected by flow cytometry. Finally, we investigated the effect of knockdown of eIF4E on the chemosensitivity of ovarian cancer cells to cisplatin in vitro. Our results show there is elevated activation of eIF4E in ovarian cancer cells compared with normal human ovarian epithelial cell line. The results of BrdU incorporation and FCM assay indicate that knockdown of eIF4E efficiently suppressed cell growth and induce cell cycle arrest in G1 phase and subsequent apoptosis in ovarian cancer cells. From Transwell assay analysis, knockdown eIF4E significantly decrease cellular migration and invasion of ovarian cancer cells. We also confirmed that knockdown eIF4E could synergistically enhance the cytotoxicity effects of cisplatin to cancer cells and sensitized cisplatin-resistant C200 cells in vitro. This study demonstrates that the activation of eIF4E gene is an essential component of the malignant phenotype in ovarian cancer, and aberration of eIF4E expression is associated with proliferation, migration, invasion and chemosensitivity to cisplatin in ovarian cancer cells. Knockdown eIF4E gene can be used as a potential therapeutic target for the treatment of human ovarian cancer.

  5. CFTR Knockdown induces proinflammatory changes in intestinal epithelial cells.

    PubMed

    Crites, Karoline St-Martin; Morin, Geneviève; Orlando, Valérie; Patey, Natacha; Cantin, Catherine; Martel, Judith; Brochiero, Emmanuelle; Mailhot, Geneviève

    2015-01-01

    Hyperinflammation is a hallmark feature of cystic fibrosis (CF) airways. However, inflammation has also been documented systemically and, more recently, in extrapulmonary CF-affected tissues such as the pancreas and intestine. The pathogenesis of CF-related inflammation and more specifically the role of the cystic fibrosis transmembrane conductance regulator (CFTR) in that respect are not entirely understood. We have tested the hypothesis that genetic depletion of CFTR will affect the inflammatory status of human intestinal epithelial cell lines. CFTR expression was genetically depleted from Caco-2/15 and HT-29 cells using short hairpin RNA interference (shRNAi). Inflammatory conditions were induced by the addition of human recombinant tumor necrosis factor (TNF) or Interleukin-1β (IL-1β) for various periods of time. Gene expression, mRNA stability and secreted levels of interleukin (IL)-6, -8 and 10 were assessed. Analysis of pro- and anti-inflammatory signaling pathways including mitogen-activated protein kinases (p38, ERK 1/2 and JNK), nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha (IκBα), and nuclear factor-kappa B (NF-κB) was also performed. Eosinophils were counted in the jejunal mucosa of Cftr-/- and Cftr+/+ mice. CFTR gene and protein knockdown caused a significant increase in basal secretion of IL-8 as well as in IL-1β-induced secretion of IL-6 and -8. Release of the anti-inflammatory cytokine, IL-10, remained unaffected by CFTR depletion. The enhanced secretion of IL-8 stems in part from increased IL8 mRNA levels and greater activation of ERK1/2 MAPK, IκBα and NF-κB in the CFTR knockdown cells. By contrast, phosphorylation levels of p38 and JNK MAPK did not differ between control and knockdown cells. We also found a higher number of infiltrating eosinophils in the jejunal mucosa of Cftr -/- females, but not males, compared to Cftr +/+ mice, thus providing in vivo support to our in vitro findings. Collectively

  6. Investigation of TbfA in Riemerella anatipestifer using plasmid-based methods for gene over-expression and knockdown

    PubMed Central

    Liu, MaFeng; Wang, MengYi; Zhu, DeKang; Wang, MingShu; Jia, RenYong; Chen, Shun; Sun, KunFeng; Yang, Qiao; Wu, Ying; Chen, XiaoYue; Biville, Francis; Cheng, AnChun

    2016-01-01

    Riemerella anatipestifer is a duck pathogen that has caused serious economic losses to the duck industry worldwide. Despite this, there are few reported studies of the physiological and pathogenic mechanisms of Riemerella anatipestifer infection. In previous study, we have shown that TonB1 and TonB2 were involved in hemin uptake. TonB family protein (TbfA) was not investigated, since knockout of this gene was not successful at that time. Here, we used a plasmid based gene over-expression and knockdown to investigate its function. First, we constructed three Escherichia-Riemerella anatipestifer shuttle vectors containing three different native Riemerella anatipestifer promoters. The shuttle plasmids were introduced into Riemerella anatipestifer ATCC11845 by conjugation at an efficiency of 5 × 10−5 antibiotic-resistant transconjugants per recipient cell. Based on the high-expression shuttle vector pLMF03, a method for gene knockdown was established. Knockdown of TbfA in Riemerella anatipestifer ATCC11845 decreased the organism’s growth ability in TSB medium but did not affect its hemin utilization. In contrast, over-expression of TbfA in Riemerella anatipestifer ATCC11845ΔtonB1ΔtonB2. Significantly promoted the organism’s growth in TSB medium but significantly inhibited its hemin utilization. Collectively, these findings suggest that TbfA is not involved in hemin utilization by Riemerella anatipestifer. PMID:27845444

  7. A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

    PubMed Central

    Huang, Liwei; Xiao, An; Wecker, Andrea; McBride, Daniel A.; Choi, Soo Young; Zhou, Weibin; Lipschutz, Joshua H.

    2014-01-01

    Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney. PMID:25489842

  8. A possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys.

    PubMed

    Huang, Liwei; Xiao, An; Wecker, Andrea; McBride, Daniel A; Choi, Soo Young; Zhou, Weibin; Lipschutz, Joshua H

    2014-12-02

    Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to "off-target" effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

  9. Knockdown of FOXO3 induces primordial oocyte activation in pigs.

    PubMed

    Moniruzzaman, Mohammad; Lee, Jibak; Zengyo, Mai; Miyano, Takashi

    2010-02-01

    Mammalian ovaries are endowed with a large number of primordial follicles, each containing a nongrowing oocyte. Only a small population of primordial oocytes (oocytes in primordial follicles) is activated to enter the growth phase throughout a female's reproductive life. Little is known about the mechanism regulating the activation of primordial oocytes. Here, we found that the primordial oocytes from infant pigs (10- to 20-day-old) grew to full size at 2 months after xenografting to immunodeficient mice, whereas those from prepubertal pigs (6-month-old) survived without initiation of their growth even after 4 months; thereafter, they started to grow and reached full size after 6 months. These results suggest that the mechanism regulating the activation of primordial oocytes in prepubertal pigs is different from that in infant pigs. In this regard, the involvement of FOXO3, a forkhead transcription factor, was studied. In prepubertal pigs, FOXO3 was detected in almost all (94+/-2%) primordial oocyte nuclei, and in infant pigs, 42+/-7% primordial oocytes were FOXO3 positive. At 4 months after xenografting, the percentage of FOXO3-positive primordial oocytes from prepubertal pigs had decreased to the infant level. Further, siRNA was designed to knock down porcine FOXO3. FOXO3-knockdown primordial follicles from prepubertal pigs developed to the antral stage accompanied by oocyte growth at 2 months after xenografting. These results suggest that primordial oocytes are dormant in prepubertal pigs by a FOXO3-related mechanism to establish a nongrowing oocyte pool in the ovary, and that a transient knockdown of the FOXO3 activates the primordial oocytes to enter the growth phase.

  10. Knockdown of long non-coding RNA KCNQ1OT1 depressed chemoresistance to paclitaxel in lung adenocarcinoma.

    PubMed

    Ren, Kaiming; Xu, Ran; Huang, Jingshan; Zhao, Jungang; Shi, Wenjun

    2017-08-01

    Lung cancer, with the highest morbidity and second highest death rates, is one of the most common cancers in both males and females worldwide. Lung adenocarcinoma (LAD) is the main lung cancer class. KCNQ1 Opposite Strand/Antisense Transcript 1 (KCNQ1OT1) gene is an lncRNA which had been reported high-expression in colorectal cancer. In this study, the expression of KCNQ1OT1 was confirmed to be highly expressed in LAD tissues and cells contrast to control tissues and cells, and high KCNQ1OT1 expression correlated to malignant behaviors of LAD, including big tumor size, poor differentiation, positive lymphatic metastasis and high TNM stages. The transfection of si-KCNQ1OT1 could effectually knockdown the expression of KCNQ1OT1 in A549 and A549/PA cells. The KCNQ1OT1 knockdown depressed the proliferation and invasion of A549 cells, and advanced cellular apoptosis of A549 cells. The expression of KCNQ1OT1 in LAD patients insensitive to paclitaxel was much higher than that in LAD patients sensitive to paclitaxel; the KCNQ1OT1 expression in A549/PA cells was also much higher than that in control A549 cells. The half maximal inhibitory concentration (IC50) of paclitaxel in A549/PA cells was depressed by KCNQ1OT1 knockdown, chemoresistance of A549/PA cells was inhibited significantly. KCNQ1OT1 knockdown also depressed the expression of multidrug resistance 1 (MDR1) protein in A549/PA cells. In summary, lncRNA KCNQ1OT1 was highly expressed in LAD and functioned as a potential oncogene to inhibit malignancy and chemoresistance of LAD cells, which might be a novel potential therapeutic target for LAD.

  11. GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines

    PubMed Central

    Chandra, Dev; Korpi, Esa R; Miralles, Celia P; De Blas, Angel L; Homanics, Gregg E

    2005-01-01

    Background Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. Results Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. Conclusion We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2

  12. The knock-down of the expression of MdMLO19 reduces susceptibility to powdery mildew (Podosphaera leucotricha) in apple (Malus domestica).

    PubMed

    Pessina, Stefano; Angeli, Dario; Martens, Stefan; Visser, Richard G F; Bai, Yuling; Salamini, Francesco; Velasco, Riccardo; Schouten, Henk J; Malnoy, Mickael

    2016-10-01

    Varieties resistant to powdery mildew (PM; caused by Podosphaera leucotricha) are a major component of sustainable apple production. Resistance can be achieved by knocking-out susceptibility S-genes to be singled out among members of the MLO (Mildew Locus O) gene family. Candidates are MLO S-genes of phylogenetic clade V up-regulated upon PM inoculation, such as MdMLO11 and 19 (clade V) and MdMLO18 (clade VII). We report the knock-down through RNA interference of MdMLO11 and 19, as well as the complementation of resistance with MdMLO18 in the Arabidopsis thaliana triple mlo mutant Atmlo2/6/12. The knock-down of MdMLO19 reduced PM disease severity by 75%, whereas the knock-down of MdMLO11, alone or in combination with MdMLO19, did not result in any reduction or additional reduction of susceptibility compared with MdMLO19 alone. The test in A. thaliana excluded a role for MdMLO18 in PM susceptibility. Cell wall appositions (papillae) were present in both PM-resistant and PM-susceptible plants, but were larger in resistant lines. No obvious negative phenotype was observed in plants with mlo genes knocked down. Apparently, MdMLO19 plays the pivotal role in apple PM susceptibility and its knock-down induces a very significant level of resistance. © 2016 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  13. Growth Hormone Receptor Knockdown Sensitizes Human Melanoma Cells to Chemotherapy by Attenuating Expression of ABC Drug Efflux Pumps.

    PubMed

    Basu, Reetobrata; Baumgaertel, Nicholas; Wu, Shiyong; Kopchick, John J

    2017-03-14

    Melanoma remains one of the most therapy-resistant forms of human cancer despite recent introductions of highly efficacious targeted therapies. The intrinsic therapy resistance of human melanoma is largely due to abundant expression of a repertoire of xenobiotic efflux pumps of the ATP-binding cassette (ABC) transporter family. Here, we report that GH action is a key mediator of chemotherapeutic resistance in human melanoma cells. We investigated multiple ABC efflux pumps (ABCB1, ABCB5, ABCB8, ABCC1, ABCC2, ABCG1, and ABCG2) reportedly associated with melanoma drug resistance in different human melanoma cells and tested the efficacy of five different anti-cancer compounds (cisplatin, doxorubicin, oridonin, paclitaxel, vemurafenib) with decreased GH action. We found that GH treatment of human melanoma cells upregulates expression of multiple ABC transporters and increases the EC50 of melanoma drug vemurafenib. Also, vemurafenib-resistant melanoma cells had upregulated levels of GH receptor (GHR) expression as well as ABC efflux pumps. GHR knockdown (KD) using siRNA in human melanoma cells treated with sub-EC50 doses of anti-tumor compounds resulted in significantly increased drug retention, decreased cell proliferation and increased drug efficacy, compared to mock-transfected controls. Our set of findings identify an unknown mechanism of GH regulation in mediating melanoma drug resistance and validates GHR as a unique therapeutic target for sensitizing highly therapy-resistant human melanoma cells to lower doses of anti-cancer drugs.

  14. Knockdown of Leptin A Expression Dramatically Alters Zebrafish Development

    PubMed Central

    Liu, Qin; Dalman, Mark; Chen, Yun; Akhter, Mashal; Brahmandam, Sravya; Patel, Yesha; Lowe, Josef; Thakkar, Mitesh; Gregory, Akil-Vuai; Phelps, Daryllanae; Riley, Caitlin; Londraville, Richard L.

    2012-01-01

    Using morpholino antisense oligonucleotide (MO) technology, we blocked leptin A or leptin receptor expression in embryonic zebrafish, and analyzed consequences of leptin knock-down on fish development. Embryos injected with leptin A or leptin receptor MOs (leptin A or leptin receptor morphants) had smaller bodies and eyes, undeveloped inner ear, enlarged pericardial cavity, curved body and/or tail and larger yolk compared to control embryos of the same stages. The defects persisted in 6-9 day old larvae. We found that blocking leptin A function had little effect on the development of early brain (1 day old), but differentiation of both the morphant dorsal brain and retinal cells was severely disrupted in older (2 day old) embryos. Despite the enlarged pericardial cavity, differentiation of cardiac cells appeared to be similar to control embryos. Formation of the morphants’ inner ear is also severely disrupted, which corroborates existing reports of leptin receptor expression in inner ear of both zebrafish and mammals. Co-injection of leptin A MO and recombinant leptin results in partial rescue of the wild-type phenotype. Our results suggest that leptin A plays distinct roles in zebrafish development. PMID:22841760

  15. RNAi knockdown of parafusin inhibits the secretory pathway.

    PubMed

    Liu, Li; Wyroba, Elzbieta; Satir, Birgit H

    2011-10-01

    Several glycolytic enzymes and their isoforms have been found to be important in cell signaling unrelated to glycolysis. The involvement of parafusin (PFUS), a member of the phosphoglucomutase (PGM) superfamily with no phosphoglucomutase activity, in Ca(2+)-dependent exocytosis has been controversial. This protein was first described in Paramecium tetraurelia, but is widely found. Earlier work showed that parafusin is a secretory vesicle scaffold component with unusual post-translational modifications (cyclic phosphorylation and phosphoglucosylation) coupled to stages in the exocytic process. Using RNAi, we demonstrate that parafusin synthesis can be reversibly blocked, with minor or no effect on other PGM isoforms. PFUS knockdown produces an inhibition of dense core secretory vesicle (DCSV) synthesis leading to an exo(-) phenotype. Although cell growth is unaffected, vesicle content is not packaged properly and no new DCSVs are formed. We conclude that PFUS and its orthologs are necessary for proper scaffold maturation. Because of this association, parafusin is an important signaling component for regulatory control of the secretory pathway. Copyright © 2011 Elsevier GmbH. All rights reserved.

  16. Knockdown of neuropeptide Y in the dorsomedial hypothalamus reverses high-fat diet-induced obesity and impaired glucose tolerance in rats.

    PubMed

    Kim, Yonwook J; Bi, Sheng

    2016-01-15

    Neuropeptide Y (NPY) in the dorsomedial hypothalamus (DMH) plays an important role in the regulation of energy balance. While DMH NPY overexpression causes hyperphagia and obesity in rats, knockdown of NPY in the DMH via adeno-associated virus (AAV)-mediated RNAi (AAVshNPY) ameliorates these alterations. Whether this knockdown has a therapeutic effect on obesity and glycemic disorder has yet to be determined. The present study sought to test this potential using a rat model of high-fat diet (HFD)-induced obesity and insulin resistance, mimicking human obesity with impaired glucose homeostasis. Rats had ad libitum access to rodent regular chow (RC) or HFD. Six weeks later, an oral glucose tolerance test (OGTT) was performed for verifying HFD-induced glucose intolerance. After verification, obese rats received bilateral DMH injections of AAVshNPY or the control vector AAVshCTL, and OGTT and insulin tolerance test (ITT) were performed at 16 and 18 wk after viral injection (23 and 25 wk on HFD), respectively. Rats were killed at 26 wk on HFD. We found that AAVshCTL rats on HFD remained hyperphagic, obese, glucose intolerant, and insulin resistant relative to lean control RC-fed rats receiving DMH injection of AAVshCTL, whereas these alterations were reversed in NPY knockdown rats fed a HFD. NPY knockdown rats exhibited normal food intake, body weight, glucose tolerance, and insulin sensitivity, as seen in lean control rats. Together, these results demonstrate a therapeutic action of DMH NPY knockdown against obesity and impaired glucose homeostasis in rats, providing a potential target for the treatment of obesity and diabetes. Copyright © 2016 the American Physiological Society.

  17. Specific in vivo knockdown of protein function by intrabodies

    PubMed Central

    Marschall, Andrea LJ; Dübel, Stefan; Böldicke, Thomas

    2015-01-01

    Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e., their antigens). The intrabody technology is an attractive alternative to the generation of gene-targeted knockout animals, and complements knockdown techniques such as RNAi, miRNA and small molecule inhibitors, by-passing various limitations and disadvantages of these methods. The advantages of intrabodies include very high specificity for the target, the possibility to knock down several protein isoforms by one intrabody and targeting of specific splice variants or even post-translational modifications. Different types of intrabodies must be designed to target proteins at different locations, typically either in the cytoplasm, in the nucleus or in the endoplasmic reticulum (ER). Most straightforward is the use of intrabodies retained in the ER (ER intrabodies) to knock down the function of proteins passing the ER, which disturbs the function of members of the membrane or plasma proteomes. More effort is needed to functionally knock down cytoplasmic or nuclear proteins because in this case antibodies need to provide an inhibitory effect and must be able to fold in the reducing milieu of the cytoplasm. In this review, we present a broad overview of intrabody technology, as well as applications both of ER and cytoplasmic intrabodies, which have yielded valuable insights in the biology of many targets relevant for drug development, including α-synuclein, TAU, BCR-ABL, ErbB-2, EGFR, HIV gp120, CCR5, IL-2, IL-6, β-amyloid protein and p75NTR. Strategies for the generation of intrabodies and various designs of their applications are also reviewed. PMID:26252565

  18. Knockdown of phosphoethanolamine transmethylation enzymes decreases viability of Haemonchus contortus.

    PubMed

    Witola, William H; Cooks-Fagbodun, Sheritta; Ordonez, Adriana Reyes; Matthews, Kwame; Abugri, Daniel A; McHugh, Mark

    2016-06-15

    The phosphobase methylation pathway, in which phosphoethanolamine N-methyltransferases (PMTs) successively catalyze the methylation of phosphoethanolamine to phosphocholine, is essential in the free-living nematode Caenorhabditis elegans. Two PMT-encoding genes (HcPMT1 and HcPMT2) cloned from Haemonchus contortus have been shown, by in vitro assays, to possess enzymatic characteristics similar to those of C. elegans PMTs, but their physiological significance in H. contortus is yet to be elucidated. Therefore, in this study, we endeavored to determine the importance of HcPMT1 and HcPMT2 in the survival of H. contortus by adapting the use of phosphorodiamidate morpholino oligomers (PPMO) antisense approach to block the translation of HcPMT1 and HcPMT2 in the worms. We found that PPMOs targeting HcPMT1 and HcPMT2 down-regulated the expression of HcPMT1 and HcPMT2 proteins in adult H. contortus. Analysis of the effect of HcPMT1 and HcPMT2 knockdown showed that it significantly decreased worm motility and viability, thus validating HcPMT1 and HcPMT2 as essential enzymes for survival of H. contortus. Studies of gene function in H. contortus have been constrained by limited forward and reverse genetic technologies for use in H. contortus. Thus, our success in adaptation of use of PPMO antisense approach in H. contortus provides an important reverse genetic technological advance for studying this parasitic nematode of veterinary significance. Copyright © 2016 Elsevier B.V. All rights reserved.

  19. Differential Effects of Histone Acetyltransferase GCN5 or PCAF Knockdown on Urothelial Carcinoma Cells

    PubMed Central

    Koutsogiannouli, Evangelia A.; Hader, Christiane; Pinkerneil, Maria; Hoffmann, Michèle J.; Schulz, Wolfgang A.

    2017-01-01

    Disturbances in histone acetyltransferases (HATs) are common in cancers. In urothelial carcinoma (UC), p300 and CBP are often mutated, whereas the GNAT family HATs GCN5 and PCAF (General Control Nonderepressible 5, p300/CBP-Associated Factor) are often upregulated. Here, we explored the effects of specific siRNA-mediated knockdown of GCN5, PCAF or both in four UC cell lines (UCCs). Expression of various HATs and marker proteins was measured by qRT-PCR and western blot. Cellular effects of knockdowns were analyzed by flow cytometry and ATP-, caspase-, and colony forming-assays. GCN5 was regularly upregulated in UCCs, whereas PCAF was variable. Knockdown of GCN5 or both GNATs, but not of PCAF alone, diminished viability and inhibited clonogenic growth in 2/4 UCCs, inducing cell cycle changes and caspase-3/7 activity. PCAF knockdown elicited GCN5 mRNA upregulation. Double knockdown increased c-MYC and MDM2 (Mouse Double Minute 2) in most cell lines. In conclusion, GCN5 upregulation is especially common in UCCs. GCN5 knockdown impeded growth of specific UCCs, whereas PCAF knockdown elicited minor effects. The limited sensitivity towards GNAT knockdown and its variation between the cell lines might be due to compensatory effects including HAT, c-MYC and MDM2 upregulation. Our results predict that developing drugs targeting individual HATs for UC treatment may be challenging. PMID:28678170

  20. Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7

    PubMed Central

    Curtis, Helen J.; Wood, Matthew J.A.

    2017-01-01

    Abstract We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach. PMID:28575281

  1. Knockdown and replacement therapy mediated by artificial mirtrons in spinocerebellar ataxia 7.

    PubMed

    Curtis, Helen J; Seow, Yiqi; Wood, Matthew J A; Varela, Miguel A

    2017-07-27

    We evaluate a knockdown-replacement strategy mediated by mirtrons as an alternative to allele-specific silencing using spinocerebellar ataxia 7 (SCA7) as a model. Mirtrons are introns that form pre-microRNA hairpins after splicing, producing RNAi effectors not processed by Drosha. Mirtron mimics may therefore avoid saturation of the canonical processing pathway. This method combines gene silencing mediated by an artificial mirtron with delivery of a functional copy of the gene such that both elements of the therapy are always expressed concurrently, minimizing the potential for undesirable effects and preserving wild-type function. This mutation- and single nucleotide polymorphism-independent method could be crucial in dominant diseases that feature both gain- and loss-of-function pathologies or have a heterogeneous genetic background. Here we develop mirtrons against ataxin 7 with silencing efficacy comparable to shRNAs, and introduce silent mutations into an ataxin 7 transgene such that it is resistant to their effect. We successfully express the transgene and one mirtron together from a single construct. Hence, we show that this method can be used to silence the endogenous allele of ataxin 7 and replace it with an exogenous copy of the gene, highlighting the efficacy and transferability across patient genotypes of this approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  2. Knockdown of homeobox A5 by small hairpin RNA inhibits proliferation and enhances cytarabine chemosensitivity of acute myeloid leukemia cells.

    PubMed

    Li, Na; Jia, Xiuhong; Wang, Jianyong; Li, Youjie; Xie, Shuyang

    2015-11-01

    Homeobox genes encode transcription factors that are essential for embryonic morphogenesis and differentiation. Transcription factors containing the highly conserved homeobox motif show considerable promise as potential regulators of hematopoietic maturation events. Previous studies have suggested that the increased expression levels of homeobox (HOX)A genes was correlated with the cytogenetic findings associated with poor prognosis in acute myeloid leukemia and mixed lineage leukemia. The aim of the present study was to investigate the role of HOXA5 in leukemia. The U937 human leukemia cell line was transfected with a HOXA5‑targeted short hairpin RNA (shRNA) to determine the effects of downregulation of the HOXA5 on proliferation, apoptosis, cell cycle distribution and chemoresistance in leukemia cells. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses demonstrated that the mRNA and protein expression levels of HOXA5 were markedly suppressed following transfection with an shRNA‑containing vector. Knockdown of HOXA5 significantly inhibited cell proliferation, as determined by Cell Counting kit‑8 assay. Flow cytometry revealed that reduced HOXA5 expression levels resulted in cell cycle arrest at the G1 phase, and induced apoptosis. In addition, western blot analysis demonstrated that HOXA5 knockdown increased the expression levels of caspase‑3, and reduced the expression levels of survivin in the U937 cells. Furthermore, knockdown of HOXA5 in the U937 cells enhanced their chemosensitivity to cytarabine. The results of the present study suggested that downregulation of HOXA5 by shRNA may trigger apoptosis and overcome drug resistance in leukemia cells. Therefore, HOXA5 may serve as a potential target for developing novel therapeutic strategies for leukemia.

  3. PlGF Knockdown Decreases Tumorigenicity and Stemness Properties of Spheroid Body Cells Derived from Gastric Cancer Cells.

    PubMed

    Mahmoodi, Fatemeh; Akrami, Hassan

    2017-04-01

    Placental growth factor (PlGF) a member of the vascular endothelial growth factor family regulates some cell processes such as survival, growth of vascular endothelial cells, invasiveness, and also involves in pathological angiogenesis and metastasis in most cancers. Cancer stem cells are believed to be the main reason for the tumor relapse and resistance to therapy. These cells have various characteristics as same as normal tissue-specific adult stem cells including self-renewability and potent to differentiate into various cell types. However, the function of PlGF in gastric cancer stem cells is not well understood. We have investigated the effect of PlGF knockdown on the tumorigenicity and stem cell properties of spheroid body cells derived from two human gastric cancer cell lines. In this study, we isolated spheroid body cells which have stemness properties from MKN-45 and AGS without using growth factors. Validation of spheroid body cells was confirmed by various methods. Then the effects of PlGF knockdown were investigated on in vitro tumorigenicity, differentiation, migration, angiogenesis, and transcription levels of stemness markers of spheroid body cells. Our findings indicated that isolation of spheroid body cells from MKN-45 and AGS cells without using growth factors is an easy and inexpensive method to isolate cancer stem cells and knockdown of PlGF in spheroid body cells reduced in vitro tumorigenicity and stemness properties of spheroid body cells such as Self-renewal ability, colony forming, migratory, and MMPs activities and decreased ability to differentiation and angiogenesis. J. Cell. Biochem. 118: 851-859, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  4. The effect of radixin knockdown on the expression and efflux function of MRP2 in SGC-7901 cells.

    PubMed

    He, Xiao-Jie; Wang, Wei-Rong; Zhang, Yun; Yang, Qing

    2012-08-15

    Multidrug resistance-associated protein 2 (MRP2, ABCC2) is the second member of the MRP transporter family and functions physiologically as an organic anion transporter. Earlier studies have confirmed that radixin, which is a member of the ERM (ezrin/radixin/moesin) family, modulates MRP2 localization at the canalicular membrane in hepatocytes. The relationship between radixin and MRP2 - particularly, the effect of radixin on the expression and function of MRP2 in cells or tissues that co-express all three ERM proteins - has not been well studied. To examine the role of radixin in the expression and function of MRP2 and other MRPs, we chose human gastric carcinoma SGC-7901 cells that express all three ERM proteins rather than hepatocytes, which predominantly express radixin. Radixin stable knockdown SGC-7901 cells, which were constructed by RNAi, exhibited no compensatory up-regulation of ezrin or moesin. The mRNA expression profiles of MRPs in the radixin knockdown cells were primarily evaluated by RT-PCR. Real time quantitative RT-PCR and western blot analysis revealed that the radixin deficiency caused the mRNA and protein expression levels of MRP2 to be reduced by about 50%, respectively. Accordingly, efflux and MTT assays showed that the radixin knockdown cells exhibited lower efflux ability with respect to calcein but no significant change in cell viability. In conclusion, among the MRP1-6 family members, radixin selectively modulates the expression and function of MRP2 in a system co-expressing all three ERM proteins.

  5. MRP4 knockdown enhances migration, suppresses apoptosis, and produces aggregated morphology in human retinal vascular endothelial cells.

    PubMed

    Tagami, Mizuki; Kusuhara, Sentaro; Imai, Hisanori; Uemura, Akiyoshi; Honda, Shigeru; Tsukahara, Yasutomo; Negi, Akira

    2010-10-01

    The multidrug resistance protein (MRP) MRP4/ABCC4 is an ATP-binding cassette transporter that actively effluxes endogenous and xenobiotic substrates out of cells. In the rodent retina, Mrp4 mRNA and protein are exclusively expressed in vascular endothelial cells, but the angiogenic properties of Mrp4 are poorly understood so far. This study aims to explore the angiogenic properties of MRP4 in human retinal microvascular endothelial cells (HRECs) utilizing the RNA interference (RNAi) technique. MRP4 expression was decreased at the mRNA and protein levels after stimulation with exogenous vascular endothelial growth factor in a dose-dependent manner. RNAi-mediated MRP4 knockdown in HRECs do not affect cell proliferation but enhances cell migration. Moreover, cell apoptosis induced by serum starvation was less prominent in MRP4 siRNA-treated HRECs as compared to control siRNA-treated HRECs. In a Matrigel-based tube-formation assay, although MRP4 knockdown did not lead to a significant change in the total tube length, MRP4 siRNA-treated HRECs assembled and aggregated into a massive tube-like structure, which was not observed in control siRNA-treated HRECs. These results suggest that MRP4 is uniquely involved in retinal angiogenesis.

  6. Knockdown of miR-27a sensitizes colorectal cancer stem cells to TRAIL by promoting the formation of Apaf-1-caspase-9 complex

    PubMed Central

    Zhang, Rui; Xu, Jian; Zhao, Jian; Bai, Jinghui

    2017-01-01

    MicroRNAs have been proved to participate in multiple biological processes in cancers. For developing resistance to cytotoxic drug, cancer cells, especially the cancer stem cells, usually change their microRNA expression profile to survive in hostile environments. In the present study, we found that expression of microRNA-27a was increased in colorectal cancer stem cells. High level of microRNA-27a was indicated to induce the resistance to TNF-related apoptosis-inducing ligand (TRAIL). Knockdown of microRNA-27a resensitized colorectal cancer stem cells to TRAIL-induced cell death. Mechanically, the gene of Apaf-1, which is associated with the mitochondrial apoptosis, was demonstrated to be the target of microRNA-27a in colorectal cancer stem cells. Knockdown of microRNA-27a increased the expression level of Apaf-1, thus enhancing the formation of Apaf-1-caspase-9 complex and subsequently promoting the TRAIL-induced apoptosis in colorectal cancer stem cells. These findings suggested that knockdown of microRNA-27a in colorectal cancer stem cells by the specific antioligonucleotides was potential to reverse the chemoresistance to TRAIL. It may represent a novel therapeutic strategy for treating the colorectal cancer more effectively. PMID:28423356

  7. Knockdown of dual specificity phosphatase 4 enhances the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin

    SciTech Connect

    Liu, Yu; Du, Feiya; Chen, Wei; Yao, Minya; Lv, Kezhen; Fu, Peifen

    2013-12-10

    Background: Breast cancer is the major cause of cancer-related deaths in females world-wide. Doxorubicin-based therapy has limited efficacy in breast cancer due to drug resistance, which has been shown to be associated with the epithelial-to-mesenchymal transition (EMT). However, the molecular mechanisms linking the EMT and drug resistance in breast cancer cells remain unclear. Dual specificity phosphatase 4 (DUSP4), a member of the dual specificity phosphatase family, is associated with cellular proliferation and differentiation; however, its role in breast cancer progression is controversial. Methods: We used cell viability assays, Western blotting and immunofluorescent staining, combined with siRNA interference, to evaluate chemoresistance and the EMT in MCF-7 and adriamycin-resistant MCF-7/ADR breast cancer cells, and investigate the underlying mechanisms. Results: Knockdown of DUSP4 significantly increased the chemosensitivity of MCF-7 and MCF-7/ADR breast cancer cells to doxorubicin, and MCF-7/ADR cells which expressed high levels of DUSP4 had a mesenchymal phenotype. Furthermore, knockdown of DUSP4 reversed the EMT in MCF-7/ADR cells, as demonstrated by upregulation of epithelial biomarkers and downregulation of mesenchymal biomarkers, and also increased the chemosensitivity of MCF-7/ADR cells to doxorubicin. Conclusions: DUSP4 might represent a potential drug target for inhibiting drug resistance and regulating the process of the EMT during the treatment of breast cancer. - Highlights: • We used different technologies to prove our conclusion. • DUSP4 knockdown increased doxorubicin chemosensitivity in breast cancer cells. • DUSP4 is a potential target for combating drug resistance in breast cancer. • DUSP4 is a potential target for regulating the EMT in breast cancer.

  8. RNAi-mediated gene knockdown and in vivo diuresis assay in adult female Aedes aegypti mosquitoes.

    PubMed

    Drake, Lisa L; Price, David P; Aguirre, Sarah E; Hansen, Immo A

    2012-07-14

    This video protocol demonstrates an effective technique to knockdown a particular gene in an insect and conduct a novel bioassay to measure excretion rate. This method can be used to obtain a better understanding of the process of diuresis in insects and is especially useful in the study of diuresis in blood-feeding arthropods that are able to take up huge amounts of liquid in a single blood meal. This RNAi-mediated gene knockdown combined with an in vivo diuresis assay was developed by the Hansen lab to study the effects of RNAi-mediated knockdown of aquaporin genes on Aedes aegypti mosquito diuresis. The protocol is setup in two parts: the first demonstration illustrates how to construct a simple mosquito injection device and how to prepare and inject dsRNA into the thorax of mosquitoes for RNAi-mediated gene knockdown. The second demonstration illustrates how to determine excretion rates in mosquitoes using an in vivo bioassay.

  9. Effective heritable gene knockdown in zebrafish using synthetic microRNAs

    PubMed Central

    Giacomotto, Jean; Rinkwitz, Silke; Becker, Thomas S.

    2015-01-01

    Although zebrafish is used to model human diseases through mutational and morpholino-based knockdown approaches, there are currently no robust transgenic knockdown tools. Here we investigate the knockdown efficiency of three synthetic miRNA-expressing backbones and show that these constructs can downregulate a sensor transgene with different degrees of potency. Using this approach, we reproduce spinal muscular atrophy (SMA) in zebrafish by targeting the smn1 gene. We also generate different transgenic lines, with severity and age of onset correlated to the level of smn1 inhibition, recapitulating for the first time the different forms of SMA in zebrafish. These lines are proof-of-concept that miRNA-based approaches can be used to generate potent heritable gene knockdown in zebrafish. PMID:26051838

  10. Knock-down of Kaiso induces proliferation and blocks granulocytic differentiation in blast crisis of chronic myeloid leukemia

    PubMed Central

    2012-01-01

    Background Kaiso protein has been identified as a new member of the POZ-ZF subfamily of transcription factors that are involved in development and cancer. There is consistent evidence of the role of Kaiso and its involvement in human tumorigenesis but there is no evidence about its role in hematopoietic differentiation or establishment of chronic myeloid leukemia (CML). We used, normal K562 cell line, established from a CML patient in blast crisis, and imatinib-resistant K562 cell line, to investigate the specific distribution of Kaiso and their contribution to the cell differentiation status of the blast crisis of CML (CML-BP). Results We found cytoplasmic expression of Kaiso, in K562 cells and patients, confirmed by immunofluorescence, immunohistochemistry and western blot of cytoplasmic protein fraction. Kaiso was weakly expressed in the imatinib-resistant K562 cell line confirmed by immunofluorescence and western blot. The cytoplasmic expression of Kaiso was not modified when the K562 cells were treated for 16 h with imatinib 0.1 and 1 μM. In our study, small interfering RNA (siRNA) was introduced to down regulate the expression of Kaiso and p120ctn in K562 cell line. Kaiso and p120ctn were down regulated individually (siRNA-Kaiso or siRNA-p120ctn) or in combination using a simultaneous co-transfection (siRNA-Kaiso/p120ctn). We next investigated whether knockdown either Kaiso or p120ctn alone or in combination affects the cell differentiation status in K562 cells. After down regulation we analyzed the expression of hematopoietic cell differentiation and proliferation genes: SCF, PU-1, c-MyB, C/EBPα, Gata-2 and maturation markers of hematopoietic cells expressed in the plasma membrane: CD15, CD11b, CD33, CD117. The levels of SCF and c-MyB were increased by 1000% and 65% respectively and PU-1, Gata-2 and C/EBPα were decreased by 66%, 50% and 80% respectively, when Kaiso levels were down regulated by siRNA. The results were similar when both Kaiso and p120

  11. Essential role of Jun family transcription factors in PU.1 knockdown-induced leukemic stem cells.

    PubMed

    Steidl, Ulrich; Rosenbauer, Frank; Verhaak, Roel G W; Gu, Xuesong; Ebralidze, Alexander; Otu, Hasan H; Klippel, Steffen; Steidl, Christian; Bruns, Ingmar; Costa, Daniel B; Wagner, Katharina; Aivado, Manuel; Kobbe, Guido; Valk, Peter J M; Passegué, Emmanuelle; Libermann, Towia A; Delwel, Ruud; Tenen, Daniel G

    2006-11-01

    Knockdown of the transcription factor PU.1 (encoded by Sfpi1) leads to acute myeloid leukemia (AML) in mice. We examined the transcriptome of preleukemic hematopoietic stem cells (HSCs) in which PU.1 was knocked down (referred to as 'PU.1-knockdown HSCs') to identify transcriptional changes preceding malignant transformation. Transcription factors c-Jun and JunB were among the top-downregulated targets. Restoration of c-Jun expression in preleukemic cells rescued the PU.1 knockdown-initiated myelomonocytic differentiation block. Lentiviral restoration of JunB at the leukemic stage led to loss of leukemic self-renewal capacity and prevented leukemia in NOD-SCID mice into which leukemic PU.1-knockdown cells were transplanted. Examination of human individuals with AML confirmed the correlation between PU.1 and JunB downregulation. These results delineate a transcriptional pattern that precedes leukemic transformation in PU.1-knockdown HSCs and demonstrate that decreased levels of c-Jun and JunB contribute to the development of PU.1 knockdown-induced AML by blocking differentiation and increasing self-renewal. Therefore, examination of disturbed gene expression in HSCs can identify genes whose dysregulation is essential for leukemic stem cell function and that are targets for therapeutic interventions.

  12. Lentivirus-mediated Knockdown of HDAC1 Uncovers Its Role in Esophageal Cancer Metastasis and Chemosensitivity

    PubMed Central

    Song, Min; He, Gang; Wang, Yan; Pang, Xueli; Zhang, Bo

    2016-01-01

    Histone deacetylationase 1 (HDAC1) is ubiquitously expressed in various cell lines and tissues and play an important role of regulation gene expression. Overexpression of HDAC1 has been observed in various types of cancers, which indicated that it might be a target for cancer therapy. To test HDAC1 inhibition for cancer treatment, the gene expression of HDAC1 was knockdown mediated by a lentivirus system. Our data showed the gene expression of HDAC1 could be efficiently knockdown by RNAi mediated by lentivirus in esophageal carcinoma EC109 cells. Knockdown of HDAC1 led to significant decrease of cell growth and altered cell cycle distribution. The result of transwell assay showed that the numbers of cells travelled through the micropore membrane was significantly decreased as HDAC1 expression was knockdown. Moreover, HDAC1 knockdown inhibited the migration of EC109 cells as determining by scratch test. Additionally, enhancement of cisplatin-stimulated apoptosis was detected by HDAC1 knockdown. Our data suggested inhibition of HDAC1 expression by lentivirus mediated shRNA might be further applied for esophageal cancer chemotherapy. PMID:27698906

  13. HuR knockdown changes the oncogenic potential of oral cancer cells.

    PubMed

    Kakuguchi, Wataru; Kitamura, Tetsuya; Kuroshima, Takeshi; Ishikawa, Makoto; Kitagawa, Yoshimasa; Totsuka, Yasunori; Shindoh, Masanobu; Higashino, Fumihiro

    2010-04-01

    HuR binds to AU-rich element-containing mRNA to protect them from rapid degradation. Here, we show that knockdown of HuR changes the oncogenic properties of oral cancer cells. Oral squamous cell carcinoma cell lines, HSC-3 and Ca9.22, which express HuR protein and cytoplasmic AU-rich element mRNA more abundantly than normal cells, were subjected to HuR knockdown. In the HuR-knockdown cancer cells, the cytoplasmic expression of c-fos, c-myc, and COX-2 mRNAs was inhibited compared with those in cells that had been transfected with a control small interfering RNA, and the half-lives of these mRNAs were shorter than those of their counterparts in the control cells. HuR-knockdown cells failed to make colonies in soft agar, suggesting that the cells had lost their ability for anchorage-independent cell growth. Additionally, the motile and invasive activities of the cells decreased remarkably by HuR knockdown. Furthermore, the expression of cell cycle-related proteins, such as cyclin A, cyclin B1, cyclin D1, and cyclin-dependent kinase 1, was reduced in HuR-knockdown cancer cells, and HuR bound to cdk1 mRNA to stabilize it. These findings suggest that HuR knockdown changes the features of oral cancer cells, at least in part, by affecting their cell cycle and shows potential as an effective therapeutic approach.

  14. ZEB1 knockdown mediated using polypeptide cationic micelles inhibits metastasis and effects sensitization to a chemotherapeutic drug for cancer therapy

    NASA Astrophysics Data System (ADS)

    Fang, Shengtao; Wu, Lei; Li, Mingxing; Yi, Huqiang; Gao, Guanhui; Sheng, Zonghai; Gong, Ping; Ma, Yifan; Cai, Lintao

    2014-08-01

    Metastasis and drug resistance are the main causes for the failure in clinical cancer therapy. Emerging evidence suggests an intricate role of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) in metastasis and drug resistance. The EMT-activator ZEB1 is crucial in malignant tumor progression by linking EMT-activation and stemness-maintenance. Here, we used multifunctional polypeptide micelle nanoparticles (NP) as nanocarriers for the delivery of ZEB1 siRNA and doxorubicin (DOX). The nanocarriers could effectively deliver siRNA to the cytoplasm and knockdown the target gene in H460 cells and H460 xenograft tumors, leading to reduced EMT and repressed CSC properties in vitro and in vivo. The complex micelle nanoparticles with ZEB1 siRNA (siRNA-NP) significantly reduced metastasis in the lung. When DOX and siRNA were co-delivered by the nanocarriers (siRNA-DOX-NP), a synergistic therapeutic effect was observed, resulting in dramatic inhibition of tumor growth in a H460 xenograft model. These results demonstrated that the siRNA-NP or siRNA-DOX-NP complex targeting ZEB1 could be developed into a new therapeutic approach for non-small cell lung cancer (NSCLC) treatment.Metastasis and drug resistance are the main causes for the failure in clinical cancer therapy. Emerging evidence suggests an intricate role of epithelial-mesenchymal transition (EMT) and cancer stem cells (CSCs) in metastasis and drug resistance. The EMT-activator ZEB1 is crucial in malignant tumor progression by linking EMT-activation and stemness-maintenance. Here, we used multifunctional polypeptide micelle nanoparticles (NP) as nanocarriers for the delivery of ZEB1 siRNA and doxorubicin (DOX). The nanocarriers could effectively deliver siRNA to the cytoplasm and knockdown the target gene in H460 cells and H460 xenograft tumors, leading to reduced EMT and repressed CSC properties in vitro and in vivo. The complex micelle nanoparticles with ZEB1 siRNA (siRNA-NP) significantly reduced

  15. Knockdown of TWIST1 enhances arsenic trioxide- and ionizing radiation-induced cell death in lung cancer cells by promoting mitochondrial dysfunction

    SciTech Connect

    Seo, Sung-Keum; Kim, Jae-Hee; Choi, Ha-Na; Choe, Tae-Boo; Hong, Seok-Il; Yi, Jae-Youn; Hwang, Sang-Gu; Lee, Hyun-Gyu; Lee, Yun-Han; Park, In-Chul

    2014-07-11

    Highlights: • Knockdown of TWIST1 enhanced ATO- and IR-induced cell death in NSCLCs. • Intracellular ROS levels were increased in cells treated with TWIST1 siRNA. • TWIST1 siRNA induced MMP loss and mitochondrial fragmentation. • TWIST1 siRNA upregulated the fission-related proteins FIS1 and DRP1. - Abstract: TWIST1 is implicated in the process of epithelial mesenchymal transition, metastasis, stemness, and drug resistance in cancer cells, and therefore is a potential target for cancer therapy. In the present study, we found that knockdown of TWIST1 by small interfering RNA (siRNA) enhanced arsenic trioxide (ATO)- and ionizing radiation (IR)-induced cell death in non-small-cell lung cancer cells. Interestingly, intracellular reactive oxygen species levels were increased in cells treated with TWIST1 siRNA and further increased by co-treatment with ATO or IR. Pretreatment of lung cancer cells with the antioxidant N-acetyl-cysteine markedly suppressed the cell death induced by combined treatment with TWIST1 siRNA and ATO or IR. Moreover, treatment of cells with TWIST1 siRNA induced mitochondrial membrane depolarization and significantly increased mitochondrial fragmentation (fission) and upregulated the fission-related proteins FIS1 and DRP1. Collectively, our results demonstrate that siRNA-mediated TWIST1 knockdown induces mitochondrial dysfunction and enhances IR- and ATO-induced cell death in lung cancer cells.

  16. Knockdown of HIF-1α by siRNA-expressing plasmid delivered by attenuated Salmonella enhances the antitumor effects of cisplatin on prostate cancer.

    PubMed

    Gu, Junlian; Li, Yang; Zeng, Jun; Wang, Bo; Ji, Kun; Tang, Yufeng; Sun, Qing

    2017-08-08

    Resistance to cisplatin (DDP) and dose-related toxicity remain two important obstacles in the treatment of prostate cancer (PCa) patients with DDP-based chemotherapy. We have investigated whether the knockdown of hypoxia-inducible factor-1 alpha (HIF-1α) by siRNA could enhance the antitumor activity of DDP, and aimed to determine the underlying mechanisms. Intravenous injection of attenuated Salmonella carrying a HIF-1α siRNA-expressing plasmid was used to knockdown HIF-1α in a PC-3 xenograft model. The in vitro and in vivo effects of HIF-1α siRNA treatment and/or DPP on PCa cell proliferation, apoptosis, glycolysis, and production of reactive oxygen species (ROS) were assessed by examining molecular markers specific to each process. The results demonstrated that the treatment of tumor-bearing mice with attenuated Salmonella carrying the HIF-1α siRNA plasmid greatly enhanced the antitumor effects of low-dose DDP. Further mechanistic studies demonstrated that knockdown of HIF-1α improved the response of PCa cells to DDP by redirecting aerobic glycolysis toward mitochondrial oxidative phosphorylation, leading to cell death through overproduction of ROS. Our findings indicate that DDP-based chemotherapy combined with targeting the HIF-1α-regulated cancer metabolism pathway might be an ideal strategy to treat PCa.

  17. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion

    SciTech Connect

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-10-10

    Highlights: • We employed RNA interference to knockdown SET expression in breast cancer cells. • Knockdown of SET expression inhibits cell proliferation, migration and invasion. • Knockdown of SET expression increases the activity and expression of PP2A. • Knockdown of SET expression decreases the expression of MMP-9. - Abstract: Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  18. Knockdown of p53 suppresses Nanog expression in embryonic stem cells

    SciTech Connect

    Abdelalim, Essam Mohamed; Tooyama, Ikuo

    2014-01-10

    Highlights: •We investigate the role of p53 in ESCs in the absence of DNA damage. •p53 knockdown suppresses ESC proliferation. •p53 knockdown downregulates Nanog expression. •p53 is essential for mouse ESC self-renewal. -- Abstract: Mouse embryonic stem cells (ESCs) express high levels of cytoplasmic p53. Exposure of mouse ESCs to DNA damage leads to activation of p53, inducing Nanog suppression. In contrast to earlier studies, we recently reported that chemical inhibition of p53 suppresses ESC proliferation. Here, we confirm that p53 signaling is involved in the maintenance of mouse ESC self-renewal. RNA interference-mediated knockdown of p53 induced downregulation of p21 and defects in ESC proliferation. Furthermore, p53 knockdown resulted in a significant downregulation in Nanog expression at 24 and 48 h post-transfection. p53 knockdown also caused a reduction in Oct4 expression at 48 h post-transfection. Conversely, exposure of ESCs to DNA damage caused a higher reduction of Nanog expression in control siRNA-treated cells than in p53 siRNA-treated cells. These data show that in the absence of DNA damage, p53 is required for the maintenance of mouse ESC self-renewal by regulating Nanog expression.

  19. Knockdown of cullin 4A inhibits growth and increases chemosensitivity in lung cancer cells.

    PubMed

    Hung, Ming-Szu; Chen, I-Chuan; You, Liang; Jablons, David M; Li, Ya-Chin; Mao, Jian-Hua; Xu, Zhidong; Lung, Jr-Hau; Yang, Cheng-Ta; Liu, Shih-Tung

    2016-07-01

    Cullin 4A (Cul4A) has been observed to be overexpressed in various cancers. In this study, the role of Cul4A in the growth and chemosensitivity in lung cancer cells were studied. We showed that Cul4A is overexpressed in lung cancer cells and tissues. Knockdown of the Cul4A expression by shRNA in lung cancer cells resulted in decreased cellular proliferation and growth in lung cancer cells. Increased sensitivity to gemcitabine, a chemotherapy drug, was also noted in those Cul4A knockdown lung cancer cells. Moreover, increased expression of p21, transforming growth factor (TGF)-β inducible early gene-1 (TIEG1) and TGF beta-induced (TGFBI) was observed in lung cancer cells after Cul4A knockdown, which may be partially related to increased chemosensitivity to gemcitabine. G0/G1 cell cycle arrest was also noted after Cul4A knockdown. Notably, decreased tumour growth and increased chemosensitivity to gemcitabine were also noted after Cul4A knockdown in lung cancer xenograft nude mice models. In summary, our study showed that targeting Cul4A with RNAi or other techniques may provide a possible insight to the development of lung cancer therapy in the future.

  20. Mitochondria-Targeted Antioxidant Prevents Cardiac Dysfunction Induced by Tafazzin Gene Knockdown in Cardiac Myocytes

    PubMed Central

    He, Quan; Harris, Nicole; Ren, Jun; Han, Xianlin

    2014-01-01

    Tafazzin, a mitochondrial acyltransferase, plays an important role in cardiolipin side chain remodeling. Previous studies have shown that dysfunction of tafazzin reduces cardiolipin content, impairs mitochondrial function, and causes dilated cardiomyopathy in Barth syndrome. Reactive oxygen species (ROS) have been implicated in the development of cardiomyopathy and are also the obligated byproducts of mitochondria. We hypothesized that tafazzin knockdown increases ROS production from mitochondria, and a mitochondria-targeted antioxidant prevents tafazzin knockdown induced mitochondrial and cardiac dysfunction. We employed cardiac myocytes transduced with an adenovirus containing tafazzin shRNA as a model to investigate the effects of the mitochondrial antioxidant, mito-Tempo. Knocking down tafazzin decreased steady state levels of cardiolipin and increased mitochondrial ROS. Treatment of cardiac myocytes with mito-Tempo normalized tafazzin knockdown enhanced mitochondrial ROS production and cellular ATP decline. Mito-Tempo also significantly abrogated tafazzin knockdown induced cardiac hypertrophy, contractile dysfunction, and cell death. We conclude that mitochondria-targeted antioxidant prevents cardiac dysfunction induced by tafazzin gene knockdown in cardiac myocytes and suggest mito-Tempo as a potential therapeutic for Barth syndrome and other dilated cardiomyopathies resulting from mitochondrial oxidative stress. PMID:25247053

  1. Knockdown of astrocyte elevated gene-1 inhibits tumor growth and modifies microRNAs expression profiles in human colorectal cancer cells

    SciTech Connect

    Huang, Sujun; Wu, Binwen; Li, Dongfeng; Zhou, Weihong; Deng, Gang; Zhang, Kaijun; Li, Youjia

    2014-02-14

    Highlights: • AEG-1 expression in CRC cell lines and down-regulation or upregulation of AEG-1 in vitro. • Knockdown of AEG-1 inhibits cell proliferation, colony formation and invasion. • Upregulation of AEG-1 enhances proliferation, invasion and colony formation. • Knockdown of AEG-1 accumulates G0/G1-phase cells and promotes apoptosis in CRC cells. • AEG-1 knockdown increases 5-FU cytotoxicity. - Abstract: Astrocyte elevated gene-1 (AEG-1), upregulated in various types of malignancies including colorectal cancer (CRC), has been reported to be associated with the carcinogenesis. MicroRNAs (miRNAs) are widely involved in the initiation and progression of cancer. However, the functional significance of AEG-1 and the relationship between AEG-1 and microRNAs in human CRC remains unclear. The aim of this study was to investigate whether AEG-1 could serve as a potential therapeutic target of human CRC and its possible mechanism. We adopted a strategy of ectopic overexpression or RNA interference to upregulate or downregulate expression of AEG-1 in CRC models. Their phenotypic changes were analyzed by Western blot, MTT and transwell matrix penetration assays. MicroRNAs expression profiles were performed using microarray analysis followed by validation using qRT-PCR. Knockdown of AEG-1 could significantly inhibit colon cancer cell proliferation, colony formation, invasion and promotes apoptosis. Conversely, upregulation of AEG-1 could significantly enhance cell proliferation, invasion and reduced apoptisis. AEG-1 directly contributes to resistance to chemotherapeutic drug. Targeted downregulation of AEG-1 might improve the expression of miR-181a-2{sup ∗}, -193b and -193a, and inversely inhibit miR-31 and -9{sup ∗}. Targeted inhibition of AEG-1 can lead to modification of key elemental characteristics, such as miRNAs, which may become a potential effective therapeutic strategy for CRC.

  2. Nodes-and-connections RNAi knockdown screening: identification of a signaling molecule network involved in fulvestrant action and breast cancer prognosis

    PubMed Central

    Miyoshi, N; Wittner, B S; Shioda, K; Hitora, T; Ito, T; Ramaswamy, S; Isselbacher, K J; Sgroi, D C; Shioda, T

    2015-01-01

    Although RNA interference (RNAi) knockdown screening of cancer cell cultures is an effective approach to predict drug targets or therapeutic/prognostic biomarkers, interactions among identified targets often remain obscure. Here, we introduce the nodes-and-connections RNAi knockdown screening that generates a map of target interactions through systematic iterations of in silico prediction of targets and their experimental validation. An initial RNAi knockdown screening of MCF-7 human breast cancer cells targeting 6560 proteins identified four signaling molecules required for their fulvestrant-induced apoptosis. Signaling molecules physically or functionally interacting with these four primary node targets were computationally predicted and experimentally validated, resulting in identification of four second-generation nodes. Three rounds of further iterations of the prediction–validation cycle generated third, fourth and fifth generation of nodes, completing a 19-node interaction map that contained three predicted nodes but without experimental validation because of technical limitations. The interaction map involved all three members of the death-associated protein kinases (DAPKs) as well as their upstream and downstream signaling molecules (calmodulins and myosin light chain kinases), suggesting that DAPKs play critical roles in the cytocidal action of fulvestrant. The in silico Kaplan–Meier analysis of previously reported human breast cancer cohorts demonstrated significant prognostic predictive power for five of the experimentally validated nodes and for three of the prediction-only nodes. Immunohistochemical studies on the expression of 10 nodal proteins in human breast cancer tissues not only supported their prognostic prediction power but also provided statistically significant evidence of their synchronized expression, implying functional interactions among these nodal proteins. Thus, the Nodes-and-Connections approach to RNAi knockdown screening yields

  3. Knockdown of the MAPK p38 pathway increases the susceptibility of Chilo suppressalis larvae to Bacillus thuringiensis Cry1Ca toxin

    PubMed Central

    Qiu, Lin; Fan, Jinxing; Liu, Lang; Zhang, Boyao; Wang, Xiaoping; Lei, Chaoliang; Lin, Yongjun; Ma, Weihua

    2017-01-01

    The bacterium Bacillus thuringiensis (Bt) produces a wide range of toxins that are effective against a number of insect pests. Identifying the mechanisms responsible for resistance to Bt toxin will improve both our ability to control important insect pests and our understanding of bacterial toxicology. In this study, we investigated the role of MAPK pathways in resistance against Cry1Ca toxin in Chilo suppressalis, an important lepidopteran pest of rice crops. We first cloned the full-length of C. suppressalis mitogen-activated protein kinase (MAPK) p38, ERK1, and ERK2, and a partial sequence of JNK (hereafter Csp38, CsERK1, CsERK2 and CsJNK). We could then measure the up-regulation of these MAPK genes in larvae at different times after ingestion of Cry1Ca toxin. Using RNA interference to knockdown Csp38, CsJNK, CsERK1 and CsERK2 showed that only knockdown of Csp38 significantly increased the mortality of larvae to Cry1Ca toxin ingested in either an artificial diet, or after feeding on transgenic rice expressed Cry1Ca. These results suggest that MAPK p38 is responsible for the resistance of C. suppressalis larvae to Bt Cry1Ca toxin. PMID:28262736

  4. Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

    PubMed Central

    Nakagaki, Brenda Naemi; Mendonça-Neto, Rondon Pessoa; Canavaci, Adriana Monte Cassiano; Souza Melo, Normanda; Martinelli, Patrícia Massara; Fernandes, Ana Paula; daRocha, Wanderson Duarte; Teixeira, Santuza M. R.

    2015-01-01

    Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole. PMID:26641088

  5. FTO knockdown in rat ventromedial hypothalamus does not affect energy balance.

    PubMed

    van Gestel, Margriet A; Sanders, Loek E; de Jong, Johannes W; Luijendijk, Mieneke C M; Adan, Roger A H

    2014-12-01

    Single nucleotide polymorphisms (SNPs) clustered in the first intron of the fat mass and obesity-associated (FTO) gene has been associated with obesity. FTO expression is ubiquitous, with particularly high levels in the hypothalamic area of the brain. To investigate the region-specific role of FTO, AAV technology was applied to knockdown FTO in the ventromedial hypothalamus (VMH). No effect of FTO knockdown was observed on bodyweight or parameters of energy balance. Animals were exposed twice to an overnight fast, followed by a high-fat high-sucrose (HFHS) diet for 1 week. FTO knockdown did not result in a different response to the diets. A region-specific role for FTO in the VMH in the regulation of energy balance could not be found.

  6. Transcriptional response to mitochondrial protease IMMP2L knockdown in human primary astrocytes.

    PubMed

    Gokoolparsadh, Akira; Fang, Zhiming; Braidy, Nady; Lin, Peijie; Pardy, Christopher J; Eapen, Valsamma; Clarke, Raymond; Voineagu, Irina

    2017-01-22

    IMMP2L encodes the inner membrane peptidase subunit 2, a mitochondrial protease involved in cleaving the space-sorting signals of mitochondrial membrane proteins. IMMP2L has been implicated in Tourette syndrome, but how its dysfunction contributes to the neurodevelopmental phenotype remains unclear. Here we show that IMMP2L transcription requires Topoisomerase I in human primary astrocytes, and characterize the downstream effects of IMMP2L knockdown on gene expression. We demonstrate that IMMP2L knockdown leads to dysregulation of genes involved in central nervous system development. We also find that the transcriptional response to IMMP2L knockdown partially overlaps the one induced by mitochondrial complex III inhibition. Overall, these data bring further insight into the molecular consequences of IMMP2L dysfunction in the brain. Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.

  7. Knockdown of GALNT1 suppresses malignant phenotype of hepatocellular carcinoma by suppressing EGFR signaling

    PubMed Central

    Huang, Miao-Juei; Hu, Rey-Heng; Chou, Chih-Hsing; Hsu, Chia-Lang; Liu, Ya-Wen; Huang, John; Hung, Ji-Shiang; Lai, I-Rue; Juan, Hsueh-Fen; Yu, Sung-Liang; Wu, Yao-Ming; Huang, Min-Chuan

    2015-01-01

    O-glycosylation is a common protein modification. Aberrant O-glycosylation is associated with many cancers. GALNT1 is a GalNAc-transferase that initiates protein O-glycosylation. We found that GALNT1 is frequently up-regulated in hepatocellular carcinoma (HCC) and is associated with poor patient survival. Overexpression of GALNT1 increased and knockdown decreased HCC cell migration and invasion. Knockdown of GALNT1 inhibited EGF-induced migration and invasion. Knockdown of GALNT1 decreased EGFR activation and increased EGFR degradation, by decreasing EGFR O-glycosylation. This study demonstrates that down-regulation of GALNT1 is sufficient to suppress malignant phenotype of HCC cells by decreasing EGFR signaling. Thus, GALNT1 is a potential target in HCC. PMID:25730904

  8. Merlin Knockdown in human Schwann cells: Clues to Vestibular Schwannoma Tumorigenesis

    PubMed Central

    Ahmad, Zana; Brown, Carrie Maiorana; Patel, Andrew K.; Ryan, Allen F.; Ongkeko, Rutherford; Doherty, Joni K.

    2010-01-01

    Hypothesis To investigate the early events in molecular progression towards schwannoma tumorigenesis, we developed an in vitro model of human Schwann cell tumorigenesis by merlin knockdown. Background Neurofibromatosis 2 (NF2)-related and sporadic vestibular schwannoma (VS) exhibit loss of functional merlin (schwannomin). Following loss of merlin expression in the Schwann cell, the initial steps toward vestibular schwannoma (VS) tumorigenesis are unknown. Merlin, a putative tumor suppressor protein, interacts with many cellular proteins, regulating their function. Among these are receptor tyrosine kinases, including the ErbB family receptors, EGFR and ErbB2. Functional merlin interacts with and internalizes these growth factor receptors, silencing their proliferation and survival signaling. Deregulation of CD44, the cell adhesion/signaling molecule and cancer stem cell marker has also been implicated in VS tumorigenesis. Methods Merlin knockdown was performed using small interfering RNA (siRNA) transfection into human Schwann cell primary cultures. Knockdown was confirmed by real-time quantitative PCR (qPCR), immunofluorescence, and Western analysis. Expression profiles of ErbB, merlin, and the stem cell markers, nestin and CD44, were examined in knockdowns. Proliferation rate was assessed with BrdU incorporation and radiation sensitivity was assessed using the Annexin assay in knockdowns versus controls. Results Merlin knockdowns demonstrated increased proliferation rate, upregulation of EGFR, ErbB2, and ErbB3, CD44, and nestin. Short-term merlin depletion had no effect on gamma irradiation sensitivity compared with controls. Conclusions Merlin depletion results in deregulation of ErbB receptor signaling, promotes a dedifferentiated state, and increases Schwann cell proliferation, suggesting critical steps towards schwannoma tumorigenesis. PMID:20195187

  9. Effect of AURKA Gene Expression Knockdown on Angiogenesis and Tumorigenesis of Human Ovarian Cancer Cell Lines.

    PubMed

    Wang, Cong; Yan, Qin; Hu, Minmin; Qin, Di; Feng, Zhenqing

    2016-12-01

    Ovarian cancer is one of the most common malignant gynecological cancers. Higher expression of AURKA has been found in immortalized human ovarian epithelial cells in previous studies, implying the relationship between AURKA and ovarian cancer pathogenesis. We investigated the effect of AURKA on angiogenesis and tumorigenesis of human ovarian cancer cells. Firstly, the expression of AURKA in HO8910 and SKOV3 ovarian cancer cell lines was knocked down using a vector expressing a short hairpin small interfering RNA (shRNA). Next, the effect of knockdown of AURKA on cell angiogenesis, proliferation, migration, and invasion was determined by microtubule formation assay, proliferation assay, transwell migration, and invasion assays. In addition, the effect of AURKA knockdown on angiogenesis and tumorigenesis was also determined in a chicken chorioallantoic membrane (CAM) model and in nude mice. The results of the microtubule formation assay indicated that knockdown of AURKA significantly inhibited ovarian cancer cell-induced angiogenesis of endothelial cells compared to its control (P < 0.001). Knockdown of AURKA also significantly inhibited cell proliferation, migration, and invasion of HO8910 and SKOV3 cells in vitro. Furthermore, the Matrigel plug assay showed that knockdown of AURKA significantly repressed ovarian cancer cell-induced angiogenesis in nude mice (P < 0.05), and the CAMs model also showed that AURKA knockdown significantly attenuated the angiogenesis (P < 0.001) and tumorigenesis (P < 0.001) of HO8910 cells compared to the control. Finally, the tumorigenicity assay in vivo further indicated that AURKA shRNA reduced tumorigenesis in nude mice inoculated with ovarian cancer cells (P < 0.001). These results suggest the potential role of AURKA in angiogenesis and tumorigenesis of ovarian cancer, which may provide a potential therapeutic target for the disease.

  10. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    PubMed Central

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  11. Knockdown of lymphoid enhancer factor 1 inhibits colon cancer progression in vitro and in vivo.

    PubMed

    Wang, Wen-Juan; Yao, Yu; Jiang, Li-Li; Hu, Ting-Hua; Ma, Jie-Qun; Liao, Zi-Jun; Yao, Jun-Tao; Li, Dong-Fan; Wang, Shu-Hong; Nan, Ke-Jun

    2013-01-01

    Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy.

  12. Knockdown of Lymphoid Enhancer factor 1 Inhibits Colon Cancer Progression In Vitro and In Vivo

    PubMed Central

    Jiang, Li-Li; Hu, Ting-Hua; Ma, Jie-Qun; Liao, Zi-Jun; Yao, Jun-Tao; Li, Dong-Fan; Wang, Shu-Hong; Nan, Ke-Jun

    2013-01-01

    Expression of lymphoid enhancer factor 1 (LEF1) is frequently altered in different human cancers. This study aimed to assess LEF1 expression in colon cancer tissues and to explore changed phenotypes, gene expressions, and the possible mechanism after knocked down LEF1 expression in colon cancer cell lines. A total of 106 colon cancer and matched paratumorous normal tissues were used to assess LEF1 expression using immunohistochemistry and qRT-PCR. LEF1 lentivirus was used to knockdown LEF1 expression for the assessment of cell viability, cell cycle distribution, apoptosis, and gene expressions. The nude mouse xenograft assay was performed to detect the effects of LEF1 knockdown in vivo. The data showed that the levels of LEF1 mRNA and protein were significantly increased in human colon cancer tissues compared to the matched paratumorous normal tissues and were associated with infiltration depth, lymph node and distant metastases, advanced TNM (tumor-node-metastasis) stages, and shorter overall survival. Furthermore, LEF1 knockdown reduced tumor cell viability, invasion capacity, MMP2 and MMP-9 expression, but induced apoptosis. Nude mouse xenograft assay showed that LEF1 knockdown suppressed tumor formation and growth in vivo. In addition, the expression of Notch pathway-related proteins RBP-jκ and Hes1 was reduced in LEF1 knockdown cells. Taken together, LEF1 protein was overexpressed in colon cancer tissues and knockdown of LEF1 expression inhibited colon cancer growth in vitro and in vivo. These data suggest that targeting of LEF1 expression should be further evaluated for colon cancer prevention and therapy. PMID:24098538

  13. Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

    PubMed Central

    Alsayegh, Khaled N.; Gadepalli, Venkat S.; Iyer, Shilpa; Rao, Raj R.

    2015-01-01

    Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether

  14. Conditional knockdown of target gene expression by tetracycline regulated transcription of double strand RNA.

    PubMed

    Hou, Xubin; Omi, Minoru; Harada, Hidekiyo; Ishii, Shunsuke; Takahashi, Yoshiko; Nakamura, Harukazu

    2011-01-01

    In vivo electroporation has served as an effective tool for the study of developmental biology. Here we report tetracycline inducible gene knockdown by electroporation. Our system consists of genome integration of a cassette encoding long double strand RNA (dsRNA) of a gene of interest by electroporation, transcription of which is assured by RNA polymerase II, and induction of transcription of dsRNA by tetracyclin. Long dsRNA decapped by ribozyme in the cassette and without poly A tail is processed into siRNA within nuclei. We could successfully induce knockdown of En2 and Coactosin by Dox administration.

  15. Gene knockdown by ihpRNA-triggering in the ectomycorrhizal basidiomycete fungus Laccaria bicolor.

    PubMed

    Kemppainen, Minna J; Pardo, Alejandro G

    2010-01-01

    Ectomycorrhiza (ECM) is a mutualistic association between fungi and the roots of the vast majority of trees. These include numerous ecologically and economically relevant species and the participating fungal symbionts are predominantly filamentous basidiomycetes. In natural ecosystems the plant nutrient uptake from soil takes place via the extraradical mycelia of these ECM mycosimbionts as a trade for plant photosyntates. The symbiotic phase in the life cycle of ECM basidiomycetes is the dikaryotic hyphae. Therefore, studies on symbiotic relevant gene functions require the inactivation of both gene copies in these dikaryotic fungi. RNA silencing is a eukaryotic sequence homology-dependent degradation of target RNAs which is believed to have evolved as a protection mechanism against invading nucleic acids. In different eukaryotic organisms, including fungi, the RNA silencing pathway can be artificially triggered to target and degrade gene transcripts of interest, resulting in gene knock-down. Most importantly, RNA silencing can act at the cytosolic level affecting mRNAs originating from several gene copies and different nuclei thus offering an efficient means of altering gene expression in dikaryotic organisms. Therefore, the pHg/pSILBAγ silencing vector was constructed for efficient RNA silencing triggering in the model mycorrhizal fungus Laccaria bicolor. This cloning vector carries the Agaricus bisporus gpdII-promoter, two multiple cloning sites separated by a L. bicolor nitrate reductase intron and the Aspergillus nidulans trpC terminator. pSILBAγ allows an easy two-step PCR-cloning of hairpin sequences to be expressed in basidiomycetes. With one further cloning step into pHg, a pCAMBIA1300-based binary vector carrying a hygromycin resistance cassette, makes the pHg/pSILBAγ plasmid compatible with Agrobacterium-mediated transformation. The pHg/pSILBAγ-system results in predominantly single integrations of RNA silencing triggering T-DNAs in the fungal genome

  16. Brain gene expression changes elicited by peripheral vitellogenin knockdown in the honey bee.

    PubMed

    Wheeler, M M; Ament, S A; Rodriguez-Zas, S L; Robinson, G E

    2013-10-01

    Vitellogenin (Vg) is best known as a yolk protein precursor. Vg also functions to regulate behavioural maturation in adult honey bee workers, but the underlying molecular mechanisms by which it exerts this novel effect are largely unknown. We used abdominal vitellogenin (vg) knockdown with RNA interference (RNAi) and brain transcriptomic profiling to gain insights into how Vg influences honey bee behavioural maturation. We found that vg knockdown caused extensive gene expression changes in the bee brain, with much of this transcriptional response involving changes in central biological functions such as energy metabolism. vg knockdown targeted many of the same genes that show natural, maturation-related differences, but the direction of change for the genes in these two contrasts was not correlated. By contrast, vg knockdown targeted many of the same genes that are regulated by juvenile hormone (JH) and there was a significant correlation for the direction of change for the genes in these two contrasts. These results indicate that the tight coregulatory relationship that exists between JH and Vg in the regulation of honey bee behavioural maturation is manifest at the genomic level and suggest that these two physiological factors act through common pathways to regulate brain gene expression and behaviour. © 2013 Royal Entomological Society.

  17. Strength knock-down assessment of porosity in composites: modelling, characterising and specimen manufacture

    NASA Astrophysics Data System (ADS)

    McMillan, Alison J.; Archer, Edward; McIlhagger, Alistair; Lelong, Guillaume

    2012-08-01

    Porosity and inclusion of foreign material is known to reduce the strength of materials, and this paper addresses the particular problem of strength knock-down assessment due to porosity in composite materials. Porosity is often measured in terms of percentage of voids per unit volume of a component, because this can be related directly to ultra-sound absorption. Nevertheless, this is a poor indicator of actual strength knock-down, as it provides little information about void size, shape, orientation and whether they are evenly distributed or are clustered. Characterisation of void clustering enables a link between a cluster characteristic and the strength knock-down. Laboratory based testing achieves controlled porosity in specimens by introducing pin-holes into the RTM in-flow pipework, which entrains voids into the body of the preform within mould tooling. Specimens are manufactured to create resin regions bounded by a fibre reinforced picture frame, to allow for easy load application. Strength knock-downs from test are related to the theoretical expectations.

  18. The functional genetic link of NLGN4X knockdown and neurodevelopment in neural stem cells.

    PubMed

    Shi, Lingling; Chang, Xiao; Zhang, Peilin; Coba, Marcelo P; Lu, Wange; Wang, Kai

    2013-09-15

    Genetic mutations in NLGN4X (neuroligin 4), including point mutations and copy number variants (CNVs), have been associated with susceptibility to autism spectrum disorders (ASDs). However, it is unclear how mutations in NLGN4X result in neurodevelopmental defects. Here, we used neural stem cells (NSCs) as in vitro models to explore the impacts of NLGN4X knockdown on neurodevelopment. Using two shRNAmir-based vectors targeting NLGN4X and one control shRNAmir vector, we modulated NLGN4X expression and differentiated these NSCs into mature neurons. We monitored the neurodevelopmental process at Weeks 0, 0.5, 1, 2, 4 and 6, based on morphological analysis and whole-genome gene expression profiling. At the cellular level, in NSCs with NLGN4X knockdown, we observed increasingly delayed neuronal development and compromised neurite formation, starting from Week 2 through Week 6 post differentiation. At the molecular level, we identified multiple pathways, such as neurogenesis, neuron differentiation and muscle development, which are increasingly disturbed in cells with NLGN4X knockdown. Notably, several postsynaptic genes, including DLG4, NLGN1 and NLGN3, also have decreased expression. Based on in vitro models, NLGN4X knockdown directly impacts neurodevelopmental process during the formation of neurons and their connections. Our functional genomics study highlights the utility of NSCs models in understanding the functional roles of CNVs in affecting neurodevelopment and conferring susceptibility to neurodevelopmental diseases.

  19. PEG-PLA nanoparticles facilitate siRNA knockdown in adult zebrafish heart.

    PubMed

    Diao, Jupeng; Wang, Hongxia; Chang, Nannan; Zhou, Xiao-Hai; Zhu, Xiaojun; Wang, Jun; Xiong, Jing-Wei

    2015-10-15

    The remarkable regenerative capacity of the zebrafish has made it an important model organism for studying heart regeneration. However, current loss-of-function studies are limited by a lack of conditional-knockout and effective gene-knockdown methods for the adult heart. Here, we report a novel siRNA knockdown method facilitated by poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) nanoparticles. The siRNA-encapsulated nanoparticles successfully entered cells and resulted in remarkable gene-specific knockdown in the adult heart. This effect was demonstrated by down-regulation of the Aldh1a2 and Dusp6 proteins after intrapleural delivery of nanoparticle-encapsulated siRNAs. Furthermore, siRNA-mediated knockdown of Aldh1a2 was sufficient to inhibit myocardial proliferation and decrease the numbers of Gata4-positive cardiomyocytes after ventricular resection. Therefore, the results of this work demonstrate that nanoparticle-facilitated siRNA delivery provides an alternative tool for loss-of-function studies of genes in the adult heart in particular and other organs in general in the adult zebrafish. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Acat1 knockdown gene therapy decreases amyloid-β in a mouse model of Alzheimer's disease.

    PubMed

    Murphy, Stephanie R; Chang, Catherine Cy; Dogbevia, Godwin; Bryleva, Elena Y; Bowen, Zachary; Hasan, Mazahir T; Chang, Ta-Yuan

    2013-08-01

    Both genetic inactivation and pharmacological inhibition of the cholesteryl ester synthetic enzyme acyl-CoA:cholesterol acyltransferase 1 (ACAT1) have shown benefit in mouse models of Alzheimer's disease (AD). In this study, we aimed to test the potential therapeutic applications of adeno-associated virus (AAV)-mediated Acat1 gene knockdown in AD mice. We constructed recombinant AAVs expressing artificial microRNA (miRNA) sequences, which targeted Acat1 for knockdown. We demonstrated that our AAVs could infect cultured mouse neurons and glia and effectively knockdown ACAT activity in vitro. We next delivered the AAVs to mouse brains neurosurgically, and demonstrated that Acat1-targeting AAVs could express viral proteins and effectively diminish ACAT activity in vivo, without inducing appreciable inflammation. We delivered the AAVs to the brains of 10-month-old AD mice and analyzed the effects on the AD phenotype at 12 months of age. Acat1-targeting AAV delivered to the brains of AD mice decreased the levels of brain amyloid-β and full-length human amyloid precursor protein (hAPP), to levels similar to complete genetic ablation of Acat1. This study provides support for the potential therapeutic use of Acat1 knockdown gene therapy in AD.

  1. Goat activin receptor type IIB knockdown by muscle specific promoter driven artificial microRNAs.

    PubMed

    Patel, Amrutlal K; Shah, Ravi K; Patel, Utsav A; Tripathi, Ajai K; Joshi, Chaitanya G

    2014-10-10

    Activin receptor type IIB (ACVR2B) is a transmembrane receptor which mediates signaling of TGF beta superfamily ligands known to function in regulation of muscle mass, embryonic development and reproduction. ACVR2B antagonism has shown to enhance the muscle growth in several disease and transgenic models. Here, we show ACVR2B knockdown by RNA interference using muscle creatine kinase (MCK) promoter driven artificial microRNAs (amiRNAs). Among the various promoter elements tested, the ∼1.26 kb MCK promoter region showed maximum transcriptional activity in goat myoblasts cells. We observed up to 20% silencing in non-myogenic 293T cells and up to 32% silencing in myogenic goat myoblasts by MCK directed amiRNAs by transient transfection. Goat myoblasts stably integrated with MCK directed amiRNAs showed merely 8% silencing in proliferating myoblasts which was increased to 34% upon induction of differentiation at transcript level whereas up to 57% silencing at protein level. Knockdown of ACVR2B by 5'-UTR derived amiRNAs resulted in decreased SMAD2/3 signaling, increased expression of myogenic regulatory factors (MRFs) and enhanced proliferation and differentiation of myoblasts. Unexpectedly, knockdown of ACVR2B by 3'-UTR derived amiRNAs resulted in increased SMAD2/3 signaling, reduced expression of MRFs and suppression of myogenesis. Our study offers muscle specific knockdown of ACVR2B as a potential strategy to enhance muscle mass in the farm animal species.

  2. Knockdown of DDX46 Inhibits the Invasion and Tumorigenesis in Osteosarcoma Cells.

    PubMed

    Jiang, Feng; Zhang, Dengfeng; Li, Guojun; Wang, Xiao

    2017-03-13

    DDX46, a member of the DEAD-box (DDX) helicase family, is involved in the development of several tumors. However, the exact role of DDX46 in osteosarcoma and the underlying mechanisms in tumorigenesis remain poorly understood. Thus, in the present study, we explored the role of DDX46 in osteosarcoma and the underlying mechanisms. Our results demonstrated that the expression levels of DDX46 in both mRNA and protein were greatly elevated in human osteosarcoma tissues and cell lines. Knockdown of DDX46 obviously inhibited osteosarcoma cell proliferation and tumor growth in vivo. In addition, knockdown of DDX46 also significantly suppressed migration and invasion in osteosarcoma cells. Furthermore, knockdown of DDX46 substantially downregulated the phosphorylation levels of PI3K and Akt in SaOS2 cells. In summary, the present results have revealed that DDX46 plays an important role in osteosarcoma growth and metastasis. Knockdown of DDX46 inhibited osteosarcoma cell proliferation, migration, and invasion in vitro and tumor growth in vivo. Therefore, DDX46 may be a potential therapeutic target for the treatment of osteosarcoma.

  3. Transcriptional and phenotypic comparisons of Ppara knockout and siRNA knockdown mice

    PubMed Central

    De Souza, Angus T.; Dai, Xudong; Spencer, Andrew G.; Reppen, Tom; Menzie, Ann; Roesch, Paula L.; He, Yudong; Caguyong, Michelle J.; Bloomer, Sherri; Herweijer, Hans; Wolff, Jon A.; Hagstrom, James E.; Lewis, David L.; Linsley, Peter S.; Ulrich, Roger G.

    2006-01-01

    RNA interference (RNAi) has great potential as a tool for studying gene function in mammals. However, the specificity and magnitude of the in vivo response to RNAi remains to be fully characterized. A molecular and phenotypic comparison of a genetic knockout mouse and the corresponding knockdown version would help clarify the utility of the RNAi approach. Here, we used hydrodynamic delivery of small interfering RNA (siRNA) to knockdown peroxisome proliferator activated receptor alpha (Ppara), a gene that is central to the regulation of fatty acid metabolism. We found that Ppara knockdown in the liver results in a transcript profile and metabolic phenotype that is comparable to those of Ppara−/− mice. Combining the profiles from mice treated with the PPARα agonist fenofibrate, we confirmed the specificity of the RNAi response and identified candidate genes proximal to PPARα regulation. Ppara knockdown animals developed hypoglycemia and hypertriglyceridemia, phenotypes observed in Ppara−/− mice. In contrast to Ppara−/− mice, fasting was not required to uncover these phenotypes. Together, these data validate the utility of the RNAi approach and suggest that siRNA can be used as a complement to classical knockout technology in gene function studies. PMID:16945951

  4. Kin5 knockdown in Tetrahymena thermophila using RNAi blocks cargo transport of Gef1.

    PubMed

    Awan, Aashir; Bell, Aaron J; Satir, Peter

    2009-01-01

    A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality.

  5. Kin5 Knockdown in Tetrahymena thermophila Using RNAi Blocks Cargo Transport of Gef1

    PubMed Central

    Awan, Aashir; Bell, Aaron J.; Satir, Peter

    2009-01-01

    A critical process that builds and maintains the eukaryotic cilium is intraflagellar transport (IFT). This process utilizes members of the kinesin-2 superfamily to transport cargo into the cilium (anterograde transport) and a dynein motor for the retrograde traffic. Using a novel RNAi knockdown method, we have analyzed the function of the homodimeric IFT kinesin-2, Kin5, in Tetrahymena ciliary transport. In RNAi transformants, Kin5 was severely downregulated and disappeared from the cilia, but cilia did not resorb, although tip structure was affected. After deciliation of the knockdown cell, cilia regrew and cells swam, which suggested that Kin5 is not responsible for the trafficking of axonemal precursors to build the cilium, but could be transporting molecules that act in ciliary signal transduction, such as guanine nucleotide exchange proteins (GEFs). Gef1 is a Tetrahymena ciliary protein, and current coimmunoprecipitation and immunofluorescence studies showed that it is absent in regrowing cilia of the knockdown cells lacking ciliary Kin5. We suggest that one important cargo of Kin5 is Gef1 and knockdown of Kin5 results in cell lethality. PMID:19290045

  6. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells

    PubMed Central

    Liu, Tuoen; Krysiak, Kilannin; Shirai, Cara Lunn; Kim, Sanghyun; Shao, Jin; Ndonwi, Matthew; Walter, Matthew J.

    2017-01-01

    Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS. PMID:28178280

  7. Knockdown of HSPA9 induces TP53-dependent apoptosis in human hematopoietic progenitor cells.

    PubMed

    Liu, Tuoen; Krysiak, Kilannin; Shirai, Cara Lunn; Kim, Sanghyun; Shao, Jin; Ndonwi, Matthew; Walter, Matthew J

    2017-01-01

    Myelodysplastic syndromes (MDS) are the most common adult myeloid blood cancers in the US. Patients have increased apoptosis in their bone marrow cells leading to low peripheral blood counts. The full complement of gene mutations that contribute to increased apoptosis in MDS remains unknown. Up to 25% of MDS patients harbor and acquired interstitial deletion on the long arm of chromosome 5 [del(5q)], creating haploinsufficiency for a large set of genes including HSPA9. Knockdown of HSPA9 in primary human CD34+ hematopoietic progenitor cells significantly inhibits growth and increases apoptosis. We show here that HSPA9 knockdown is associated with increased TP53 expression and activity, resulting in increased expression of target genes BAX and p21. HSPA9 protein interacts with TP53 in CD34+ cells and knockdown of HSPA9 increases nuclear TP53 levels, providing a possible mechanism for regulation of TP53 by HSPA9 haploinsufficiency in hematopoietic cells. Concurrent knockdown of TP53 and HSPA9 rescued the increased apoptosis observed in CD34+ cells following knockdown of HSPA9. Reduction of HSPA9 below 50% results in severe inhibition of cell growth, suggesting that del(5q) cells may be preferentially sensitive to further reductions of HSPA9 below 50%, thus providing a genetic vulnerability to del(5q) cells. Treatment of bone marrow cells with MKT-077, an HSPA9 inhibitor, induced apoptosis in a higher percentage of cells from MDS patients with del(5q) compared to non-del(5q) MDS patients and normal donor cells. Collectively, these findings indicate that reduced levels of HSPA9 may contribute to TP53 activation and increased apoptosis observed in del(5q)-associated MDS.

  8. Knockdown of lecithin retinol acyltransferase increases all-trans retinoic acid levels and restores retinoid sensitivity in malignant melanoma cells.

    PubMed

    Amann, Philipp M; Czaja, Katharina; Bazhin, Alexandr V; Rühl, Ralph; Skazik, Claudia; Heise, Ruth; Marquardt, Yvonne; Eichmüller, Stefan B; Merk, Hans F; Baron, Jens M

    2014-11-01

    Retinoids such as all-trans retinoic acid (ATRA) influence cell growth, differentiation and apoptosis and may play decisive roles in tumor development and progression. An essential retinoid-metabolizing enzyme known as lecithin retinol acyltransferase (LRAT) is expressed in melanoma cells but not in melanocytes catalysing the esterification of all-trans retinol (ATRol). In this study, we show that a stable LRAT knockdown (KD) in the human melanoma cell line SkMel23 leads to significantly increased levels of the substrate ATRol and biologically active ATRA. LRAT KD restored cellular sensitivity to retinoids analysed in cell culture assays and melanoma 3D skin models. Furthermore, ATRA-induced gene regulatory mechanisms drive depletion of added ATRol in LRAT KD cells. PCR analysis revealed a significant upregulation of retinoid-regulated genes such as CYP26A1 and STRA6 in LRAT KD cells, suggesting their possible involvement in mediating retinoid resistance in melanoma cells. In conclusion, LRAT seems to be important for melanoma progression. We propose that reduction in ATRol levels in melanoma cells by LRAT leads to a disturbance in cellular retinoid level. Balanced LRAT expression and activity may provide protection against melanoma development and progression. Pharmacological inhibition of LRAT activity could be a promising strategy for overcoming retinoid insensitivity in human melanoma cells.

  9. Knockdown of SALL4 Protein Enhances All-trans Retinoic Acid-induced Cellular Differentiation in Acute Myeloid Leukemia Cells*

    PubMed Central

    Liu, Li; Liu, Liang; Leung, Lai-Han; Cooney, Austin J.; Chen, Changyi; Rosengart, Todd K.; Ma, Yupo; Yang, Jianchang

    2015-01-01

    All-trans retinoic acid (ATRA) is a differentiation agent that revolutionized the treatment of acute promyelocytic leukemia. However, it has not been useful for other types of acute myeloid leukemia (AML). Here we explored the effect of SALL4, a stem cell factor, on ATRA-induced AML differentiation in both ATRA-sensitive and ATRA-resistant AML cells. Aberrant SALL4 expression has been found in nearly all human AML cases, whereas, in normal bone marrow and peripheral blood cells, its expression is only restricted to hematopoietic stem/progenitor cells. We reason that, in AMLs, SALL4 activation may prevent cell differentiation and/or protect self-renewal that is seen in normal hematopoietic stem/progenitor cells. Indeed, our studies show that ATRA-mediated myeloid differentiation can be largely blocked by exogenous expression of SALL4, whereas ATRA plus SALL4 knockdown causes significantly increased AML differentiation and cell death. Mechanistic studies indicate that SALL4 directly associates with retinoic acid receptor α and modulates ATRA target gene expression. SALL4 is shown to recruit lysine-specific histone demethylase 1 (LSD1) to target genes and alter the histone methylation status. Furthermore, coinhibition of LSD1 and SALL4 plus ATRA treatment exhibited the strongest anti-AML effect. These findings suggest that SALL4 plays an unfavorable role in ATRA-based regimes, highlighting an important aspect of leukemia therapy. PMID:25737450

  10. Knockdown of asparagine synthetase by RNAi suppresses cell growth in human melanoma cells and epidermoid carcinoma cells.

    PubMed

    Li, Hui; Zhou, Fusheng; Du, Wenhui; Dou, Jinfa; Xu, Yu; Gao, Wanwan; Chen, Gang; Zuo, Xianbo; Sun, Liangdan; Zhang, Xuejun; Yang, Sen

    2016-05-01

    Melanoma, the most aggressive form of skin cancer, causes more than 40,000 deaths each year worldwide. And epidermoid carcinoma is another major form of skin cancer, which could be studied together with melanoma in several aspects. Asparagine synthetase (ASNS) gene encodes an enzyme that catalyzes the glutamine- and ATP-dependent conversion of aspartic acid to asparagine, and its expression is associated with the chemotherapy resistance and prognosis in several human cancers. The present study aims to explore the potential role of ASNS in melanoma cells A375 and human epidermoid carcinoma cell line A431. We applied a lentivirus-mediated RNA interference (RNAi) system to study its function in cell growth of both cells. The results revealed that inhibition of ASNS expression by RNAi significantly suppressed the growth of melanoma cells and epidermoid carcinoma cells, and induced a G0/G1 cell cycle arrest in melanoma cells. Knockdown of ASNS in A375 cells remarkably downregulated the expression levels of CDK4, CDK6, and Cyclin D1, and upregulated the expression of p21. Therefore, our study provides evidence that ASNS may represent a potential therapeutic target for the treatment of melanoma.

  11. TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport.

    PubMed

    Zhang, Yushuo; Chen, Fei; Tai, Guomei; Wang, Jiaojiao; Shang, Jun; Zhang, Bing; Wang, Ping; Huang, Baoxing; Du, Jie; Yu, Jiahua; Zhang, Haowen; Liu, Fenju

    2017-03-24

    The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours.

  12. TIGAR knockdown radiosensitizes TrxR1-overexpressing glioma in vitro and in vivo via inhibiting Trx1 nuclear transport

    PubMed Central

    Zhang, Yushuo; Chen, Fei; Tai, Guomei; Wang, Jiaojiao; Shang, Jun; Zhang, Bing; Wang, Ping; Huang, Baoxing; Du, Jie; Yu, Jiahua; Zhang, Haowen; Liu, Fenju

    2017-01-01

    The up-regulation of thioredoxin reductase-1 (TrxR1) is detected in more than half of gliomas, which is significantly associated with increased malignancy grade and recurrence rate. The biological functions of NADPH-dependent TrxR1 are mainly associated with reduced thioredoxin-1 (Trx1) which plays critical roles in cellular redox signaling and tumour radio-resistance. Our previous work has proved that TP53 induced glycolysis and apoptosis regulator (TIGAR) knockdown could notably radiosensitize glioma cells. However, whether TrxR1-overexpressing glioma cells could be re-radiosensitized by TIGAR silence is still far from clear. In the present study, TrxR1 was stably over-expressed in U-87MG and T98G glioma cells. Both in vitro and in vivo data demonstrated that the radiosensitivity of glioma cells was considerably diminished by TrxR1 overexpression. TIGAR abrogation was able to radiosensitize TrxR1-overexpressing gliomas by inhibiting IR-induced Trx1 nuclear transport. Post-radiotherapy, TIGAR low-expression predicted significant longer survival time for animals suffering from TrxR1-overexpessing xenografts, which suggested that TIGAR abrogation might be a promising strategy for radiosensitizing TrxR1-overexpressing glial tumours. PMID:28338004

  13. Effects of Shell-Buckling Knockdown Factors in Large Cylindrical Shells

    NASA Technical Reports Server (NTRS)

    Hrinda, Glenn A.

    2012-01-01

    Shell-buckling knockdown factors (SBKF) have been used in large cylindrical shell structures to account for uncertainty in buckling loads. As the diameter of the cylinder increases, achieving the manufacturing tolerances becomes increasingly more difficult. Knockdown factors account for manufacturing imperfections in the shell geometry by decreasing the allowable buckling load of the cylinder. In this paper, large-diameter (33 ft) cylinders are investigated by using various SBKF's. An investigation that is based on finite-element analysis (FEA) is used to develop design sensitivity relationships. Different manufacturing imperfections are modeled into a perfect cylinder to investigate the effects of these imperfections on buckling. The analysis results may be applicable to large- diameter rockets, cylindrical tower structures, bulk storage tanks, and silos.

  14. Lentivirus-Mediated knockdown of tectonic family member 1 inhibits medulloblastoma cell proliferation

    PubMed Central

    Jing, Junjie; Wang, Chengfeng; Liang, Qinchuan; Zhao, Yang; Zhao, Qingshuang; Wang, Shousen; Ma, Jie

    2015-01-01

    Tectonic family member 1 (TCTN1) encodes a member of the tectonic family which are evolutionarily conserved secreted and transmembrane proteins, involving in a diverse variety of developmental processes. It has been demonstrated that tectonics expressed in regions that participate in Hedgehog (Hh) signaling during mouse embryonic development and was imperative for Hh-mediated patterning of the ventral neural tube. However, the expression and regulation of tectonics in human tumor is still not clear. In this study, shRNA-expressing lentivirus was constructed to knockdown TCTN1 in medulloblastoma cell line Daoy. The results showed that knockdown of TCTN1 inhibited cell proliferation and colony formation in Daoy cell line, also caused cell cycle arrest at the G2/M boundary. Taken all together, our data suggest that TCTN1 might play an important role in the progression of medulloblastoma. PMID:26550235

  15. Lentivirus-Mediated knockdown of tectonic family member 1 inhibits medulloblastoma cell proliferation.

    PubMed

    Jing, Junjie; Wang, Chengfeng; Liang, Qinchuan; Zhao, Yang; Zhao, Qingshuang; Wang, Shousen; Ma, Jie

    2015-01-01

    Tectonic family member 1 (TCTN1) encodes a member of the tectonic family which are evolutionarily conserved secreted and transmembrane proteins, involving in a diverse variety of developmental processes. It has been demonstrated that tectonics expressed in regions that participate in Hedgehog (Hh) signaling during mouse embryonic development and was imperative for Hh-mediated patterning of the ventral neural tube. However, the expression and regulation of tectonics in human tumor is still not clear. In this study, shRNA-expressing lentivirus was constructed to knockdown TCTN1 in medulloblastoma cell line Daoy. The results showed that knockdown of TCTN1 inhibited cell proliferation and colony formation in Daoy cell line, also caused cell cycle arrest at the G2/M boundary. Taken all together, our data suggest that TCTN1 might play an important role in the progression of medulloblastoma.

  16. C/EBPβ knockdown protects cardiomyocytes from hypertrophy via inhibition of p65-NFκB.

    PubMed

    Zou, Jian; Li, Hong; Chen, Xi; Zeng, Siyu; Ye, Jiantao; Zhou, Changhua; Liu, Min; Zhang, Luankun; Yu, Na; Gan, Xiaohong; Zhou, Houfeng; Xian, Zhiwei; Chen, Shaorui; Liu, Peiqing

    2014-06-05

    C/EBPβ, a member of the bHLH gene family of DNA-binding transcription factors, has been indicated as a central signal in physiologic hypertrophy. However, the role of C/EBPβ in pathological cardiac hypertrophy remains to be elucidated. In this study, we revealed that C/EBPβ is involved in cardiac hypertrophy, the expression of C/EBPβ were significantly increased in response to hypertrophic stimulation in vitro and in vivo. C/EBPβ knockdown inhibited PE-induced cardiac hypertrophy, and diminished the nuclear translocation and DNA binding activity of p65-NFκB. These results suggested that C/EBPβ knockdown protected cardiomyocytes from hypertrophy, which may be attributed to inhibition of NFκB-dependent transcriptional activity. These findings shed new light on the understanding of C/EBPβ-related cardiomyopathy, and suggest the potential application of C/EBPβ inhibitors in cardiac hypertrophy.

  17. Transposon-mediated targeted and specific knockdown of maternally expressed transcripts in the ascidian Ciona intestinalis.

    PubMed

    Iitsuka, Takako; Mita, Kaoru; Hozumi, Akiko; Hamada, Mayuko; Satoh, Nori; Sasakura, Yasunori

    2014-05-23

    Maternal mRNAs play crucial roles during early embryogenesis of ascidians, but their functions are largely unknown. In this study, we developed a new method to specifically knockdown maternal mRNAs in Ciona intestinalis using transposon-mediated transgenesis. We found that GFP expression is epigenetically silenced in Ciona intestinalis oocytes and eggs, and this epigenetic silencing of GFP was used to develop the knockdown method. When the 5' upstream promoter and 5' untranslated region (UTR) of a maternal gene are used to drive GFP in eggs, the maternal gene is specifically knocked down together with GFP. The 5' UTR of the maternal gene is the major element that determines the target gene silencing. Zygotic transcription of the target gene is unaffected, suggesting that the observed phenotypes specifically reflect the maternal function of the gene. This new method can provide breakthroughs in studying the functions of maternal mRNAs.

  18. Knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells.

    PubMed

    D'Anjou, François; Couture, Frédéric; Desjardins, Roxane; Day, Robert

    2014-01-01

    Gene silencing strategies targeting mRNA are suitable methods to validate the functions of specific genes. In this chapter, we sought to compare two knockdown strategies for the study of proprotein convertases and proliferation in prostate cancer cells. We used both SOFA-HDV ribozyme and lentiviral-mediated shRNA delivery system to reduce PACE4 mRNA levels and validate its implication in the proliferation of DU145 prostate cancer cells. The cellular effects of PACE4 knockdown were assessed (1) in vitro using two tetrazolium salts (MTT and XTT assays) and (2) in vivo using a tumor xenograft approach in immunodeficient mice (Nu/Nu). Our results confirm the unique role of the proprotein convertase PACE4 in prostate cancer cell proliferation while demonstrating advantages and disadvantages of each approach. Achieving target validation in an effective manner is critical, as further development using a drug development approach is highly laborious and requires enormous resources.

  19. Knockdown of ERM family member moesin in host cells increases HIV type 1 replication.

    PubMed

    Capalbo, Gianni; Mueller-Kuller, Thea; Markovic, Sandra; Klein, Stefan A; Dietrich, Ursula; Hoelzer, Dieter; Ottmann, Oliver G; Scheuring, Urban J

    2011-12-01

    Moesin is a member of the ERM (ezrin, radixin, moesin) family of cytoskeleton/membrane structure organizing and signal transduction proteins. Previously, we found an increased expression of moesin during HIV-1 infection. Moesin was also reported to be incorporated into HIV-1 virions. To analyze whether moesin is a host factor affecting the replication cycle of human immunodeficiency virus type 1 (HIV-1), we used small interfering RNAs (siRNAs) to evaluate the effect of moesin knockdown on HIV-1 replication in P4-CCR5 cells. Moesin's knockdown did not affect the cell viability or cell phenotype. Interestingly, we observed a marked increase in viral replication, as demonstrated by enhanced HIV-1 RNA, p24 antigen, and ß-galactosidase reporter expression. Moesin-dependent enhancement of HIV-1 replication was confirmed in lymphocytic host cells (Jurkat). These results suggest an overall rather restrictive role of moesin for HIV-1 replication in host cells in vitro.

  20. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    SciTech Connect

    Cui, Ju; Jin, Guoxiang; Yu, Bin; Wang, Zai; Lin, Raozhou; Huang, Jian-Dong

    2015-07-17

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.

  1. CHIP Knockdown Reduced Heat Shock Response and Protein Quality Control Capacity in Lens Epithelial Cells.

    PubMed

    Zhang, W; Liu, Z; Bao, X; Qin, Y; Taylor, A; Shang, F; Wu, M

    2015-01-01

    Protein quality control (PQC) systems, including molecular chaperones and ubiquitin-proteasome pathway (UPP), plays an important role in maintaining intracellular protein homeostasis. Carboxyl terminus of Hsc70- interacting protein (CHIP) links the chaperone and UPPs, thus contributing to the repair or removal of damaged proteins. Over-expression of CHIP had previously been used to protect cells from environmental stress. In order to gain a more physiologic mechanism of the advantage conferred by CHIP, we induced a CHIP knockdown and monitored the ability of cells to cope with environmental stress. To knockdown CHIP, the human lens epithelial cell line HLE B3 was transfected with lentiviral particles that encode a CHIP short hairpin RNA (shRNA) or negative control lentiviral particles. Stable CHIP-knock down cells (KD) and negative control cells (NC) were selected with puromycin. After exposure to heat shock stress, there was no change observed in the expression of Hsp90. In contrast, Hsp70 levels increased significantly in NC cells but less so in KD cells. Hsp27 levels also increased after heat shock, but only in NC cells. Protein ubiquitination was reduced when CHIP was knocked down. CHIP knockdown reduced the ability to clear aggregation proteins. When same levels of aggregation-prone RFP-mutant crystallin fusion protein, RFP/V76D-γD, was expressed, there was ~9- fold more aggregates in KD cells as compared to that observed in NC cells. Furthermore, KD cells were more sensitive to toxicity of amino acid analog canavanine as compared to NC cells. Together, these data indicate that CHIP is required for PQC and that CHIP knockdown diminished cellular PQC capacity in lens cells.

  2. RNAi-mediated double gene knockdown and gustatory perception measurement in honey bees (Apis mellifera).

    PubMed

    Wang, Ying; Baker, Nicholas; Amdam, Gro V

    2013-07-25

    This video demonstrates novel techniques of RNA interference (RNAi) which downregulate two genes simultaneously in honey bees using double-stranded RNA (dsRNA) injections. It also presents a protocol of proboscis extension response (PER) assay for measuring gustatory perception. RNAi-mediated gene knockdown is an effective technique downregulating target gene expression. This technique is usually used for single gene manipulation, but it has limitations to detect interactions and joint effects between genes. In the first part of this video, we present two strategies to simultaneously knock down two genes (called double gene knockdown). We show both strategies are able to effectively suppress two genes, vitellogenin (vg) and ultraspiracle (usp), which are in a regulatory feedback loop. This double gene knockdown approach can be used to dissect interrelationships between genes and can be readily applied in different insect species. The second part of this video is a demonstration of proboscis extension response (PER) assay in honey bees after the treatment of double gene knockdown. The PER assay is a standard test for measuring gustatory perception in honey bees, which is a key predictor for how fast a honey bee's behavioral maturation is. Greater gustatory perception of nest bees indicates increased behavioral development which is often associated with an earlier age at onset of foraging and foraging specialization in pollen. In addition, PER assay can be applied to identify metabolic states of satiation or hunger in honey bees. Finally, PER assay combined with pairing different odor stimuli for conditioning the bees is also widely used for learning and memory studies in honey bees.

  3. Effect of vegf gene knockdown on growth of the murine sarcoma cell line MS-K.

    PubMed

    Zhong, Xiu Y; Yoshioka, Asami; Mashio, Yuka; Ikeda, Toru; Jiang, Huijie; Touma, Maki; Wu, Qiong; Wang, ChangLiu; Sugimoto, Kenkichi

    2011-06-01

    The murine sarcoma cell line MS-K was previously established as a Ki-ras-positive cell line. Inoculation of this cell line under the flank of C3H/HeN mice results in the growth of large tumors with well-developed blood vessels within day 30 of transplantation without any metastasis because MS-K cells produce vascular endothelial growth factor (VEGF). To elucidate the role of VEGF in tumor formation in vivo, stable vegf-knockdown-MS-K clones were obtained using plasmid-based knockdown vectors. Interestingly, tumorigenesis was completely suppressed in a vegf-A-knockdown-MS-K clone [designated MS-K (A-KD)]. Proliferation and colony formation capacity of the MS-K (A-KD) cells in a semi-solid medium under low serum conditions was significantly lower than that of control MS-K (SCR) cells; however, the expression of vegf-receptor 1 (vegf-r-1) was not changed. Addition of the recombinant VEGF-A(165) partially restored the colony formation capacity of MS-K (A-KD) cells and caused the phosphorylation of VEGF-r-1 (Flt-1) in MS-K (Normal) cells. Furthermore, tumorigenicity of the vegf-r-1-knockdown-MS-K clone [designated MS-K (R1-KD)] had obviously delayed or strongly suppressed compared with the MS-K (Normal). These results indicate that Vascular endothelial growth factor-A, produced from MS-K, acts as a growth factor for MS-K cells itself and supports tumor formation in vivo by inducing the blood vessel formation. © 2011 The Authors. Journal compilation © 2011 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.

  4. Brain-Targeted (Pro)Renin Receptor Knockdown attenuates Angiotensin II-Dependent Hypertension

    PubMed Central

    Li, Wencheng; Peng, Hua; Cao, Theresa; Sato, Ryosuke; McDaniels, Sarah. J.; Kobori, Hiroyuki; Navar, L. Gabriel; Feng, Yumei

    2012-01-01

    The (pro)renin receptor is a newly discovered member of the brain renin-angiotensin system. To investigate the role of brain (pro)renin receptor in hypertension, adeno-associated virus-mediated (pro)renin receptor shRNA was used to knockdown (pro)renin receptor expression in the brain of non-transgenic normotensive and human renin-angiotensinogen double transgenic hypertensive mice. Blood pressure was monitored using implanted telemetric probes in conscious animals. Real-time PCR and immunostaining were performed to determine (pro)renin receptor, angiotensin II type 1 receptor and vasopressin mRNA levels. Plasma vasopressin levels were determined by Enzyme-Linked Immuno Sorbent Assay. Double transgenic mice exhibited higher blood pressure, elevated cardiac and vascular sympathetic tone, and impaired spontaneous baroreflex sensitivity. Intracerebroventricular delivery of (pro)renin receptor shRNA significantly reduced blood pressure, cardiac and vasomotor sympathetic tone, and improved baroreflex sensitivity compared to the control virus treatment in double transgenic mice. (Pro)renin receptor knockdown significantly reduced angiotensin II type 1 receptor and vasopressin levels in double transgenic mice. These data indicate that (pro)renin receptor knockdown in the brain attenuates angiotensin II-dependent hypertension and is associated with a decrease insympathetic tone and an improvement of the baroreflex sensitivity. In addition, brain-targeted (pro)renin receptor knockdown is associated with down-regulation of angiotensin II type 1 receptor and vasopressin levels. We conclude that central (pro)renin receptor contributes to the pathogenesis of hypertension in human renin-angiotensinogen transgenic mice. PMID:22526255

  5. Interaction of ionophore and vitamin E in knockdown syndrome of turkeys.

    PubMed

    Crespo, Rocio; Shivaprasad, H L; Sommer, Franz; Puschner, Birgit

    2008-07-01

    Monensin and vitamin E concentrations, as well as histopathology of skeletal muscles and myocardium, were evaluated in broad-breasted white turkeys kept in commercial facilities. Turkeys with knockdown syndrome had myopathy of skeletal muscles, but no lesions in the myocardium. Generally, concentration of monensin in serum was highest in turkeys diagnosed with knockdown syndrome given more than 90 mg/kg of monensin in the diet, followed by turkeys diagnosed with knockdown syndrome given <90 mg/kg of monensin in the diet, healthy turkeys fed a diet that contained <90 mg/kg of monensin, and finally healthy turkeys fed a diet free of monensin (not detectable). However, the concentration of monensin was highly variable within each group, and the median was lower than the average. Vitamin E concentrations in the livers varied from low-normal to below normal and were statistically higher in healthy turkeys fed a diet free of monensin than in the livers of birds from the 3 groups exposed to monensin. This suggests that the concentration of monensin in serum positively correlates to the severity of clinical signs and pathology and to the amount of monensin in the feed. Although the methodology developed to detect serum monensin concentrations is beneficial and accurate for case investigations, it is recommended that several samples from each flock be evaluated because of variation within a flock. The current study also suggests that monensin in the feed could induce lower concentrations of vitamin E in the liver of turkeys and can predispose the turkeys to knockdown syndrome.

  6. Selective knockdown of ceramide synthases reveals complex interregulation of sphingolipid metabolism.

    PubMed

    Mullen, Thomas D; Spassieva, Stefka; Jenkins, Russell W; Kitatani, Kazuyuki; Bielawski, Jacek; Hannun, Yusuf A; Obeid, Lina M

    2011-01-01

    Mammalian ceramide synthases 1 to 6 (CerS1-6) generate Cer in an acyl-CoA-dependent manner, and expression of individual CerS has been shown to enhance the synthesis of ceramides with particular acyl chain lengths. However, the contribution of each CerS to steady-state levels of specific Cer species has not been evaluated. We investigated the knockdown of individual CerS in the MCF-7 human breast adenocarcinoma cell line by using small-interfering RNA (siRNA). We found that siRNA-induced downregulation of each CerS resulted in counter-regulation of nontargeted CerS. Additionally, each CerS knockdown produced unique effects on the levels of multiple sphingolipid species. For example, downregulation of CerS2 decreased very long-chain Cer but increased levels of CerS4, CerS5, and CerS6 expression and upregulated long-chain and medium-long-chain sphingolipids. Conversely, CerS6 knockdown decreased C16:0-Cer but increased CerS5 expression and caused non-C16:0 sphingolipids to be upregulated. Knockdown of individual CerS failed to decrease total sphingolipids or upregulate sphingoid bases. Treatment with siRNAs targeting combined CerS, CerS2, CerS5, and CerS6, did not change overall Cer or sphingomyelin mass but caused upregulation of dihydroceramide and hexosyl-ceramide and promoted endoplasmic reticulum stress. These data suggest that sphingolipid metabolism is robustly regulated by both redundancy in CerS-mediated Cer synthesis and counter-regulation of CerS expression.

  7. Knockdown of pre-mRNA cleavage factor Im 25 kDa promotes neurite outgrowth

    SciTech Connect

    Fukumitsu, Hidefumi; Soumiya, Hitomi; Furukawa, Shoei

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer CFIm25 knockdown promoted NGF-induced neurite out growth from PC12 cells. Black-Right-Pointing-Pointer Depletion of CFIm25 did not influence the morphology of proliferating PC12 cells. Black-Right-Pointing-Pointer CFIm regulated NGF-induced neurite outgrowth via coordinating RhoA activity. Black-Right-Pointing-Pointer CFIm25 knockdown increase the number of primary dendrites of hippocampal neurons. -- Abstract: Mammalian precursor mRNA (pre-mRNA) cleavage factor I (CFIm) plays important roles in the selection of poly(A) sites in a 3 Prime -untranslated region (3 Prime -UTR), producing mRNAs with variable 3 Prime ends. Because 3 Prime -UTRs often contain cis elements that impact stability or localization of mRNA or translation, alternative polyadenylation diversifies utilization of primary transcripts in mammalian cells. However, the physiological role of CFIm remains unclear. CFIm acts as a heterodimer comprising a 25 kDa subunit (CFIm25) and one of the three large subunits-CFIm59, CFIm68, or CFIm72. CFIm25 binds directly to RNA and introduces and anchors the larger subunit. To examine the physiological roles of CFIm, we knocked down the CFIm25 gene in neuronal cells using RNA interference. Knockdown of CFIm25 increased the number of primary dendrites of developing hippocampal neurons and promoted nerve growth factor (NGF)-induced neurite extension from rat pheochromocytoma PC12 cells without affecting the morphology of proliferating PC12 cells. On the other hand, CFIm25 knockdown did not influence constitutively active or dominantly negative RhoA suppression or promotion of NGF-induced neurite extension from PC12 cells, respectively. Taken together, our results indicate that endogenous CFIm may promote neuritogenesis in developing neurons by coordinating events upstream of NGF-induced RhoA inactivation.

  8. Sustained miRNA-mediated knockdown of mutant AAT with simultaneous augmentation of wild-type AAT has minimal effect on global liver miRNA profiles.

    PubMed

    Mueller, Christian; Tang, Qiushi; Gruntman, Alisha; Blomenkamp, Keith; Teckman, Jeffery; Song, Lina; Zamore, Phillip D; Flotte, Terence R

    2012-03-01

    α-1 antitrypsin (AAT) deficiency can exhibit two pathologic states: a lung disease that is primarily due to the loss of AAT's antiprotease function, and a liver disease resulting from a toxic gain-of-function of the PiZ-AAT (Z-AAT) mutant protein. We have developed several recombinant adeno-associated virus (rAAV) vectors that incorporate microRNA (miRNA) sequences targeting the AAT gene while also driving the expression of miRNA-resistant wild-type AAT-PiM (M-AAT) gene, thus achieving concomitant Z-AAT knockdown in the liver and increased expression of M-AAT. Transgenic mice expressing the human PiZ allele treated with dual-function rAAV9 vectors showed that serum PiZ was stably and persistently reduced by an average of 80%. Treated animals showed knockdown of Z-AAT in liver and serum with concomitant increased serum M-AAT as determined by allele-specific enzyme-linked immunosorbent assays (ELISAs). In addition, decreased globular accumulation of misfolded Z-AAT in hepatocytes and a reduction in inflammatory infiltrates in the liver was observed. Results from microarray studies demonstrate that endogenous miRNAs were minimally affected by this treatment. These data suggests that miRNA mediated knockdown does not saturate the miRNA pathway as has been seen with viral vector expression of short hairpin RNAs (shRNAs). This safe dual-therapy approach can be applied to other disorders such as amyotrophic lateral sclerosis, Huntington disease, cerebral ataxia, and optic atrophies.

  9. Knockdown of REGγ inhibits proliferation by inducing apoptosis and cell cycle arrest in prostate cancer

    PubMed Central

    Chen, Shaojun; Wang, Longsheng; Xu, Chen; Chen, Hui; Peng, Bo; Xu, Yunfei; Yao, Xudong; Li, Lei; Zheng, Junhua

    2017-01-01

    Prostate cancer (PCa) is the most common malignant tumor and the second leading cause of cancer related death among men in western countries. REGγ, a proteasome activator, is reported to play important roles in various human cancers. However, the expression patterns and potential roles of REGγ in prostate cancer have never been reported. In this study, we found for the first time that REGγ is overexpressed in prostate cancer tissues and cell lines at both protein and mRNA levels. In addition, we demonstrated that knockdown of REGγ significantly inhibited cell proliferation and induced apoptosis and cell cycle arrest in PCa cell lines PC-3 and DU145. Moreover, we observed that the expressions of P21 were increased while the levels of cycinD1 and bcl-2 were decreased after knockdown of REGγ in PCa cells. And the stabilization of P21 was enhanced after REGγ knockdown in PC-3 cells. In summary, our findings suggest that REGγ may play important roles in prostate cancer and may serve as a novel therapeutic target in the treatment of PCa patients. PMID:28861169

  10. Effects of ezrin knockdown on the structure of gastric glandular epithelia.

    PubMed

    Yoshida, Saori; Yamamoto, Hiroto; Tetsui, Takahito; Kobayakawa, Yuka; Hatano, Ryo; Mukaisho, Ken-ichi; Hattori, Takanori; Sugihara, Hiroyuki; Asano, Shinji

    2016-01-01

    Ezrin, an adaptor protein that cross-links plasma membrane-associated proteins with the actin cytoskeleton, is concentrated on apical surfaces of epithelial cells, especially in microvilli of the small intestine and stomach. In the stomach, ezrin is predominantly expressed on the apical canalicular membrane of parietal cells. Transgenic ezrin knockdown mice in which the expression level of ezrin was reduced to <7% compared with the wild-type suffered from achlorhydria because of impairment of membrane fusion between tubulovesicles and apical membranes. We observed, for the first time, hypergastrinemia and foveolar hyperplasia in the gastric fundic region of the knockdown mice. Dilation of fundic glands was observed, the percentage of parietal and chief cells was reduced, and that of mucous-secreting cells was increased. The parietal cells of knockdown mice contained dilated tubulovesicles and abnormal mitochondria, and subsets of these cells contained abnormal vacuoles and multilamellar structures. Therefore, lack of ezrin not only causes achlorhydria and hypergastrinemia but also changes the structure of gastric glands, with severe perturbation of the secretory membranes of parietal cells.

  11. Knockdown of HOXA5 inhibits the tumorigenesis in esophageal squamous cell cancer.

    PubMed

    Zhang, Hui; Zhao, Jiang-Hai; Suo, Zhi-Min

    2017-02-01

    Homeobox A5 (HOXA5) is a member of the homeobox (HOX) family and was upregulated in many types of tumors. However, its expression and role in esophageal squamous cell carcinoma (ESCC) remain unclear. In this study, the aim of this study was to investigate the expression and function of HOXA5 in ESCC. Our results showed that HOXA5 was highly expressed in ESCC cell lines. The in vitro experiments demonstrated that knockdown of HOXA5 significantly inhibited the proliferation, migration and invasion of ESCC cells. Furthermore, the in vivo experiments showed that knockdown of HOXA5 significantly inhibited the tumor growth of ESCC in mice xenograft model. Finally, sh-HOXA5 inhibited the expression of β-catenin, cyclin D1 and c-Myc in ESCC cells. Taken together, these data revealed that knockdown of HOXA5 suppressed the proliferation and metastasis partly by interfering with Wnt/β-catenin signaling pathway in ESCC cells. Therefore, these findings suggest that HOXA5 may be a potential therapeutic target for the treatment of ESCC.

  12. Knockdown of Neuropeptide Y in the Dorsomedial Hypothalamus Promotes Hepatic Insulin Sensitivity in Male Rats.

    PubMed

    Li, Lin; de La Serre, C Barbier; Zhang, Ni; Yang, Liang; Li, Hong; Bi, Sheng

    2016-12-01

    Recent evidence has shown that alterations in dorsomedial hypothalamic (DMH) neuropeptide Y (NPY) signaling influence glucose homeostasis, but the mechanism through which DMH NPY acts to affect glucose homeostasis remains unclear. Here we report that DMH NPY descending signals to the dorsal motor nucleus of the vagus (DMV) modulate hepatic insulin sensitivity to control hepatic glucose production in rats. Using the hyperinsulinemic-euglycemic clamp, we revealed that knockdown of NPY in the DMH by adeno-associated virus-mediated NPY-specific RNAi promoted insulin's action on suppression of hepatic glucose production. This knockdown silenced DMH NPY descending signals to the DMV, leading to an elevation of hepatic vagal innervation. Hepatic vagotomy abolished the inhibitory effect of DMH NPY knockdown on hepatic glucose production, but this glycemic effect was not affected by vagal deafferentation. Together, these results demonstrate a distinct role for DMH NPY in the regulation of glucose homeostasis through the hepatic vagal efferents and insulin action on hepatic glucose production.

  13. PNUTS knockdown potentiates the apoptotic effect of Roscovitine in breast and colon cancer cells

    PubMed Central

    De Leon, Gabriel; Cavino, Margaret; D’Angelo, Mikilyn; Krucher, Nancy A

    2013-01-01

    The phosphorylation state of Retinoblastoma protein (Rb) plays a role in cell proliferation and apoptosis. Within cells, cyclin dependent kinases (cdks) phosphorylate Rb in response to growth stimulatory signals, whereas protein phosphatase 1 (PP1) dephosphorylates Rb when cells stop proliferating or undergo apoptosis in response to anti-proliferative or stress signals. Stimulation of PP1 activity via siRNA mediated knockdown of its interacting protein PNUTS (Phosphatase Nuclear Targeting Subunit) leads to Rb dephosphorylation and apoptosis in cancer cells. Here we utilize two separate methods to modulate the phosphorylation state of Rb in cancer cells. Kinase activity toward Rb is inhibited by the clinically relevant cdk inhibitor, Roscovitine. In addition, siRNA mediated PNUTS knockdown stimulates phosphatase activity toward Rb. Either of these treatments in cancer cells causes a two-fold stimulation of apoptosis. When activation of phosphatase activity is combined with inhibition of cdk activity toward Rb, however, cells exhibit a 4-fold increase in apoptosis. The mechanism by which PNUTS knockdown mediated PP1 activation leads to apoptosis was determined to be dependent on the activity of the transcription factor E2F1. The Rb phosphorylation profiles resulting from each treatment were analyzed and found to be similar but not identical. In addition, the two treatments differentially effect the expression of bcl-2 family proteins. Thus inhibition of cdk activity and activation of PP1 activity toward pRb are functionally distinct processes that together increase the apoptotic effect in cells. PMID:20372802

  14. Promising Noninvasive Cellular Phenotype in Prostate Cancer Cells Knockdown of Matrix Metalloproteinase 9

    PubMed Central

    Gupta, Aditi; Cao, Wei; Sadashivaiah, Kavitha; Chen, Wantao; Schneider, Abraham; Chellaiah, Meenakshi A.

    2013-01-01

    Cell surface interaction of CD44 and MMP9 increases migration and invasion of PC3 cells. We show here that stable knockdown of MMP9 in PC3 cells switches CD44 isoform expression from CD44s to CD44v6 which is more glycosylated. These cells showed highly adhesive morphology with extensive cell spreading which is due to the formation of focal adhesions and well organized actin-stress fibers. MMP9 knockdown blocks invadopodia formation and matrix degradation activity as well. However, CD44 knockdown PC3 cells failed to develop focal adhesions and stress fibers; hence these cells make unstable adhesions. A part of the reason for these changes could be caused by silencing of CD44v6 as well. Immunostaining of prostate tissue microarray sections illustrated significantly lower levels of CD44v6 in adenocarcinoma than normal tissue. Our results suggest that interaction between CD44 and MMP9 is a potential mechanism of invadopodia formation. CD44v6 expression may be essential for the protection of non-invasive cellular phenotype. CD44v6 decrease may be a potential marker for prognosis and therapeutics. PMID:23476138

  15. Fruitless RNAi knockdown in the desert locust, Schistocerca gregaria, influences male fertility.

    PubMed

    Boerjan, Bart; Tobback, Julie; Vandersmissen, Hans Peter; Huybrechts, Roger; Schoofs, Liliane

    2012-02-01

    In Drosophila melanogaster, the male-specific splice isoform of the fruitless gene (Fru(M)) encodes a set of transcription factors that are involved in the regulation of male courtship and copulation. Recent insights from non-drosophilid insects suggest a conserved evolutionary role for the transcription factor Fruitless. In the desert locust, Schistocerca gregaria and the German cockroach, Blatella germanica, both orthopteran insects, a conserved functional role for fruitless has been proposed. Fru specific RNAi knockdown in the third nymphal stage of male Schistocera gregaria delays copulation initiation and results in reduced progeny. In order to identify the origin of the observed phenotypic effects following a fruitless RNAi treatment in the male, we show that the fru knockdown has no detectable effect on spermio- or spermatogenesis and on the transfer of spermatozoa during copulation. Nevertheless, it is clear that the male seminal vesicles contain significantly less spermatozoa after fru RNAi as compared to gfp RNAi controls. We conclude that a lowered male fertility, caused by the fru knockdown in male desert locusts may be the direct cause for the reduction of the progeny numbers in their naïve female copulation partners.

  16. ETV4 and Myeov knockdown impairs colon cancer cell line proliferation and invasion

    SciTech Connect

    Moss, Alan C. . E-mail: amoss@bidmc.harvard.edu; Lawlor, Garrett; Murray, David; Tighe, Donal; Madden, Stephen F.; Mulligan, Anne-Marie; Keane, Conor O.; Brady, Hugh R.; Doran, Peter P.; MacMathuna, Padraic

    2006-06-23

    We have identified novel colorectal cancer-associated genes using NCBI's UNIGENE cDNA libraries. Colon cancer libraries were examined using Digital Differential Display and disease-associated genes were selected. Among these were ETV4 and MYEOV, novel colorectal cancer-associated genes. Samples of matched normal and neoplastic colon were obtained from human subjects and gene expression was quantified using real-time PCR. ETV4 gene expression was significantly increased in colonic neoplasia in comparison to matched normal colonic tissue (p < 0.05). Myeov expression was also increased in colon neoplasia in comparison to matched normal tissue. The effect of siRNA-mediated knockdown of ETV4 and Myeov on cell proliferation and invasion was assessed. ETV4 knockdown resulted in a 90% decrease in cell proliferation (p < 0.05) and a 67% decrease in cell invasion. Myeov knockdown resulted in a 48% decrease in cell proliferation (p < 0.05) and a 36% decrease in cell invasion. These data suggest that ETV4 and Myeov may provide novel targets for therapeutic intervention.

  17. Genetic and chemical knockdown: a complementary strategy for evaluating an anti-infective target

    PubMed Central

    Ramachandran, Vasanthi; Singh, Ragini; Yang, Xiaoyu; Tunduguru, Ragadeepthi; Mohapatra, Subrat; Khandelwal, Swati; Patel, Sanjana; Datta, Santanu

    2013-01-01

    The equity of a drug target is principally evaluated by its genetic vulnerability with tools ranging from antisense- and microRNA-driven knockdowns to induced expression of the target protein. In order to upgrade the process of antibacterial target identification and discern its most effective type of inhibition, an in silico toolbox that evaluates its genetic and chemical vulnerability leading either to stasis or cidal outcome was constructed and validated. By precise simulation and careful experimentation using enolpyruvyl shikimate-3-phosphate synthase and its specific inhibitor glyphosate, it was shown that genetic knockdown is distinct from chemical knockdown. It was also observed that depending on the particular mechanism of inhibition, viz competitive, uncompetitive, and noncompetitive, the antimicrobial potency of an inhibitor could be orders of magnitude different. Susceptibility of Escherichia coli to glyphosate and the lack of it in Mycobacterium tuberculosis could be predicted by the in silico platform. Finally, as predicted and simulated in the in silico platform, the translation of growth inhibition to a cidal effect was able to be demonstrated experimentally by altering the carbon source from sorbitol to glucose. PMID:23413046

  18. Nociception, neurogenic inflammation and thermoregulation in TRPV1 knockdown transgenic mice.

    PubMed

    Tóth, Dániel Márton; Szoke, Eva; Bölcskei, Kata; Kvell, Krisztián; Bender, Balázs; Bosze, Zsuzsanna; Szolcsányi, János; Sándor, Zoltán

    2011-08-01

    Transgenic mice with a small hairpin RNA construct interfering with the expression of transient receptor potential vanilloid 1 (TRPV1) were created by lentiviral transgenesis. TRPV1 expression level in transgenic mice was reduced to 8% while the expression of ankyrin repeat domain 1 (TRPA1) was unchanged. Ear oedema induced by topical application of TRPV1 agonist capsaicin was completely absent in TRPV1 knockdown mice. Thermoregulatory behaviour in relation to environmental thermopreference (30 vs. 35°C) was slightly impaired in male knockdown mice, but the reduction of TRPV1 function was not associated with enhanced hyperthermia. TRPV1 agonist resiniferatoxin induced hypothermia and tail vasodilatation was markedly inhibited in knockdown mice. In conclusion, shRNA-mediated knock down of the TRPV1 receptor in mice induced robust inhibition of the responses to TRPV1 agonists without altering the expression, gating function or neurogenic oedema provoked by TRPA1 activation. Thermoregulatory behaviour in response to heat was inhibited, but enhanced hyperthermia was not observed.

  19. The exocyst protein Sec10 interacts with Polycystin-2 and knockdown causes PKD-phenotypes.

    PubMed

    Fogelgren, Ben; Lin, Shin-Yi; Zuo, Xiaofeng; Jaffe, Kimberly M; Park, Kwon Moo; Reichert, Ryan J; Bell, P Darwin; Burdine, Rebecca D; Lipschutz, Joshua H

    2011-04-01

    Autosomal dominant polycystic kidney disease (ADPKD) is characterized by formation of renal cysts that destroy the kidney. Mutations in PKD1 and PKD2, encoding polycystins-1 and -2, cause ADPKD. Polycystins are thought to function in primary cilia, but it is not well understood how these and other proteins are targeted to cilia. Here, we provide the first genetic and biochemical link between polycystins and the exocyst, a highly-conserved eight-protein membrane trafficking complex. We show that knockdown of exocyst component Sec10 yields cellular phenotypes associated with ADPKD, including loss of flow-generated calcium increases, hyperproliferation, and abnormal activation of MAPK. Sec10 knockdown in zebrafish phenocopies many aspects of polycystin-2 knockdown-including curly tail up, left-right patterning defects, glomerular expansion, and MAPK activation-suggesting that the exocyst is required for pkd2 function in vivo. We observe a synergistic genetic interaction between zebrafish sec10 and pkd2 for many of these cilia-related phenotypes. Importantly, we demonstrate a biochemical interaction between Sec10 and the ciliary proteins polycystin-2, IFT88, and IFT20 and co-localization of the exocyst and polycystin-2 at the primary cilium. Our work supports a model in which the exocyst is required for the ciliary localization of polycystin-2, thus allowing for polycystin-2 function in cellular processes.

  20. Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line.

    PubMed

    Cleveland, Beth M; Leonard, Stephen S; Klandorf, Hillar; Blemings, Kenneth P

    2009-01-01

    Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX) and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B). Urate oxidase mRNA was reduced 66% (p < 0.05) compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI)) or 3-morpholinosydnonimine hydrochloride (SIN-1). Compared to wild type, cells with UOX knockdown exhibited a 37.2 +/- 3.5% reduction (p < 0.05) in the electron spin resonance (ESR) signal after being exposed to Cr(VI) and displayed less DNA fragmentation (p < 0.05) following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05), but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress.

  1. Urate oxidase knockdown decreases oxidative stress in a murine hepatic cell line

    PubMed Central

    Cleveland, Beth M; Leonard, Stephen S; Klandorf, Hillar

    2009-01-01

    Humans, birds, and some primates do not express the uric acid degrading enzyme urate oxidase (UOX) and, as a result, have plasma uric acid concentrations higher than UOX expressing animals. Although high uric acid concentrations are suggested to increase the antioxidant defense system and provide a health advantage to animals without UOX, knockout mice lacking UOX develop pathological complications including gout and kidney failure. As an alternative to the knockout model, RNA interference was used to decrease UOX expression using stable transfection in a mouse hepatic cell line (ATCC, FL83B). Urate oxidase mRNA was reduced 66% (p < 0.05) compared to wild type, as measured by real time RT-PCR. To determine if UOX knockdown resulted in enhanced protection against oxidative stress, cells were challenged with hexavalent chromium (Cr(VI)) or 3-morpholinosydnonimine hydrochloride (SIN-1). Compared to wild type, cells with UOX knockdown exhibited a 37.2 ± 3.5% reduction (p < 0.05) in the electron spin resonance (ESR) signal after being exposed to Cr(VI) and displayed less DNA fragmentation (p < 0.05) following SIN-1 treatment. Cell viability decreased in wild type cells (p < 0.05), but not cells with UOX knockdown, after treatment with SIN-1. These results are consistent with an increased intracellular uric acid concentration and an increased defense against oxidative stress. PMID:20357931

  2. Knockdown of FAM3B triggers cell apoptosis through p53-dependent pathway.

    PubMed

    Mou, Haiwei; Li, Zongmeng; Yao, Pengle; Zhuo, Shu; Luan, Wei; Deng, Bo; Qian, Lihua; Yang, Mengmei; Mei, Hong; Le, Yingying

    2013-03-01

    FAM3B, also named PANDER, is a cytokine-like protein identified in 2002. Previous studies showed that FAM3B regulates glucose and lipid metabolism through interaction with liver and endocrine pancreas. FAM3B is also expressed by other tissues but its basic function is unclear. In this study, we found that FAM3B was expressed in mouse colon, intestine, liver and lung tissues and multiple types of cell lines, including murine pancreatic β-cell (Min6), microglia (N9) and muscle cell (C2C12); human colon cancer cells (HCT8, HCT116, HT29), hepatocyte (HL-7702), hepatocellular carcinoma cell (SMMC-7721) and lung carcinoma cell (A549). Inhibition of FAM3B expression by RNA interference induced apoptotic cell death of HCT8, HCT116, A549, N9, C2C12 and Min6 cells and decreased cell viability of HL-7702 and murine primary hepatocytes. Further studies with HCT8 cells showed that knockdown of FAM3B increased the protein levels of membrane-bound Fas and Bax, reduced the expression of Bcl-2, promoted the cleavage of caspases-8, -3, -9 and PARP, and the nuclear translocation of cleaved PARP. These results suggest that FAM3B silencing activates both extrinsic and intrinsic apoptotic pathways. Mechanistic studies showed that neutralizing antibody against Fas or silencing Fas-associated death domain had no effect on, while caspase inhibitors could significantly reverse FAM3B knockdown induced apoptosis, suggesting Fas and death receptor mediated extrinsic apoptotic pathway is not involved in FAM3B silencing induced apoptosis. Further studies showed that p53 was significantly upregulated after FAM3B knockdown. Silencing p53 could almost completely reverse FAM3B knockdown induced upregulation of Bax, downregulation of Bcl-2, cleavage of caspases-8, -9, -3, and apoptotic cell death, suggesting p53-dependent pathway plays critical roles in FAM3B silencing induced apoptosis. Studies with HCT116 cells confirmed that inhibition of FAM3B expression induced apoptosis through p53-dependent

  3. Quantitative proteomics reveals direct and indirect alterations in the histone code following methyltransferase knockdown.

    PubMed

    Plazas-Mayorca, Mariana D; Bloom, Joshua S; Zeissler, Ulrike; Leroy, Gary; Young, Nicolas L; DiMaggio, Peter A; Krugylak, Leonid; Schneider, Robert; Garcia, Benjamin A

    2010-09-01

    Histones are highly conserved proteins that organize cellular DNA. These proteins, especially their N-terminal domains, are adorned with many post-translational modifications (PTMs) such as lysine methylation, which are associated with active or repressed transcriptional states. The lysine methyltransferase G9a and its interaction partner Glp1 can mono- or dimethylate histone H3 on lysine (H3K9me1 or me2); possible cross-talk between these modifications and other PTMs on the same or other histone molecules is currently uncharacterized. In this study, we comprehensively analyze the effects of G9a/Glp1 knockdown on the most abundant histone modifications through both Bottom Up and Middle Down mass spectrometry-based proteomics. In addition to the expected decrease in H3K9me1/me2 we find that other degrees of methylation on K9 are affected by the reduction of G9a/Glp1 activity, particularly when K9 methylation occurs in combination with K14 acetylation. In line with this, an increase in K14 acetylation upon G9a knockdown was observed across all H3 variants (H3.1, H3.2 and H3.3), hinting at the potential existence of a binary switch between K9 methylation and K14 acetylation. Interestingly, we also detect changes in the abundance of other modifications (such as H3K79me2) in response to lowered levels of G9a/Glp1 suggesting histone PTM cross-talk amongst the H3 variants. In contrast, we find that G9a/Glp1 knockdown produces little effect on the levels of histone H4 PTMs, indicating low to no trans-histone PTM crosstalk. Lastly, we determined gene expression profiles of control and G9a/Glp1 knockdown cells, and we find that the G9a/Glp1 knockdown influences several genes, including DNA binding proteins and key factors in chromatin. Our results provide new insights into the intra- and inter- histone cross-regulation of histone K9 methylation and its potential downstream gene targets.

  4. siRNA-knockdown of ADAMTS-13 modulates endothelial cell angiogenesis.

    PubMed

    Tang, Huiyuan; Lee, Manfai; Kim, Eun Ho; Bishop, Daniel; Rodgers, George M

    2017-09-01

    ADAMTS-13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13, is a zinc-containing metalloprotease that cleaves von Willebrand factor (vWf). Previous publications by our laboratory have shown that ADAMTS-13 may also be involved in angiogenesis. For this study, we report the successful transient knockdown of endogenous ADAMTS-13 in human umbilical vein endothelial cells (HUVEC) via siRNA and the effects of reduced endogenous ADAMTS-13 on HUVEC angiogenesis functions. 15nM of ADAMTS-13 siRNA reduced HUVEC ADAMTS-13 protein levels by 90% after 24h incubation, whereas control siRNA did not affect endogenous ADAMTS-13 levels. Furthermore, this transfection did not affect the HUVEC endogenous protein level of ADAMTS-1, a related family member of ADAMTS-13 indicating the specificity of the siRNA. Transfection of HUVEC with 15nM of ADAMTS-13 siRNA resulted in a 21% decrease in proliferation after 24h incubation. The effects of ADAMTS-13 knockdown on migration of HUVEC across a scratch wound were also evaluated. 24h after transfection with control siRNA, there was increased cell migration across the scratch wound. This dramatic migration did not occur with ADAMTS-13 knockdown cells. Decreased protein levels of endogenous ADAMTS-13 also affected angiogenesis as measured by endothelial cell tube formation using a Matrigel matrix method. The tube lengths, sizes and junction numbers of the ADAMTS-13 knockdown cells were all significantly lower compared to control cells by about 40%. The protein level of vascular endothelial growth factor (VEGF), a well-known regulator of angiogenesis, was significantly decreased by 45% upon knockdown of ADAMTS-13. Moreover, activity of the AKT pathway, one of the VEGF angiogenesis downstream signaling pathways was down-regulated by ADAMTS-13 siRNA. These data indicate that in cultured endothelial cells, one role of endogenous ADAMTS-13 is regulation of angiogenesis, mediated through VEGF and AKT signaling pathway

  5. Nuclear factor E2-related factor 2 knockdown enhances glucose uptake and alters glucose metabolism in AML12 hepatocytes.

    PubMed

    Yuan, Xiaoyang; Huang, Huijing; Huang, Yi; Wang, Jinli; Yan, Jinhua; Ding, Ling; Zhang, Cuntai; Zhang, Le

    2017-05-01

     vivo studies employing either Nrf2-knockout or Nrf2-activated mice have achieved a similar endpoint: protection against an obese and insulin-resistant phenotype that includes impaired lipogenesis and gluconeogenesis in the liver. These apparently paradoxical observations led us to evaluate the impact of Nrf2 in liver cells in the absence of any influence from the systemic environment, including changes in the secretion of adipokines and proinflammatory cytokines by adipose tissues. In the present study, Nrf2 knockdown was sufficient to induce fundamental changes in the glucose metabolism of AML12 hepatocytes in addition to its classical cytoprotective functions. We also discuss similarities and differences between our in vitro study and previous in vivo studies, which may be helpful to dissect and better understand in vivo data that represents the culmination of both local and systemic alterations.

  6. Enhanced radiosensitivity and radiation-induced apoptosis in glioma CD133-positive cells by knockdown of SirT1 expression

    SciTech Connect

    Chang, C.-J.; Hsu, C.-C.; Yung, M.-C.; Chen, K.-Y.; Tzao Ching; Wu, W.-F.; Chou, H.-Y.; Lee, Y.-Y.; Lu, K.-H.; Chiou, S.-H.; Ma, H.-I

    2009-03-06

    CD133-expressing glioma cells play a critical role in tumor recovery after treatment and are resistant to radiotherapy. Herein, we demonstrated that glioblastoma-derived CD133-positive cells (GBM-CD133{sup +}) are capable of self-renewal and express high levels of embryonic stem cell genes and SirT1 compared to GBM-CD133{sup -} cells. To evaluate the role of SirT1 in GBM-CD133{sup +}, we used a lentiviral vector expressing shRNA to knock-down SirT1 expression (sh-SirT1) in GBM-CD133{sup +}. Silencing of SirT1 significantly enhanced the sensitivity of GBM-CD133{sup +} to radiation and increased the level of radiation-mediated apoptosis. Importantly, knock-down of SirT1 increased the effectiveness of radiotherapy in the inhibition of tumor growth in nude mice transplanted with GBM-CD133{sup +}. Kaplan-Meier survival analysis indicated that the mean survival rate of GBM-CD133{sup +} mice treated with radiotherapy was significantly improved by Sh-SirT1 as well. In sum, these results suggest that SirT1 is a potential target for increasing the sensitivity of GBM and glioblastoma-associated cancer stem cells to radiotherapy.

  7. Knockdown of Pyruvate Carboxylase or Fatty Acid Synthase Lowers Numerous Lipids and Glucose-Stimulated Insulin Release in Insulinoma Cells

    PubMed Central

    MacDonald, Michael J.; Hasan, Noaman M.; Dobrzyn, Agnieszka; Stoker, Scott W.; Ntambi, James M.; Liu, Xueqing; Sampath, Harini

    2013-01-01

    We previously showed that knockdown of the anaplerotic enzyme pyruvate carboxylase in the INS-1 832/13 insulinoma cell line inhibited glucose-stimulated insulin release and glucose carbon incorporation into lipids. We now show that knockdown of fatty acid synthase (FAS) mRNA and protein also inhibits glucose-stimulated insulin release in this cell line. Levels of numerous phospholipids, cholesterol esters, diacylglycerol, triglycerides and individual fatty acids with C14-C24 side chains were acutely lowered about 20% in glucose-stimulated pyruvate carboxylase knockdown cells over a time course that coincides with insulin secretion. In FAS knockdown cells glucose carbon incorporation into lipids and the levels of the subclasses of phospholipids and cholesterol ester species were lower by 20–30% without inhibition of glucose oxidation. These studies suggest that rapid lipid modification is essential for normal glucose-stimulated insulin secretion. PMID:23357280

  8. HP1 knockdown is associated with abnormal condensation of almost all chromatin types in a grasshopper (Eyprepocnemis plorans).

    PubMed

    Ruiz-Estévez, Mercedes; Bakkali, Mohammed; Cabrero, Josefa; Camacho, Juan Pedro M; López-León, María Dolores

    2014-09-01

    Heterochromatin protein 1 (HP1) is a highly conserved family of eukaryotic proteins required for heterochromatic gene silencing and euchromatic gene transcription regulation. In addition, HP1 is involved in chromatin organization and protection of chromosome integrity during cell division. Here, we present a cytological and molecular analysis of the effects of HP1 knockdown in Eyprepocnemis plorans, a grasshopper species polymorphic for supernumerary heterochromatic chromosomes. Our results revealed contrasting effects of HP1 knockdown on gene activity. While the Bub1 gene decreased in expression level in HP1 knockdown animals, NOR activity, rRNA and, contrarily to previous reports in Drosophila, Hsp70 gene expression remained unchanged. Furthermore, HP1 knockdown resulted in abnormal chromatin condensation, chromosomal bridges, higher frequency of macrospermatids, loss of muscle mass and hemolymph amount as well as a low number of dividing cells and survival reduction. All these phenotypes are very likely due to the chromatin condensation disruption observed for almost all kinds of chromatin.

  9. [Knockdown of Puma protects cord blood CD34(+) cells against γ- irradiation].

    PubMed

    Zhao, Lei; Zhang, Hong-Yan; Pang, Ya-Kun; Gu, Hai-Hui; Xu, Jing; Yuan, Wei-Ping; Cheng, Tao

    2014-04-01

    Puma (P53 upregulated modulator of apoptosis) is a BCL-2 homology 3 (BH3)-only BCL-1 family member and a critical mediator of P53-dependent and -independent apoptosis. Puma plays an essential role in the apoptosis of hematopoietic stem cells exposed to irradiation without an increased risk of malignancies. This study was purposed to develop an effective lentiviral vector to target Puma in human hematopoietic cells and to investigate the effect of Puma gene knockdown on the biological function of human cord blood CD34(+) cells. SF-LV-shPuma-EGFP and control vectors were constructed, and packaged with the pSPAX2/pMD2.G packaging plasmids via 293T cells to produce pseudo-type lentiviruses. SF-LV-shPuma-EGFP or control lentiviruses were harvested within 72 hours after transfection and then were used to transduce human cord blood CD34(+) cells. GFP(+) transduced cells were sorted by flow cytometry (FCM) for subsequent studies. Semi-quantitative real time RT PCR, Western blot, FCM with Annexin V-PE/7-AAD double staining, Ki67 staining, colony forming cell assay (CFC), CCK-8 assay and BrdU incorporation were performed to determine the expression of Puma and its effect on the cord blood CD34(+) cells. The results showed that Puma was significantly knocked down in cord blood CD34(+) cells and the low expression of Puma conferred a radio-protective effect on the cord blood CD34(+) cells. This effect was achieved through reduced apoptosis and sustained quiescence after irradiation due to Puma knockdown. It is concluded that knockdown of puma gene in CD34(+) hematopoietic stem cells of human cord blood possesses the radioprotective effect, maintains the cells in silence targeting Puma in human hematopoietic cells may have a similar effect with that on mouse hematopoietic cells as previously shown, and our lentiviral targeting system for Puma provides a valuable tool for future translational studies with human cells.

  10. Nrf2 Knockdown Disrupts the Protective Effect of Curcumin on Alcohol-Induced Hepatocyte Necroptosis.

    PubMed

    Lu, Chunfeng; Xu, Wenxuan; Zhang, Feng; Shao, Jiangjuan; Zheng, Shizhong

    2016-12-05

    It has emerged that hepatocyte necroptosis plays a critical role in chronic alcoholic liver disease (ALD). Our previous study has identified that the beneficial therapeutic effect of curcumin on alcohol-caused liver injury might be attributed to activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2), whereas the role of curcumin in regulating necroptosis and the underlying mechanism remain to be determined. We first found that chronic alcohol consumption triggered obvious hepatocyte necroptosis, leading to increased expression of receptor-interacting protein 1, receptor-interacting protein 3, high-mobility group box 1, and phosphorylated mixed lineage kinase domain-like in murine livers. Curcumin dose-dependently ameliorated hepatocyte necroptosis and alleviated alcohol-caused decrease in hepatic Nrf2 expression in alcoholic mice. Then Nrf2 shRNA lentivirus was introduced to generate Nrf2-knockdown mice. Our results indicated that Nrf2 knockdown aggravated the effects of alcohol on liver injury and necroptosis and even abrogated the inhibitory effect of curcumin on necroptosis. Further, activated Nrf2 by curcumin inhibited p53 expression in both livers and cultured hepatocytes under alcohol stimulation. The next in vitro experiments, similar to in vivo ones, revealed that although Nrf2 knockdown abolished the suppression of curcumin on necroptosis of hepatocytes exposed to ethanol, p53 siRNA could clearly rescued the relative effect of curcumin. In summary, for the first time, we concluded that curcumin attenuated alcohol-induced hepatocyte necroptosis in a Nrf2/p53-dependent mechanism. These findings make curcumin an excellent candidate for ALD treatment and advance the understanding of ALD mechanisms associated with hepatocyte necroptosis.

  11. Neuron-specific Sumo1-3 knockdown in mice impairs episodic and fear memories.

    PubMed

    Wang, Liangli; Rodriguiz, Ramona M; Wetsel, William C; Sheng, Huaxin; Zhao, Shengli; Liu, Xiaozhi; Paschen, Wulf; Yang, Wei

    2014-07-01

    Growing evidence suggests that small ubiquitin-like modifier (SUMO) conjugation plays a key role in brain plasticity by modulating activity-dependent synaptic transmission. However, these observations are based largely on cell culture experiments. We hypothesized that episodic and fear memories would be affected by silencing SUMO1-3 expression. To investigate the role of SUMO conjugation in neuronal functioning in vivo, we generated a novel Sumo transgenic mouse model in which a Thy1 promoter drives expression of 3 distinct microRNAs to silence Sumo1-3 expression, specifically in neurons. Wild-type and Sumo1-3 knockdown mice were subjected to a battery of behavioural tests to elucidate whether Sumoylation is involved in episodic and emotional memory. Expression of Sumo1-3 microRNAs and the corresponding silencing of Sumo expression were particularly pronounced in hippocampal, amygdala and layer V cerebral cortex neurons. The Sumo knockdown mice displayed anxiety-like responses and were impaired in episodic memory processes, contextual and cued fear conditioning and fear-potentiated startle. Since expression of Sumo1-3 was silenced in this mouse model, we need to verify in future studies which of the SUMO paralogues play the pivotal role in episodic and emotional memory. Our results indicate that a functional SUMO conjugation pathway is essential for emotionality and cognition. This novel Sumo knockdown mouse model and the technology used in generating this mutant may help to reveal novel mechanisms that underlie a variety of neuropsychiatric conditions associated with anxiety and impairment of episodic and emotional memory.

  12. In Vivo Knockdown of Pathogenic Proteins via Specific and Nongenetic IAP-dependent Protein Erasers (SNIPERs).

    PubMed

    Ohoka, Nobumichi; Okuhira, Keiichiro; Ito, Masahiro; Nagai, Katsunori; Shibata, Norihito; Hattori, Takayuki; Ujikawa, Osamu; Shimokawa, Kenichiro; Sano, Osamu; Koyama, Ryokichi; Fujita, Hisashi; Teratani, Mika; Matsumoto, Hirokazu; Imaeda, Yasuhiro; Nara, Hiroshi; Cho, Nobuo; Naito, Mikihiko

    2017-02-02

    Many diseases, especially cancers, result from aberrant or over-expression of pathogenic proteins. Specific inhibitors against these proteins have shown remarkable therapeutic effects, but are limited mainly to enzymes. An alternative approach that may have utility in drug development relies on selective degradation of pathogenic proteins via small, chimeric molecules linking an E3 ubiquitin ligase to the targeted protein for proteasomal degradation. To this end, we recently developed a protein-knockdown system based on hybrid small-molecule SNIPERs (Specific and Nongenetic IAP-dependent Protein Erasers) that recruit inhibitor of apoptosis protein (IAP) ubiquitin ligases to specifically degrade targeted proteins. Here, we extend our prior study to show a proof of concept of the SNIPER technology in vivo. By incorporating a high affinity IAP ligand, we developed a novel SNIPER against estrogen receptor α (ERα), SNIPER(ER)-87, that has a potent protein knockdown activity. The SNIPER(ER) reduced ERα levels in tumor xenografts and suppressed the growth of ERα-positive breast tumors in mice. Mechanistically, it preferentially recruits X-linked inhibitor of apoptosis protein (XIAP), rather than cellular inhibitor of apoptosis protein 1 (cIAP1), to degrade ERα via the ubiquitin-proteasome pathway. With this IAP ligand, potent SNIPERs against other pathogenic proteins, BCR-ABL, bromodomain-containing protein 4 (BRD4), and phosphodiesterase-4 (PDE4) could also be developed. These results indicate that forced ubiquitylation by SNIPERs is a useful method to achieve efficient protein knockdown with a potential therapeutic activities and could also be applied to study the role of ubiquitylation in many cellular processes.

  13. β-catenin knockdown inhibits the proliferation of human glioma cells in vitro and in vivo

    PubMed Central

    WANG, ZHONG; CHEN, QIANXUE

    2016-01-01

    β-catenin is a crucial oncogene that is capable of regulating cancer progression. The aim of the present study was to clarify whether β-catenin was associated with the proliferation and progress of glioma. In order to knockdown the expression of β-catenin in human U251 glioma cells, three pairs of small interfering (si)RNA were designed and synthesized and the most effective siRNA was selected and used for silencing the endogenous β-catenin, which was detected by western blot analysis and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Proliferation was subsequently detected using a methylthiazolyl-tetrazolium bromide assay and the results demonstrated that knockdown of β-catenin significantly inhibited the proliferation of U251 cells in a time- and dose-dependent manner (P<0.01). Cell apoptosis rate was analyzed using flow cytometry and Annexin V-fluorescein isothiocyanate/propidium iodide staining demonstrated that β-catenin siRNA significantly increased the apoptosis of U251 cells (P<0.01). Furthermore, the results of an in vitro scratch assay demonstrated that β-catenin silencing suppressed the proliferation of U251 cells, as compared with the control group (P<0.01). In vivo, β-catenin expression levels in U251 cells were significantly inhibited (P<0.01) following β-catenin short hairpin (sh)RNA lentiviral-vector transfection, as detected by western blot analysis and RT-qPCR. Tumorigenicity experiments demonstrated that β-catenin inhibition significantly increased the survival rate of nude mice. The results of the present study demonstrated that knockdown of β-catenin expression significantly inhibited the progression of human glioma cancer cells, in vitro and in vivo; thus suggesting that β-catenin silencing may be a novel therapy for the treatment of human glioma. PMID:26998037

  14. Lifespan and reproduction in brain-specific miR-29-knockdown mouse.

    PubMed

    Takeda, Toru; Tanabe, Hiroyuki

    2016-03-18

    The microRNA miR-29 is widely distributed and highly expressed in adult mouse brain during the mouse's lifetime. We recently created conditional mutant mice whose miR-29 was brain-specifically knocked down through overexpression of an antisense RNA transgene against miR-29. To explore a role for brain miR-29 in maximizing organismal fitness, we assessed somatic growth, reproduction, and lifespan in the miR-29-knockdown (KD) mice and their wild-type (WT) littermates. The KD mice were developmentally indistinguishable from WT mice with respect to gross morphology and physical activity. Fertility testing revealed that KD males were subfertile, whereas KD females were hyperfertile, only in terms of reproductive success, when compared to their gender-matched WT correspondents. Another phenotypic difference between KD and WT animals appeared in their lifespan data; KD males displayed an overall increasing tendency in post-reproductive survival relative to WT males. In contrast, KD females were prone to shorter lifespans than WT females. These results clarify that brain-targeted miR-29 knockdown affects both lifespan and reproduction in a gender-dependent manner, and moreover that the reciprocal responsiveness to the miR-29 knockdown between these two phenotypes in both genders closely follow life-course models based on the classical trade-off prediction wherein elaborate early-life energetic investment in reproduction entails accelerated late-life declines in survival, and vice versa. Thus, this study identified miR-29 as the first mammalian miRNA that is directly implicated in the lifetime trade-off between the two major fitness components, lifespan and reproduction.

  15. Knockdown of monocarboxylate transporter 8 (mct8) disturbs brain development and locomotion in zebrafish.

    PubMed

    de Vrieze, Erik; van de Wiel, Sandra M W; Zethof, Jan; Flik, Gert; Klaren, Peter H M; Arjona, Francisco J

    2014-06-01

    Allan-Herndon-Dudley syndrome (AHDS) is an inherited disorder of brain development characterized by severe psychomotor retardation. This X-linked disease is caused by mutations in the monocarboxylate transporter 8 (MCT8), an important thyroid hormone transporter in brain neurons. MCT8-knockout mice lack the 2 major neurological symptoms of AHDS, namely locomotor problems and cognitive impairment. The pathological mechanism explaining the symptoms is still obscure, and no cure for this condition is known. The development of an animal model that carries MCT8-related neurological symptoms is warranted. We have employed morpholino-based gene knockdown to create zebrafish deficient for mct8. Knockdown of mct8 results in specific symptoms in the thyroid axis and brain. The mct8-morphants showed impaired locomotor behavior and brain development. More specifically, we observed maldevelopment of the cerebellum and mid-hindbrain boundary and apoptotic clusters in the zebrafish brain. The mRNA expression of zebrafish orthologs of mammalian TSH, thyroid hormone transporters, and deiodinases was altered in mct8 morphants. In particular, deiodinase type 3 gene expression was consistently up-regulated in zebrafish mct8 morphants. The thyroid hormone metabolite tetrac, but not T3, partly ameliorated the affected phenotype and locomotion disability of morphant larvae. Our results show that mct8 knockdown in zebrafish larvae results in disturbances in the thyroid axis, brain, and locomotion behavior, which is congruent with the clinical aspect of impaired locomotion and cognition in patients with AHDS. Taken together, the zebrafish is a suitable animal model for the study of the pathophysiology of AHDS.

  16. Knock-down of p300 decreases the proliferation and odontogenic differentiation potentiality of HDPCs.

    PubMed

    Liu, H J; Wang, T; Li, Q M; Guan, X Y; Xu, Q

    2015-10-01

    To investigate the role of p300 in the regulation of proliferation and odontogenic differentiation of human dental pulp cells (HDPCs). The recombinant lentiviral vector pshRNA-copGFP was used to knock-down p300 expression in HDPCs. Protein level of acetylated H3 was detected. The proliferation of HDPCs was measured using the CCK8 assay. The cell cycle and apoptosis were analysed using flow cytometry and TUNEL staining, respectively. The expression levels of Cdc25A, p21(waf1) and the cleaved products of caspase 3 and caspase 7 were determined utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. The alkaline phosphatase (ALP) activity was measured, and the formation of mineralized nodules was assessed using alizarin red staining after the induction of odontogenic differentiation of HDPCs. The expression levels of the odontogenic differentiation markers DMP-1, DSPP and DSP were detected utilizing real-time quantitative polymerase chain reaction and Western blotting analysis. After p300 was knocked down in HDPCs, p300 was significantly down-regulated at both the mRNA and protein levels, and histone H3 acetylation was reduced. The proliferation capacity of HDPCs was suppressed in p300 knock-down groups. The cells were arrested in the G0/G1 phase of the cell cycle, and cell apoptosis was triggered. ALP activity, the formation of mineralized nodules and the expression levels of DMP-1, DSPP and DSP were all decreased in p300-knock-down HDPCs undergoing odontogenic differentiation. Knocking down p300 restrains the proliferation and odontogenic differentiation potentiality of HDPCs. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  17. AHR2 morpholino knockdown reduces the toxicity of total particulate matter to zebrafish embryos.

    PubMed

    Massarsky, Andrey; Bone, Audrey J; Dong, Wu; Hinton, David E; Prasad, G L; Di Giulio, Richard T

    2016-10-15

    The zebrafish embryo has been proposed as a 'bridge model' to study the effects of cigarette smoke on early development. Previous studies showed that exposure to total particulate matter (TPM) led to adverse effects in developing zebrafish, and suggested that the antioxidant and aryl hydrocarbon receptor (AHR) pathways play important roles. This study investigated the roles of these two pathways in mediating TPM toxicity. The study consisted of four experiments. In experiment I, zebrafish embryos were exposed from 6h post fertilization (hpf) until 96hpf to TPM0.5 and TPM1.0 (corresponding to 0.5 and 1.0μg/mL equi-nicotine units) in the presence or absence of an antioxidant (N-acetyl cysteine/NAC) or a pro-oxidant (buthionine sulfoximine/BSO). In experiment II, TPM exposures were performed in embryos that were microinjected with nuclear factor erythroid 2-related factor 2 (Nrf2), AHR2, cytochrome P450 1A (CYP1A), or CYP1B1 morpholinos, and deformities were assessed. In experiment III, embryos were exposed to TPM, and embryos/larvae were collected at 24, 48, 72, and 96hpf to assess several genes associated with the antioxidant and AHR pathways. Lastly, experiment IV assessed the activity and protein levels of CYP1A and CYP1B1 after exposure to TPM. We demonstrate that the incidence of TPM-induced deformities was generally not affected by NAC/BSO treatments or Nrf2 knockdown. In contrast, AHR2 knockdown reduced, while CYP1A or CYP1B1 knockdowns elevated the incidence of some deformities. Moreover, as shown by gene expression the AHR pathway, but not the antioxidant pathway, was induced in response to TPM exposure, providing further evidence for its importance in mediating TPM toxicity.

  18. Nanofibrous scaffold-mediated REST knockdown to enhance neuronal differentiation of stem cells.

    PubMed

    Low, Wei Ching; Rujitanaroj, Pim-On; Lee, Dong-Keun; Messersmith, Phillip B; Stanton, Lawrence W; Goh, Eyleen; Chew, Sing Yian

    2013-05-01

    At present, the recovery prospect for patients with chronic neurodegenerative diseases or acute trauma in the central nervous system is sub-optimal. The controlled differentiation of neural stem/progenitor cells (NPCs) to functional neurons is a possible treatment strategy. In contrast to the classical approach of biochemicals supplementation for guided stem cell commitment, this study explores the feasibility of directing neuronal differentiation through synergistic integration of three-dimensional nanofibrous topographical cues and scaffold-mediated knockdown of RE-1 silencing transcription factor (REST) in mouse NPCs. Taking advantage of the strong adhesive property and latent reactivity of mussel-inspired polydopamine (PD) coating, electrospun polycaprolactone (PCL) nanofibers were successfully functionalized with REST siRNAs (denoted as siREST PD-fiber). Sustained REST knockdown in NPCs was achieved for up to five days in vitro and the silencing efficiency was significantly higher than that mediated through siRNA adsorption onto non-PD coated sample controls. The silencing of REST, together with nanofiber topographical effect, significantly enhanced NPC neuronal commitment (57.5% Map2(+) cells in siREST PD-fiber vs. 43.5% in siREST PD-film vs. 50% in PD-fiber controls, p < 0.05) while reducing astrocytic and oligodendrocytic differentiation (10.7% O4(+) cells vs. ∼30% in siREST PD-film, p < 0.01). Taken together, the synergistic effects of scaffold-mediated REST knockdown and topographical cues from PD-modified nanofibers may be a useful strategy for generating functional neurons for therapeutic purposes. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Knockdown of the putative Lifeguard homologue CG3814 in neurons of Drosophila melanogaster.

    PubMed

    M'Angale, P G; Staveley, B E

    2016-12-19

    Lifeguard is an integral transmembrane protein that modulates FasL-mediated apoptosis by interfering with the activation of caspase 8. It is evolutionarily conserved, with homologues present in plants, nematodes, zebra fish, frog, chicken, mouse, monkey, and human. The Lifeguard homologue in Drosophila, CG3814, contains the Bax inhibitor-1 family motif of unknown function. Downregulation of Lifeguard disrupts cellular homeostasis and disease by sensitizing neurons to FasL-mediated apoptosis. We used bioinformatic analyses to identify CG3814, a putative homologue of Lifeguard, and knocked down CG3814/LFG expression under the control of the Dopa decarboxylase (Ddc-Gal4) transgene in Drosophila melanogaster neurons to investigate whether it possesses neuroprotective activity. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons resulted in a shortened lifespan and impaired locomotor ability, phenotypes that are strongly associated with the degeneration and loss of dopaminergic neurons. Lifeguard interacts with anti-apoptotic Bcl-2 proteins and possibly pro-apoptotic proteins to exert its neuroprotective function. The co-expression of Buffy, the sole anti-apoptotic Bcl-2 gene family member in Drosophila, and CG3814/LFG by stable inducible RNA interference, suppresses the shortened lifespan and the premature age-dependent loss in climbing ability. Suppression of CG3814/LFG in the Drosophila eye reduces the number of ommatidia and increases disruption of the ommatidial array. Overexpression of Buffy, along with the knockdown of CG3814/LFG, counteracts the eye phenotypes. Knockdown of CG3814/LFG in Ddc-Gal4-expressing neurons in Drosophila diminishes its neuroprotective ability and results in a shortened lifespan and loss of climbing ability, phenotypes that are improved upon overexpression of the pro-survival Buffy.

  20. OAZ1 knockdown enhances viability and inhibits ER and LHR transcriptions of granulosa cells in geese

    PubMed Central

    Ma, Rong; He, Hui; Yi, Zhixin; Chen, Ziyu

    2017-01-01

    An increasing number of studies suggest that ornithine decarboxylase antizyme 1 (OAZ1), which is regarded as a tumor suppressor gene, regulates follicular development, ovulation, and steroidogenesis. The granulosa cells in the ovary play a critical role in these ovarian functions. However, the action of OAZ1 mediating physiological functions of granulosa cells is obscure. OAZ1 knockdown in granulosa cells of geese was carried out in the current study. The effect of OAZ1 knockdown on polyamine metabolism, cell proliferation, apoptosis, and hormone receptor transcription of primary granulosa cells in geese was measured. The viability of granulosa cells transfected with the shRNA OAZ1 at 48 h was significantly higher than the control (p<0.05). The level of putrescine and spermidine in granulosa cells down-regulating OAZ1 was 7.04- and 2.11- fold higher compared with the control, respectively (p<0.05). The CCND1, SMAD1, and BCL-2 mRNA expression levels in granulosa cells down-regulating OAZ1 were each significantly higher than the control, respectively (p<0.05), whereas the PCNA and CASPASE 3 expression levels were significantly lower than the control (p<0.05). The estradiol concentration, ER and LHR mRNA expression levels were significantly lower in granulosa cells down-regulating OAZ1 compared with the control (p<0.05). Taken together, our results indicated that OAZ1 knockdown elevated the putrescine and spermidine contents and enhanced granulosa cell viability and inhibited ER and LHR transcriptions of granulosa cells in geese. PMID:28362829

  1. Regeneration of diabetic axons is enhanced by selective knockdown of the PTEN gene

    PubMed Central

    Singh, Bhagat; Singh, Vandana; Krishnan, Anand; Koshy, Kurien; Martinez, Jose A.; Cheng, Chu; Almquist, Chris

    2014-01-01

    Diabetes mellitus renders both widespread and localized irreversible damage to peripheral axons while imposing critical limitations on their ability to regenerate. A major failure of regenerative capacity thereby imposes a ‘double hit’ in diabetic patients who frequently develop focal neuropathies such as carpal tunnel syndrome in addition to generalized diffuse polyneuropathy. The mechanisms of diabetic neuron regenerative failure have been speculative and few approaches have offered therapeutic opportunities. In this work we identify an unexpected but major role for PTEN upregulation in diabetic peripheral neurons in attenuating axon regrowth. In chronic diabetic neuropathy models in mice, we identified significant PTEN upregulation in peripheral sensory neurons of messenger RNA and protein compared to littermate controls. In vitro, sensory neurons from these mice responded to PTEN knockdown with substantial rises in neurite outgrowth and branching. To test regenerative plasticity in a chronic diabetic model with established neuropathy, we superimposed an additional focal sciatic nerve crush injury and assessed morphological, electrophysiological and behavioural recovery. Knockdown of PTEN in dorsal root ganglia ipsilateral to the side of injury was achieved using a unique form of non-viral short interfering RNA delivery to the ipsilateral nerve injury site and paw. In comparison with scrambled sequence control short interfering RNA, PTEN short interfering RNA improved several facets of regeneration: recovery of compound muscle action potentials, reflecting numbers of reconnected motor axons to endplates, conduction velocities of both motor and sensory axons, reflecting their maturation during regrowth, numbers and calibre of regenerating myelinated axons distal to the injury site, reinnervation of the skin by unmyelinated epidermal axons and recovery of mechanical sensation. Collectively, these findings identify a novel therapeutic approach, potentially

  2. Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma.

    PubMed

    Mitra, Moutushy; Kandalam, Mallikarjuna; Verma, Rama Shanker; UmaMaheswari, Krishnan; Krishnakumar, Subramanian

    2010-05-11

    Previously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study, we studied the genes regulated by Ep-CAM in a retinoblastoma Y79 cell line in vitro using a combination of short interference RNA and microarray technology. Flow cytometry, quantitative reverse transcriptase PCR (Q-RT-PCR), and immunohistochemistry were performed to confirm the Ep-CAM re-expression in the Y79 cells treated with 5'-azacytidine (AZC). Ep-CAM expression in AZC-treated Y79 cells was silenced using synthetic anti-Ep-CAM short interference RNA, and whole genome microarray was performed to determine the gene expression changes post Ep-CAM knockdown. Ep-CAM inhibition was confirmed by Q-RT-PCR, western blotting, and immunofluorescence. Ep-CAM expression was significantly restored in Y79 cells on day 5 of AZC treatment. Ep-CAM inhibition significantly affected Y79 cell proliferation. We identified 465 upregulated genes (>or=1.0 fold) and 205 downregulated genes (knockdown of Ep-CAM. These genes regulate several aspects of tumor function, including cell survival/proliferation, DNA replication/transcription, apoptosis, and angiogenesis. Quantitative pathway analysis using Biointerpreter further revealed that the most pronounced effect of Ep-CAM knockdown was deregulation of pathways that include mitogen-activated protein kinase (MAP) kinase and tumor protein 53 (P53) pathways. Real-time Q-RT-PCR confirmed microarray gene expression changes for selected genes. Ep-CAM silencing significantly decreases Y79 cell proliferation and revealed a wide network of deregulated pathways in vitro. Future studies targeting Ep-CAM gene expression in vivo will help to delineate the mechanisms associated with Ep-CAM gene function in neoplastic transformation and define the potential for Ep-CAM-based molecular intervention in retinoblastoma patients.

  3. The effects of Kiaa0319 knockdown on cortical and subcortical anatomy in male rats.

    PubMed

    Szalkowski, Caitlin E; Fiondella, Christopher F; Truong, Dongnhu T; Rosen, Glenn D; LoTurco, Joseph J; Fitch, Roslyn H

    2013-04-01

    Developmental dyslexia is a disorder characterized by a specific deficit in reading despite adequate overall intelligence and educational resources. The neurological substrate underlying these significant behavioral impairments is not known. Studies of post mortem brain tissue from male and female dyslexic individuals revealed focal disruptions of neuronal migration concentrated in the left hemisphere, along with aberrant symmetry of the right and left the planum temporale, and changes in cell size distribution within the medial geniculate nucleus of the thalamus (Galaburda et al., 1985; Humphreys et al., 1990). More recent neuroimaging studies have identified several changes in the brains of dyslexic individuals, including regional changes in gray matter, changes in white matter, and changes in patterns of functional activation. In a further effort to elucidate the etiology of dyslexia, epidemiological and genetic studies have identified several candidate dyslexia susceptibility genes. Some recent work has investigated associations between some of these genetic variants and structural changes in the brain. Variants of one candidate dyslexia susceptibility gene, KIAA0319, have been linked to morphological changes in the cerebellum and functional activational changes in the superior temporal sulcus (Jamadar et al., 2011; Pinel et al., 2012). Animal models have been used to create a knockdown of Kiaa0319 (the rodent homolog of the human gene) via in utero RNA interference in order to study the gene's effects on brain development and behavior. Studies using this animal model have demonstrated that knocking down the gene leads to focal disruptions of neuronal migration in the form of ectopias and heterotopias, similar to those observed in the brains of human dyslexics. However, further changes to the structure of the brain have not been studied following this genetic disruption. The current study sought to determine the effects of embryonic Kiaa0319 knockdown on volume

  4. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

    PubMed

    Bagci, Enise; Heijlen, Marjolein; Vergauwen, Lucia; Hagenaars, An; Houbrechts, Anne M; Esguerra, Camila V; Blust, Ronny; Darras, Veerle M; Knapen, Dries

    2015-01-01

    Thyroid hormone (TH) balance is essential for vertebrate development. Deiodinase type 1 (D1) and type 2 (D2) increase and deiodinase type 3 (D3) decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray), biochemistry, morphology and physiology using morpholino (MO) knockdown. Knockdown of D1+D2 (D1D2MO) and knockdown of D3 (D3MO) both resulted in transcriptional regulation of energy metabolism and (muscle) development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct TH balance

  5. Glyoxalase 1-knockdown in human aortic endothelial cells – effect on the proteome and endothelial function estimates

    PubMed Central

    Stratmann, Bernd; Engelbrecht, Britta; Espelage, Britta C.; Klusmeier, Nadine; Tiemann, Janina; Gawlowski, Thomas; Mattern, Yvonne; Eisenacher, Martin; Meyer, Helmut E.; Rabbani, Naila; Thornalley, Paul J.; Tschoepe, Diethelm; Poschmann, Gereon; Stühler, Kai

    2016-01-01

    Methylglyoxal (MG), an arginine-directed glycating agent, is implicated in diabetic late complications. MG is detoxified by glyoxalase 1 (GLO1) of the cytosolic glyoxalase system. The aim was to investigate the effects of MG accumulation by GLO1-knockdown under hyperglycaemic conditions in human aortic endothelial cells (HAECs) hypothesizing that the accumulation of MG accounts for the deleterious effects on vascular function. SiRNA-mediated knockdown of GLO1 was performed and MG concentrations were determined. The impact of MG on the cell proteome and targets of MG glycation was analysed, and confirmed by Western blotting. Markers of endothelial function and apoptosis were assessed. Collagen content was assayed in cell culture supernatant. GLO1-knockdown increased MG concentration in cells and culture medium. This was associated with a differential abundance of cytoskeleton stabilisation proteins, intermediate filaments and proteins involved in posttranslational modification of collagen. An increase in fibrillar collagens 1 and 5 was detected. The extracellular concentration of endothelin-1 was increased following GLO1-knockdown, whereas the phosphorylation and amount of eNOS was not influenced by GLO1-knockdown. The expression of ICAM-1, VCAM-1 and of MCP-1 was elevated and apoptosis was increased. MG accumulation by GLO1-knockdown provoked collagen expression, endothelial inflammation and dysfunction and apoptosis which might contribute to vascular damage. PMID:27898103

  6. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

    DOE PAGES

    Cass, Cynthia L.; Peraldi, Antoine; Dowd, Patrick F.; ...

    2015-06-19

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants hadmore » reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. Lastly, the data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops.« less

  7. Effects of PHENYLALANINE AMMONIA LYASE (PAL) knockdown on cell wall composition, biomass digestibility, and biotic and abiotic stress responses in Brachypodium

    PubMed Central

    Cass, Cynthia L.; Peraldi, Antoine; Dowd, Patrick F.; Mottiar, Yaseen; Santoro, Nicholas; Karlen, Steven D.; Bukhman, Yury V.; Foster, Cliff E.; Thrower, Nick; Bruno, Laura C.; Moskvin, Oleg V.; Johnson, Eric T.; Willhoit, Megan E.; Phutane, Megha; Ralph, John; Mansfield, Shawn D.; Nicholson, Paul; Sedbrook, John C.

    2015-01-01

    The phenylpropanoid pathway in plants synthesizes a variety of structural and defence compounds, and is an important target in efforts to reduce cell wall lignin for improved biomass conversion to biofuels. Little is known concerning the trade-offs in grasses when perturbing the function of the first gene family in the pathway, PHENYLALANINE AMMONIA LYASE (PAL). Therefore, PAL isoforms in the model grass Brachypodium distachyon were targeted, by RNA interference (RNAi), and large reductions (up to 85%) in stem tissue transcript abundance for two of the eight putative BdPAL genes were identified. The cell walls of stems of BdPAL-knockdown plants had reductions of 43% in lignin and 57% in cell wall-bound ferulate, and a nearly 2-fold increase in the amounts of polysaccharide-derived carbohydrates released by thermochemical and hydrolytic enzymic partial digestion. PAL-knockdown plants exhibited delayed development and reduced root growth, along with increased susceptibilities to the fungal pathogens Fusarium culmorum and Magnaporthe oryzae. Surprisingly, these plants generally had wild-type (WT) resistances to caterpillar herbivory, drought, and ultraviolet light. RNA sequencing analyses revealed that the expression of genes associated with stress responses including ethylene biosynthesis and signalling were significantly altered in PAL knocked-down plants under non-challenging conditions. These data reveal that, although an attenuation of the phenylpropanoid pathway increases carbohydrate availability for biofuel, it can adversely affect plant growth and disease resistance to fungal pathogens. The data identify notable differences between the stress responses of these monocot pal mutants versus Arabidopsis (a dicot) pal mutants and provide insights into the challenges that may arise when deploying phenylpropanoid pathway-altered bioenergy crops. PMID:26093023

  8. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  9. NBS1 knockdown by small interfering RNA increases ionizing radiation mutagenesis and telomere association in human cells

    NASA Technical Reports Server (NTRS)

    Zhang, Ying; Lim, Chang U K.; Williams, Eli S.; Zhou, Junqing; Zhang, Qinming; Fox, Michael H.; Bailey, Susan M.; Liber, Howard L.

    2005-01-01

    Hypomorphic mutations which lead to decreased function of the NBS1 gene are responsible for Nijmegen breakage syndrome, a rare autosomal recessive hereditary disorder that imparts an increased predisposition to development of malignancy. The NBS1 protein is a component of the MRE11/RAD50/NBS1 complex that plays a critical role in cellular responses to DNA damage and the maintenance of chromosomal integrity. Using small interfering RNA transfection, we have knocked down NBS1 protein levels and analyzed relevant phenotypes in two closely related human lymphoblastoid cell lines with different p53 status, namely wild-type TK6 and mutated WTK1. Both TK6 and WTK1 cells showed an increased level of ionizing radiation-induced mutation at the TK and HPRT loci, impaired phosphorylation of H2AX (gamma-H2AX), and impaired activation of the cell cycle checkpoint regulating kinase, Chk2. In TK6 cells, ionizing radiation-induced accumulation of p53/p21 and apoptosis were reduced. There was a differential response to ionizing radiation-induced cell killing between TK6 and WTK1 cells after NBS1 knockdown; TK6 cells were more resistant to killing, whereas WTK1 cells were more sensitive. NBS1 deficiency also resulted in a significant increase in telomere association that was independent of radiation exposure and p53 status. Our results provide the first experimental evidence that NBS1 deficiency in human cells leads to hypermutability and telomere associations, phenotypes that may contribute to the cancer predisposition seen among patients with this disease.

  10. Ron knockdown and Ron monoclonal antibody IMC-RON8 sensitize pancreatic cancer to histone deacetylase inhibitors (HDACi).

    PubMed

    Zou, Yi; Howell, Gillian M; Humphrey, Lisa E; Wang, Jing; Brattain, Michael G

    2013-01-01

    Recepteur d'origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer.

  11. Ron Knockdown and Ron Monoclonal Antibody IMC-RON8 Sensitize Pancreatic Cancer to Histone Deacetylase Inhibitors (HDACi)

    PubMed Central

    Zou, Yi; Howell, Gillian M.; Humphrey, Lisa E.; Wang, Jing; Brattain, Michael G.

    2013-01-01

    Recepteur d’origine nantais (Ron) is overexpressed in a panel of pancreatic cancer cells and tissue samples from pancreatic cancer patients. Ron can be activated by its ligand macrophage stimulating protein (MSP), thereby activating oncogenic signaling pathways. Crosstalk between Ron and EGFR, c-Met, or IGF-1R may provide a mechanism underlying drug resistance. Thus, targeting Ron may represent a novel therapeutic strategy. IMC-RON8 is the first Ron monoclonal antibody (mAb) entering clinical trial for targeting Ron overexpression. Our studies show IMC-RON8 downmodulated Ron expression in pancreatic cancer cells and significantly blocked MSP-stimulated Ron activation, downstream Akt and ERK phosphorylation, and survivin mRNA expression. IMC-RON8 hindered MSP-induced cell migration and reduced cell transformation. Histone deacetylase inhibitors (HDACi) are reported to target expression of various genes through modification of nucleosome histones and non-histone proteins. Our work shows HDACi TSA and Panobinostat (PS) decreased Ron mRNA and protein expression in pancreatic cancer cells. PS also reduced downstream signaling of pAkt, survivin, and XIAP, as well as enhanced cell apoptosis. Interestingly, PS reduced colony formation in Ron knockdown cells to a greater extent than Ron scramble control cells in colony formation and soft agarose assays. IMC-RON8 could also sensitize pancreatic cancer cells to PS, as reflected by reduced colony numbers and size in combination treatment with IMC-RON8 and PS compared to single treatment alone. The co-treatment further reduced Ron expression and pAkt, and increased PARP cleavage compared to either treatment alone. This study suggests the potential for a novel combination approach which may ultimately be of value in treatment of pancreatic cancer. PMID:23922886

  12. Knock-down of heat-shock protein 90 and isocitrate lyase gene expression reduced root-knot nematode reproduction.

    PubMed

    Lourenço-Tessutti, Isabela Tristan; Souza Junior, José Dijair Antonino; Martins-de-Sa, Diogo; Viana, Antônio Américo Barbosa; Carneiro, Regina Maria Dechechi Gomes; Togawa, Roberto Coiti; de Almeida-Engler, Janice; Batista, João Aguiar Nogueira; Silva, Maria Cristina Mattar; Fragoso, Rodrigo Rocha; Grossi-de-Sa, Maria Fatima

    2015-05-01

    Crop losses caused by nematode infections are estimated to be valued at USD 157 billion per year. Meloidogyne incognita, a root-knot nematode (RKN), is considered to be one of the most important plant pathogens due to its worldwide distribution and the austere damage it can cause to a large variety of agronomically important crops. RNA interference (RNAi), a gene silencing process, has proven to be a valuable biotechnology alternative method for RKN control. In this study, the RNAi approach was applied, using fragments of M. incognita genes that encode for two essential molecules, heat-shock protein 90 (HSP90) and isocitrate lyase (ICL). Plant-mediated RNAi of these genes led to a significant level of resistance against M. incognita in the transgenic Nicotiana tabacum plants. Bioassays of plants expressing HSP90 dsRNA demonstrated a delay in gall formation and up to 46% reduction in eggs compared with wild-type plants. A reduction in the level of HSP90 transcripts was observed in recovered eggs from plants expressing dsRNA, indicating that gene silencing persisted and was passed along to first progeny. The ICL knock-down had no clear effect on gall formation but resulted in up to 77% reduction in egg oviposition compared with wild-type plants. Our data suggest that both genes may be involved in RKN development and reproduction. Thus, in this paper, we describe essential candidate genes that could be applied to generate genetically modified crops, using the RNAi strategy to control RKN parasitism.

  13. Diverse effects on mitochondrial and nuclear functions elicited by drugs and genetic knockdowns in bloodstream stage Trypanosoma brucei.

    PubMed

    Worthen, Christal; Jensen, Bryan C; Parsons, Marilyn

    2010-05-04

    The options for treating the fatal disease human African trypanosomiasis are limited to a few drugs that are toxic or facing increasing resistance. New drugs that kill the causative agents, subspecies of Trypanosoma brucei, are therefore urgently needed. Little is known about the cellular mechanisms that lead to death of the pathogenic bloodstream stage. We therefore conducted the first side by side comparison of the cellular effects of multiple death inducers that target different systems in bloodstream form parasites, including six drugs (pentamidine, prostaglandin D(2), quercetin, etoposide, camptothecin, and a tetrahydroquinoline) and six RNAi knockdowns that target distinct cellular functions. All compounds tested were static at low concentrations and killed at high concentrations. Dead parasites were rapidly quantified by forward and side scatter during flow cytometry, as confirmed by ethidium homodimer and esterase staining, making these assays convenient for quantitating parasite death. The various treatments yielded different combinations of defects in mitochondrial potential, reactive oxygen species, cell cycle, and genome segregation. No evidence was seen for phosphatidylserine exposure, a marker of apoptosis. Reduction in ATP levels lagged behind decreases in live cell number. Even when the impact on growth was similar at 24 hours, drug-treated cells showed dramatic differences in their ability to further proliferate, demonstrating differences in the reversibility of effects induced by the diverse compounds. Parasites showed different phenotypes depending on the treatment, but none of them were clear predictors of whether apparently live cells could go on to proliferate after drugs were removed. We therefore suggest that clonal proliferation assays may be a useful step in selecting anti-trypanosomal compounds for further development. Elucidating the genetic or biochemical events initiated by the compounds with the most profound effects on subsequent

  14. Knockdown of human deubiquitinase PSMD14 induces cell cycle arrest and senescence

    SciTech Connect

    Byrne, Ann; McLaren, Rajashree P.; Mason, Paul; Chai, Lilly; Dufault, Michael R.; Huang, Yinyin; Liang, Beirong; Gans, Joseph D.; Zhang, Mindy; Carter, Kara; Gladysheva, Tatiana B.; Teicher, Beverly A.; Biemann, Hans-Peter N.; Booker, Michael; Goldberg, Mark A.; Klinger, Katherine W.; Lillie, James; Madden, Stephen L.; Jiang, Yide

    2010-01-15

    The PSMD14 (POH1, also known as Rpn11/MPR1/S13/CepP1) protein within the 19S complex (19S cap; PA700) is responsible for substrate deubiquitination during proteasomal degradation. The role of PSMD14 in cell proliferation and senescence was explored using siRNA knockdown in carcinoma cell lines. Our results reveal that down-regulation of PSMD14 by siRNA transfection had a considerable impact on cell viability causing cell arrest in the G0-G1 phase, ultimately leading to senescence. The molecular events associated with decreased cell proliferation, cell cycle arrest and senescence include down-regulation of cyclin B1-CDK1-CDC25C, down-regulation of cyclin D1 and up-regulation of p21{sup /Cip} and p27{sup /Kip1}. Most notably, phosphorylation of the retinoblastoma protein was markedly reduced in PSMD14 knockdown cells. A comparative study with PSMB5, a subunit of the 20S proteasome, revealed that PSMB5 and PSMD14 have different effects on cell cycle, senescence and associated molecular events. These data support the view that the 19S and 20S subunits of the proteasome have distinct biological functions and imply that targeting 19S and 20S would have distinct molecular consequences on tumor cells.

  15. Effects of Buckling Knockdown Factor, Internal Pressure and Material on the Design of Stiffened Cylinders

    NASA Technical Reports Server (NTRS)

    Lovejoy, Andrew E.; Hilburger, Mark W.; Chunchu, Prasad B.

    2010-01-01

    A design study was conducted to investigate the effect shell buckling knockdown factor (SBKF), internal pressure and aluminum alloy material selection on the structural weight of stiffened cylindrical shells. Two structural optimization codes were used for the design study to determine the optimum minimum-weight design for a series of design cases, and included an in-house developed genetic algorithm (GA) code and PANDA2. Each design case specified a unique set of geometry, material, knockdown factor combinations and loads. The resulting designs were examined and compared to determine the effects of SBKF, internal pressure and material selection on the acreage design weight and controlling failure mode. This design study shows that use of less conservative SBKF values, including internal pressure, and proper selection of material alloy can result in significant weight savings for stiffened cylinders. In particular, buckling-critical cylinders with integrally machined stiffener construction can benefit from the use of thicker plate material that enables taller stiffeners, even when the stiffness, strength and density properties of these materials appear to be inferior.

  16. The knockdown of OsVIT2 and MIT affects iron localization in rice seed.

    PubMed

    Bashir, Khurram; Takahashi, Ryuichi; Akhtar, Shamim; Ishimaru, Yasuhiro; Nakanishi, Hiromi; Nishizawa, Naoko K

    2013-11-20

    The mechanism of iron (Fe) uptake in plants has been extensively characterized, but little is known about how Fe transport to different subcellular compartments affects Fe localization in rice seed. Here, we discuss the characterization of a rice vacuolar Fe transporter 2 (OsVIT2) T-DNA insertion line (osvit2) and report that the knockdown of OsVIT2 and mitochondrial Fe transporter (MIT) expression affects seed Fe localization. osvit2 plants accumulated less Fe in their shoots when grown under normal or excess Fe conditions, while the accumulation of Fe was comparable to that in wild-type (WT) plants under Fe-deficient conditions. The accumulation of zinc, copper, and manganese also changed significantly in the shoots of osvit2 plants. The growth of osvit2 plants was also slow compared to that of WT plants. The concentration of Fe increased in osvit2 polished seeds. Previously, we reported that the expression of OsVIT2 was higher in MIT knockdown (mit-2) plants, and in this study, the accumulation of Fe in mit-2 seeds decreased significantly. These results suggest that vacuolar Fe trafficking is important for plant Fe homeostasis and distribution, especially in plants grown in the presence of excess Fe. Moreover, changes in the expression of OsVIT2 and MIT affect the concentration and localization of metals in brown rice as well as in polished rice seeds.

  17. CXCL5 knockdown expression inhibits human bladder cancer T24 cells proliferation and migration

    SciTech Connect

    Zheng, Jiajia; Zhu, Xi; Zhang, Jie

    2014-03-28

    Highlights: • We first demonstrated CXCL5 is highly expressed in human bladder tumor tissues and cells. • CXCL5 knockdown inhibits proliferation, migration and promotes apoptosis in T24 cells. • CXCL5 knockdown inhibits Snail, PI3K-AKT and ERK1/2 signaling pathways in T24 cells. • CXCL5 is critical for bladder tumor growth and progression. - Abstract: CXCL5 (epithelial neutrophil activating peptide-78) which acts as a potent chemoattractant and activator of neutrophil function was reported to play a multifaceted role in tumorigenesis. To investigate the role of CXCL5 in bladder cancer progression, we examined the CXCL5 expression in bladder cancer tissues by real-time PCR and Western blot, additionally, we used shRNA-mediated silencing to generate stable CXCL5 silenced bladder cancer T24 cells and defined its biological functions. Our results demonstrated that mRNA and protein of CXCL5 is increased in human bladder tumor tissues and cell lines, down-regulation of CXCL5 in T24 cells resulted in significantly decreased cell proliferation, migration and increased cell apoptosis in vitro through Snail, PI3K-AKT and ERK1/2 signaling pathways. These data suggest that CXCL5 is critical for bladder tumor growth and progression, it may represent a potential application in cancer diagnosis and therapy.

  18. Lentivirus-mediated knockdown of NLK inhibits small-cell lung cancer growth and metastasis

    PubMed Central

    Lv, Mutian; Li, Yaming; Tian, Xin; Dai, Shundong; Sun, Jing; Jin, Guojiang; Jiang, Shenyi

    2016-01-01

    Nemo-like kinase (NLK), an evolutionarily conserved serine/threonine kinase, has been recognized as a critical regulator of various cancers. In this study, we investigated the role of NLK in human small-cell lung cancer (SCLC), which is the most aggressive form of lung cancer. NLK expression was evaluated by quantitative real-time polymerase chain reaction in 20 paired fresh SCLC tissue samples and found to be noticeably elevated in tumor tissues. Lentivirus-mediated RNAi efficiently suppressed NLK expression in NCI-H446 cells, resulting in a significant reduction in cell viability and proliferation in vitro. Moreover, knockdown of NLK led to cell cycle arrest at the S-phase via suppression of Cyclin A, CDK2, and CDC25A, which could contribute to cell growth inhibition. Furthermore, knockdown of NLK decreased the migration of NCI-H446 cells and downregulated matrix metalloproteinase 9. Treatment with NLK short hairpin RNA significantly reduced SCLC tumor growth in vivo. In conclusion, this study suggests that NLK plays an important role in the growth and metastasis of SCLC and may serve as a potential therapeutic target for the treatment of SCLC. PMID:27895463

  19. NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B.

    PubMed

    Zhang, Yanke; Chen, Guojun; Gao, Baobing; Li, Yunlin; Liang, Shuli; Wang, Xiaofei; Wang, Xuefeng; Zhu, Binglin

    2016-11-23

    Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy.

  20. Inducible Knockdown of Plasmodium Gene Expression Using the glmS Ribozyme

    PubMed Central

    Prommana, Parichat; Uthaipibull, Chairat; Wongsombat, Chayaphat; Kamchonwongpaisan, Sumalee; Yuthavong, Yongyuth; Knuepfer, Ellen; Holder, Anthony A.; Shaw, Philip J.

    2013-01-01

    Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection. PMID:24023691

  1. SiRNA-mediated in vivo gene knockdown by acid-degradable cationic nanohydrogel particles.

    PubMed

    Leber, Nadine; Kaps, Leonard; Aslam, Misbah; Schupp, Jonathan; Brose, Alexander; Schäffel, David; Fischer, Karl; Diken, Mustafa; Strand, Dennis; Koynov, Kaloian; Tuettenberg, Andrea; Nuhn, Lutz; Zentel, Rudolf; Schuppan, Detlef

    2017-02-28

    Cationic nanohydrogel particles have become an attractive tool for systemic siRNA delivery, but improvement of their in vivo tolerance is desirable, especially to prevent potential long term side effects by tissue and cellular accumulation. Here, we designed novel ketal cross-linked cationic nanohydrogel particles that were assessed for reduced tissue accumulation and robust siRNA delivery in vitro and in vivo. An oligo-amine cross-linker equipped with a ketal moiety in its core was synthesized and applied to nanohydrogel cross-linking of self-assembled reactive ester block copolymers in DMSO. The resulting acid-sensitive cationic nanoparticles spontaneously disassembled over time in acidic milieu, as investigated by dynamic light scattering. Fluorescent correlation spectroscopy showed effective complexation with siRNA as well as its release upon particle degradation at endosomal pH. These properties resulted in an enhanced in vitro gene knockdown for the acid-degradable cationic nanoparticles compared to their non-degradable spermine analogues. In a murine liver fibrosis model enhanced carrier and payload accumulation in the fibrotic tissue facilitated sequence-specific gene knockdown and prevented fibrosis progression. Long-term monitoring of the carrier in the body showed an enhanced clearance for the acid-degradable carrier, even after multiple dosing. Therefore, these acid-degradable cationic nanohydrogel particles can be considered as promising siRNA carriers for in vivo purposes towards therapeutic applications. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Knockdown of Zebrafish Blood Vessel Epicardial Substance Results in Incomplete Retinal Lamination

    PubMed Central

    Chen, Ruei-Feng; Liu, Chia-Yang; Hu, Fung Rong; Huang, Chang-Jen; Wang, I-Jong

    2014-01-01

    Cell polarity during eye development determines the normal retinal lamination and differentiation of photoreceptor cells in the retina. In vertebrates, blood vessel epicardial substance (Bves) is known to play an important role in the formation and maintenance of the tight junctions essential for epithelial cell polarity. In the current study, we generated a transgenic zebrafish Bves (zbves) promoter-EGFP zebrafish line to investigate the expression pattern of Bves in the retina and to study the role of zbves in retinal lamination. Immunostaining with different specific antibodies from retinal cells and transmission electron microscopy were used to identify the morphological defects in normal and Bves knockdown zebrafish. In normal zebrafish, Bves is located at the apical junctions of embryonic retinal neuroepithelia during retinogenesis; later, it is strongly expressed around inner plexiform layer (IPL) and retinal pigment epithelium (RPE). In contrast, a loss of normal retinal lamination and cellular polarity was found with undifferentiated photoreceptor cells in Bves knockdown zebrafish. Herein, our results indicated that disruption of Bves will result in a loss of normal retinal lamination. PMID:24741362

  3. Knockdown of GPR137 by RNAi inhibits pancreatic cancer cell growth and induces apoptosis.

    PubMed

    Cui, Xianping; Liu, Yanguo; Wang, Bo; Xian, Guozhe; Liu, Xin; Tian, Xingsong; Qin, Chengkun

    2015-01-01

    G-protein-coupled receptors (GPCRs), the largest family of cell-surface molecules involved in a number of biological and pathological processes, have recently emerged as key players in carcinogenesis and cancer progression. Orphan G protein-coupled receptors (oGPCRs) are a group of proteins lacking endogenous ligands. GPR137, one of the novel oGPCR genes, was discovered by homology screening. However, the biological role of GPR137 in cancers has not yet been discussed and is of great therapeutic interest. In this study, we knocked down GPR137 via a lentivirus system in two human pancreatic cancer cell lines BXPC-3 and PANC-1. Knockdown of GPR137 strongly inhibited cell proliferation and colony formation. Flow cytometry showed that cell cycle was arrested in the sub-G1 phase and apoptotic cells were significantly increased after GPR137 knockdown. Western blotting confirmed that GPR137 silencing induced apoptosis due to cleavage of PARP (poly ADP-ribose polymerase) and upregulation of caspase 3. Furthermore, lentivirus-mediated overexpression of GPR137 promoted the proliferation of PANC-1 cells, suggesting GPR137 as a potential oncogene in pancreatic cancer cells. Taken together, our results prove the importance of GPR137 as a crucial regulator in controlling cancer cell growth and apoptosis.

  4. Raman spectroscopic study of keratin 8 knockdown oral squamous cell carcinoma derived cells

    NASA Astrophysics Data System (ADS)

    Singh, S. P.; Alam, Hunain; Dmello, Crismita; Vaidya, Milind M.; Krishna, C. Murali

    2012-03-01

    Keratins are one of most widely used markers for oral cancers. Keratin 8 and 18 are expressed in simple epithelia and perform both mechanical and regulatory functions. Their expression are not seen in normal oral tissues but are often expressed in oral squamous cell carcinoma. Aberrant expression of keratins 8 and 18 is most common change in human oral cancer. Optical-spectroscopic methods are sensitive to biochemical changes and being projected as novel diagnostic tools for cancer diagnosis. Aim of this study was to evaluate potentials of Raman spectroscopy in detecting minor changes associated with differential level of keratin expression in tongue-cancer-derived AW13516 cells. Knockdown clones for K8 were generated and synchronized by growing under serum-free conditions. Cell pellets of three independent experiments in duplicate were used for recording Raman spectra with fiberoptic-probe coupled HE-785 Raman-instrument. A total of 123 and 96 spectra from knockdown clones and vector controls respectively in 1200-1800 cm-1 region were successfully utilized for classification using LDA. Two separate clusters with classification-efficiency of ~95% were obtained. Leave-one-out cross-validation yielded ~63% efficiency. Findings of the study demonstrate the potentials of Raman spectroscopy in detecting even subtle changes such as variations in keratin expression levels. Future studies towards identifying Raman signals from keratin in oral cells can help in precise cancer diagnosis.

  5. Disrupted Junctional Membrane Complexes and Hyperactive Ryanodine Receptors Following Acute Junctophilin Knockdown in Mice

    PubMed Central

    van Oort, Ralph J.; Garbino, Alejandro; Wang, Wei; Dixit, Sayali S.; Landstrom, Andrew P.; Gaur, Namit; De Almeida, Angela C.; Skapura, Darlene G.; Rudy, Yoram; Burns, Alan R.; Ackerman, Michael J.; Wehrens, Xander H.T.

    2011-01-01

    Background Excitation-contraction coupling in striated muscle requires proper communication of plasmalemmal voltage-activated Ca2+ channels and Ca2+ release channels on sarcoplasmic reticulum (SR) within junctional membrane complexes (JMCs). Whereas previous studies revealed a loss of JMCs and embryonic lethality in germ-line junctophilin-2 (JPH2) knockout mice, it has remained unclear whether JPH2 plays an essential role in JMC formation and the Ca2+-induced Ca2+ release process in the heart. Our recent work demonstrated loss-of-function mutations in JPH2 in patients with hypertrophic cardiomyopathy. Methods and Results To elucidate the role of JPH2 in the heart, we developed a novel approach to conditionally reduce JPH2 protein levels using RNA interference. Cardiac-specific JPH2 knockdown resulted in impaired cardiac contractility, which caused heart failure and increased mortality. JPH2 deficiency resulted in loss of excitation-contraction coupling gain, precipitated by a reduction in the number of JMCs and increased variability in the plasmalemma-SR distance. Conclusions Loss of JPH2 had profound effects on Ca2+ release channel inactivation, suggesting a novel functional role for JPH2 in regulating intracellular Ca2+ release channels in cardiac myocytes. Thus, our novel approach of cardiac-specific shRNA-mediated knockdown of junctophilin-2 has uncovered a critical role for junctophilin in intracellular Ca2+ release in the heart. PMID:21339484

  6. Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome.

    PubMed

    Narayanasamy, Suresh K; Simpson, David C; Martin, Ian; Grotewiel, Mike; Gronert, Scott

    2014-11-01

    Exposure to Paraquat and RNA interference knockdown of mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the 20 canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS-based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both Western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS-based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36).

  7. Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs.

    PubMed

    Wang, Xue; Tan, Jiying; Zhao, Jiangqin; Ragupathy, Viswannath; Haleyurgirisetty, Mohan; Hewlett, Indira

    2014-08-01

    Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.

  8. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells.

    PubMed

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forte, Trudy M; Ryan, Robert O

    2015-08-21

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry of HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid.

  9. Exogenous cardiolipin localizes to mitochondria and prevents TAZ knockdown-induced apoptosis in myeloid progenitor cells

    PubMed Central

    Ikon, Nikita; Su, Betty; Hsu, Fong-Fu; Forteand, Trudy M.; Ryan, Robert O.

    2015-01-01

    The concentration and composition of cardiolipin (CL) in mitochondria are altered in age-related heart disease, Barth Syndrome, and other rare genetic disorders, resulting in mitochondrial dysfunction. To explore whether exogenous CL can be delivered to cells, CL was combined with apolipoprotein A-I to generate water-soluble, nanoscale complexes termed nanodisks (ND). Mass spectrometry HL60 myeloid progenitor cell extracts revealed a 30-fold increase in cellular CL content following incubation with CL-ND. When CL-ND containing a fluorescent CL analogue was employed, confocal microscopy revealed CL localization to mitochondria. The ability of CL-ND to elicit a physiological response was examined in an HL60 cell culture model of Barth Syndrome neutropenia. siRNA knockdown of the phospholipid transacylase, tafazzin (TAZ), induced apoptosis in these cells. When TAZ knockdown cells were incubated with CL-ND, the apoptotic response was attenuated. Thus, CL-ND represent a potential intervention strategy for replenishment of CL in Barth Syndrome, age-related heart disease, and other disorders characterized by depletion of this key mitochondrial phospholipid. PMID:26164234

  10. Expression profiles of genes in DJ-1-knockdown and L 166 P DJ-1 mutant cells.

    PubMed

    Nishinaga, Hiromi; Takahashi-Niki, Kazuko; Taira, Takahiro; Andreadis, Athena; Iguchi-Ariga, Sanae M M; Ariga, Hiroyoshi

    2005-12-16

    DJ-1 is a novel oncogene and a causative gene for the familial form of Parkinson's disease (PD). DJ-1 has been shown to play roles in anti-oxidative stress by eliminating reactive oxygen species and in transcriptional regulation of genes. Loss of these functions of DJ-1 is thought to trigger the onset of PD. In this study, to identify genes for which expressions are regulated by DJ-1, DNA microarray analyses were carried out using two mouse NIH3T3 cell lines, DJ-1-knockdown cells and cells harboring an exogenously added L 166 P DJ-1 mutant found in PD patients. In both cell lines, drastic changes in expressions of genes, including genes related to stress, apoptosis, oxidative stress and neurotoxicity, were observed and changes in expressions were confirmed by RT-PCR. Of the genes identified, expression level of the extracellular superoxide dismutase (SOD 3) gene was found to decrease in DJ-1-knockdown cells, while expressions of SOD 1 and SOD 2 genes did not change. Furthermore, expression of the tau gene, a gene whose product gives cells neurotoxicity by aggregation, was found to increase at its promoter level in L 166 P DJ-1 cells. These findings suggest that DJ-1 regulates expressions of genes for which functions are thought to be related to cell death or neurodegeneration.

  11. Paraquat exposure and Sod2 knockdown have dissimilar impacts on the Drosophila melanogaster carbonylated protein proteome

    PubMed Central

    Narayanasamy, Suresh K.; Simpson, David C.; Martin, Ian; Grotewiel, Mike; Gronert, Scott

    2014-01-01

    Exposure to Paraquat and RNA interference knockdown of Mn or mitochondrial superoxide dismutase (Sod2) are known to result in significant lifespan reduction, locomotor dysfunction, and mitochondrial degeneration in Drosophila melanogaster. Both perturbations increase the flux of the progenitor ROS, superoxide, but the molecular underpinnings of the resulting phenotypes are poorly understood. Improved understanding of such processes could lead to advances in the treatment of numerous age-related disorders. Superoxide toxicity can act through protein carbonylation. Analysis of carbonylated proteins is attractive since carbonyl groups are not present in the twenty canonical amino acids and are amenable to labeling and enrichment strategies. Here, carbonylated proteins were labeled with biotin hydrazide and enriched on streptavidin beads. On-bead digestion was used to release carbonylated protein peptides, with relative abundance ratios versus controls obtained using the iTRAQ MS-based proteomics approach. Western blotting and biotin quantitation assay approaches were also investigated. By both western blotting and proteomics, Paraquat exposure, but not Sod2 knockdown, resulted in increased carbonylated protein relative abundance. For Paraquat exposure versus control, the median carbonylated protein relative abundance ratio (1.53) determined using MS-based proteomics was in good agreement with that obtained using a commercial biotin quantitation kit (1.36). PMID:25091824

  12. Focal adhesion kinase knockdown modulates the response of human corneal epithelial cells to topographic cues.

    PubMed

    Dreier, Britta; Raghunathan, Vijaya Krishna; Russell, Paul; Murphy, Christopher J

    2012-12-01

    A rapidly expanding literature broadly documents the impact of biophysical cues on cellular behaviors. In spite of increasing research efforts in this field, the underlying signaling processes are poorly understood. One of the candidate molecules for being involved in mechanotransduction is focal adhesion kinase (FAK). To examine the role of FAK in the response of immortalized human corneal epithelial (hTCEpi) cells to topographic cues, FAK was depleted by siRNA transfection. Contrary to expectations, FAK knockdown resulted in an enhanced response with a greater number of hTCEpi cells aligned to the long axis of anisotropically ordered surface ridges and grooves. Both underlying topographic features and FAK depletion modulated the migration of corneal epithelial cells. The impact of FAK knockdown on both migration and alignment varied depending on the topographic cues to which the cells were exposed, with the most significant change observed on the biologically relevant size scale (400nm). Additionally, a change in expression of genes encoding perinuclear Nesprins 1 and 2 (SYNE1, 2) was observed in response to topographic cues. SYNE1/2 expression was also altered by FAK depletion, suggesting that these proteins might represent a link between cytosolic and nuclear signaling processes. The data presented here have relevance to our understanding of the fundamental processes involved in corneal cell behavior to topographic cues. These results highlight the importance of incorporating biophysical cues in the conduction of in vitro studies and into the design and fabrication of implantable prosthetics.

  13. A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA.

    PubMed

    Suryawanshi, Hemant; Sarangdhar, Mayuresh Anant; Vij, Manika; Roshan, Reema; Singh, Vijay Pal; Ganguli, Munia; Pillai, Beena

    2015-12-26

    MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

  14. Knockdown mortality, repellency, and residual effects of insecticides for control of adult Bactericera cockerelli (Hemiptera: Psyllidae).

    PubMed

    Gharalari, A H; Nansen, C; Lawson, D S; Gilley, J; Munyaneza, J E; Vaughn, K

    2009-06-01

    The psyllid Bactericera cockerelli (Sulc) (Hemiptera: Psyllidae) is the vector of a bacterial pathogen causing zebra chip (ZC) disease in potato, Solanum tuberosum L. (Solanaceae). Recently, ZC has become important to potato growers and the potato chipping industry in the southwestern United States, Mexico, Central America, and New Zealand. In the current study, we evaluated the knockdown effect of 12 insecticides against adult B. cockerelli, and we examined the effects of treating potato leaves: top side only, bottom side only, or both sides. Within 48 h of exposure, abamectin showed the highest and most consistent rate of adult B. cockerelli mortality and a dosage response to three dosages within labeled field rates. Choice tests conducted in petri dishes, involving untreated potato leaves and leaves treated with abamectin, revealed that abamectin did not affect host preference of adult B. cockerelli. A residual test under field conditions revealed that, although highly effective immediately after application, abamectin-effect was nonsignificantly different from control treatments 24 and 48 h after field application. Higher adult B. cockerelli mortality was recorded from potato plants treated with ground application compared with aerial 48 h after application. Our results indicated that abamectin has a high knockdown effect on adult B. cockerelli and that it can be used effectively in insecticide rotation programs against adult B. cockerelli and ZC control in potatoes.

  15. NR4A1 Knockdown Suppresses Seizure Activity by Regulating Surface Expression of NR2B

    PubMed Central

    Zhang, Yanke; Chen, Guojun; Gao, Baobing; Li, Yunlin; Liang, Shuli; Wang, Xiaofei; Wang, Xuefeng; Zhu, Binglin

    2016-01-01

    Nuclear receptor subfamily 4 group A member 1 (NR4A1), a downstream target of CREB that is a key regulator of epileptogenesis, has been implicated in a variety of biological processes and was previously identified as a seizure-associated molecule. However, the relationship between NR4A1 and epileptogenesis remains unclear. Here, we showed that NR4A1 protein was predominantly expressed in neurons and up-regulated in patients with epilepsy as well as pilocarpine-induced mouse epileptic models. NR4A1 knockdown by lentivirus transfection (lenti-shNR4A1) alleviated seizure severity and prolonged onset latency in mouse models. Moreover, reciprocal coimmunoprecipitation of NR4A1 and NR2B demonstrated their interaction. Furthermore, the expression of p-NR2B (Tyr1472) in epileptic mice and the expression of NR2B in the postsynaptic density (PSD) were significantly reduced in the lenti-shNR4A1 group, indicating that NR4A1 knockdown partly decreased surface NR2B by promoting NR2B internalization. These results are the first to indicate that the expression of NR4A1 in epileptic brain tissues may provide new insights into the molecular mechanisms underlying epilepsy. PMID:27876882

  16. Knockdown of Mediator Complex Subunit 19 Suppresses the Growth and Invasion of Prostate Cancer Cells

    PubMed Central

    Zhao, Hongwei; Lv, Wei; Chen, Jian; Wan, Fengchun; Liu, Dongfu; Gao, Zhenli; Wu, Jitao

    2017-01-01

    Prostate cancer (PCa) is one of the most common cancers in elderly men. Mediator Complex Subunit 19 (Med19) is overexpressed and plays promotional roles in many cancers. However, the roles of Med19 in PCa are still obscure. In this study, by using immunohistochemical staining, we found higher expression level of Med19 in PCa tissues than in adjacent benign prostate tissues. We then knocked down the Med19 expression in PCa cell lines LNCaP and PC3 by using lentivirus siRNA. Cell proliferation, anchor-independent growth, migration, and invasion were suppressed in Med19 knockdown PCa cells. In nude mice xenograft model, we found that Med19 knockdown PCa cells formed smaller tumors with lower proliferation index than did control cells. In the mechanism study, we found that Med19 could regulate genes involved in cell proliferation, cell cycle, and epithelial-mesenchymal transition, including P27, pAKT, pPI3K, IGF1R, E-Cadherin, N-Cadherin, Vimentin, ZEB2, Snail-1 and Snail-2. Targeting Med19 in PCa cells could inhibit the PCa growth and metastasis, and might be a therapeutic option for PCa in the future. PMID:28125713

  17. Novel siRNA formulation to effectively knockdown mutant p53 in osteosarcoma.

    PubMed

    Kundu, Anup K; Iyer, Swathi V; Chandra, Sruti; Adhikari, Amit S; Iwakuma, Tomoo; Mandal, Tarun K

    2017-01-01

    The tumor suppressor p53 plays a crucial role in the development of osteosarcoma. The primary objective of this study is to develop and optimize lipid based nanoparticle formulations that can carry siRNA and effectively silence mutant p53 in 318-1, a murine osteosarcoma cell line. The nanoparticles were composed of a mixture of two lipids (cholesterol and DOTAP) and either PLGA or PLGA-PEG and prepared by using an EmulsiFlex-B3 high pressure homogenizer. A series of studies that include using different nanoparticles, different amount of siRNAs, cell numbers, incubation time, transfection media volume, and storage temperature was performed to optimize the gene silencing efficiency. Replacement of lipids by PLGA or PLGA-PEG decreased the particle size and overall cytotoxicity. Among all lipid-polymer nanoformulations, nanoparticles with 10% PLGA showed highest mutant p53 knockdown efficiency while maintaining higher cell viability when a nanoparticle to siRNA ratio equal to 6.8:0.66 and 75 nM siRNA was used. With long term storage the mutant p53 knockdown efficiency decreased to a greater extent. This study warrants a future evaluation of this formulation for gene silencing efficiency of mutant p53 in tissue culture and animal models for the treatment of osteosarcoma.

  18. Gene expression profiling of NB4 cells following knockdown of nucleostemin using DNA microarrays.

    PubMed

    Sun, Xiaoli; Jia, Yu; Wei, Yuanyu; Liu, Shuai; Yue, Baohong

    2016-07-01

    Nucleostemin (NS) is mainly expressed in stem and tumor cells, and is necessary for the maintenance of their self-renewal and proliferation. Originally, NS was thought to exert its effects through inhibiting p53, while recent studies have revealed that NS is also able to function independently of p53. The present study performed a gene expression profiling analysis of p53‑mutant NB4 leukeima cells following knockdown of NS in order to elucidate the p53‑independent NS pathway. NS expression was silenced using lentivirus‑mediated RNA interference technology, and gene expression profiling of NB4 cells was performed by DNA microarray analysis. A total of 1,953 genes were identified to be differentially expressed (fold change ≥2 or ≤0.5) following knockdown of NS expression. Furthermore, reverse‑transcription quantitative polymerase chain reaction analysis was used to detect the expression of certain candidate genes, and the results were in agreement with the micaroarray data. Pathway analysis indicated that aberrant genes were enhanced in endoplasmic, c‑Jun N‑terminal kinase and mineral absorption pathways. The present study shed light on the mechanisms of the p54‑independent NS pathway in NB4 cells and provided a foundation for the discovery of promising targets for the treatment of p53-mutant leukemia.

  19. Zebrafish ambra1a and ambra1b Knockdown Impairs Skeletal Muscle Development

    PubMed Central

    Skobo, Tatjana; Benato, Francesca; Grumati, Paolo; Meneghetti, Giacomo; Cianfanelli, Valentina; Castagnaro, Silvia; Chrisam, Martina; Di Bartolomeo, Sabrina; Bonaldo, Paolo; Cecconi, Francesco; Valle, Luisa Dalla

    2014-01-01

    The essential role of autophagy in muscle homeostasis has been clearly demonstrated by phenotype analysis of mice with muscle-specific inactivation of genes encoding autophagy-related proteins. Ambra1 is a key component of the Beclin 1 complex and, in zebrafish, it is encoded by two paralogous genes, ambra1a and ambra1b, both required for normal embryogenesis and larval development. In this study we focused on the function of Ambra1, a positive regulator of the autophagic process, during skeletal muscle development by means of morpholino (MO)-mediated knockdown and compared the phenotype of zebrafish Ambra1-depleted embryos with that of Ambra1gt/gt mouse embryos. Morphological analysis of zebrafish morphant embryos revealed that silencing of ambra1 impairs locomotor activity and muscle development, as well as myoD1 expression. Skeletal muscles in ATG-morphant embryos displayed severe histopathological changes and contained only small areas of organized myofibrils that were widely dispersed throughout the cell. Double knockdown of ambra1a and ambra1b resulted in a more severe phenotype whereas defects were much less evident in splice-morphants. The morphants phenotypes were effectively rescued by co-injection with human AMBRA1 mRNA. Together, these results indicate that ambra1a and ambra1b are required for the correct development and morphogenesis of skeletal muscle. PMID:24922546

  20. SMARCAD1 knockdown uncovers its role in breast cancer cell migration, invasion, and metastasis.

    PubMed

    Al Kubaisy, Elham; Arafat, Kholoud; De Wever, Olivier; Hassan, Ahmed H; Attoub, Samir

    2016-09-01

    Breast cancer is the most common cancer seen in women worldwide and breast cancer patients are at high risk of recurrence in the form of metastatic disease. Identification of genes associated with invasion and metastasis is crucial in order to develop novel anti-metastasis targeted therapy. It has been demonstrated that the DEAD-BOX helicase DP103 was implicated in breast cancer invasion and metastasis. SMARCAD1 is also a DEAD/H box-containing helicase, suggested to play a role in genetic instability. However, its involvement in cancer migration, invasion, and metastasis has never been explored. Using two different designs of shRNA targeting SMARCAD1, we investigated the impact of SMARCAD1 knockdown on the migration, invasion, and metastasis potential of the breast cancer cells MDA-MB-231 and T47D. We observed that SMARCAD1 knockdown in the invasive breast cancer cells MDA-MB-231, unlike in the non-invasive breast cancer cells T47D, was associated with an increased cell-cell adhesion and a significant decrease in cell migration, invasion, and metastasis due at least in part to a strong inhibition of STAT3 phosphorylation. These results indicate that SMARCAD1 is involved in breast cancer metastasis and can be a promising target for metastatic breast cancer therapy.

  1. Kir6.1 knockdown aggravates cerebral ischemia/reperfusion-induced neural injury in mice.

    PubMed

    Dong, Yin-Feng; Wang, Lin-Xiao; Huang, Xu; Cao, Wen-Jing; Lu, Ming; Ding, Jian-Hua; Sun, Xiu-Lan; Hu, Gang

    2013-08-01

    ATP-sensitive potassium (K-ATP) channels couple energy metabolism with electric activity, which play important roles in brain diseases including stroke. However, the impacts of Kir6.1-containing K-ATP channels that mainly expressed on glia in stroke remain unclear. In this study, we found that expression of Kir6.1 was significantly decreased in the ischemic brain area of C57BL/6J mice after 1-h middle cerebral artery occlusion (MCAO) and 24-h reperfusion. Then, we subjected Kir6.1 heterozygote knockout (Kir6.1(+/-) ) mice to cerebral ischemia/reperfusion (I/R) injury and found that Kir6.1(+/-) mice exhibited exacerbated neurological disorder and enlarged infarct size, companied by glial over-activation and blood-brain barrier (BBB) damages. Furthermore, we showed that Kir6.1 knockdown aggravated endoplasmic reticulum (ER) stress and thereby increased the levels of proinflammatory factors tumor necrosis factor-α and interleukin-1β (TNF-α and IL-1β) in mouse brain. Our findings reveal that Kir6.1 knockdown exacerbates cerebral I/R-induced brain damages via increasing ER stress and inflammatory response, indicating that Kir6.1-containing K-ATP channels may be a potential therapeutic target for stroke. © 2013 John Wiley & Sons Ltd.

  2. Acute sterol o-acyltransferase 2 (SOAT2) knockdown rapidly mobilizes hepatic cholesterol for fecal excretion.

    PubMed

    Marshall, Stephanie M; Gromovsky, Anthony D; Kelley, Kathryn L; Davis, Matthew A; Wilson, Martha D; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Rudel, Lawrence L; Brown, J Mark; Temel, Ryan E

    2014-01-01

    The primary risk factor for atherosclerotic cardiovascular disease is LDL cholesterol, which can be reduced by increasing cholesterol excretion from the body. Fecal cholesterol excretion can be driven by a hepatobiliary as well as a non-biliary pathway known as transintestinal cholesterol efflux (TICE). We previously showed that chronic knockdown of the hepatic cholesterol esterifying enzyme sterol O-acyltransferase 2 (SOAT2) increased fecal cholesterol loss via TICE. To elucidate the initial events that stimulate TICE, C57Bl/6 mice were fed a high cholesterol diet to induce hepatic cholesterol accumulation and were then treated for 1 or 2 weeks with an antisense oligonucleotide targeting SOAT2. Within 2 weeks of hepatic SOAT2 knockdown (SOAT2HKD), the concentration of cholesteryl ester in the liver was reduced by 70% without a reciprocal increase in hepatic free cholesterol. The rapid mobilization of hepatic cholesterol stores resulted in a ∼ 2-fold increase in fecal neutral sterol loss but no change in biliary cholesterol concentration. Acute SOAT2HKD increased plasma cholesterol carried primarily in lipoproteins enriched in apoB and apoE. Collectively, our data suggest that acutely reducing SOAT2 causes hepatic cholesterol to be swiftly mobilized and packaged onto nascent lipoproteins that feed cholesterol into the TICE pathway for fecal excretion.

  3. Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection.

    PubMed

    Bredow, Melissa; Vanderbeld, Barbara; Walker, Virginia K

    2016-01-01

    Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first 'IBP-knockdowns'. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03-0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to -10°C, showing a 13-22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to -8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops.

  4. Long Noncoding RNA-Sox2OT Knockdown Alleviates Diabetes Mellitus-Induced Retinal Ganglion Cell (RGC) injury.

    PubMed

    Li, Chao-Peng; Wang, Shu-Hong; Wang, Wen-Qi; Song, Shu-Guang; Liu, Xiu-Ming

    2017-03-01

    Retinal ganglion cell (RGC) injury is one of the important pathological features of diabetes-induced retinal neurodegeneration. Increasing attention has been paid to find strategies for protecting against RGC injury. Long noncoding RNAs (lncRNAs) have emerged as the key regulators of many cell functions. Here, we show that Sox2OT expression is significantly down-regulated in the retinas of STZ-induced diabetic mice and in the RGCs upon high glucose or oxidative stress. SOX2OT knockdown protects RGCs against high glucose-induced injury in vitro. Moreover, Sox2OT knockdown plays a neuroprotective role in diabetes-related retinal neurodegeneration in vivo. Sox2OT knockdown could regulate oxidative stress response in RGCs and diabetic mouse retinas. Sox2OT knockdown plays an anti-oxidative role via regulating NRF2/HO-1 signaling activity. Taken together, Sox2OT knockdown may be a therapeutic strategy for the prevention and treatment of diabetes-induced retinal neurodegeneration.

  5. The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster.

    PubMed

    Fedotov, Sergey A; Bragina, Julia V; Besedina, Natalia G; Danilenkova, Larisa V; Kamysheva, Elena A; Panova, Anna A; Kamyshev, Nikolai G

    2014-01-01

    Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination.

  6. The effect of neurospecific knockdown of candidate genes for locomotor behavior and sound production in Drosophila melanogaster

    PubMed Central

    Fedotov, Sergey A; Bragina, Julia V; Besedina, Natalia G; Danilenkova, Larisa V; Kamysheva, Elena A; Panova, Anna A; Kamyshev, Nikolai G

    2014-01-01

    Molecular mechanisms underlying the functioning of central pattern generators (CPGs) are poorly understood. Investigations using genetic approaches in the model organism Drosophila may help to identify unknown molecular players participating in the formation or control of motor patterns. Here we report Drosophila genes as candidates for involvement in the neural mechanisms responsible for motor functions, such as locomotion and courtship song. Twenty-two Drosophila lines, used for gene identification, were isolated from a previously created collection of 1064 lines, each carrying a P element insertion in one of the autosomes. The lines displayed extreme deviations in locomotor and/or courtship song parameters compared with the whole collection. The behavioral consequences of CNS-specific RNAi-mediated knockdowns for 10 identified genes were estimated. The most prominent changes in the courtship song interpulse interval (IPI) were seen in flies with Sps2 or CG15630 knockdown. Glia-specific knockdown of these genes produced no effect on the IPI. Estrogen-induced knockdown of CG15630 in adults reduced the IPI. The product of the CNS-specific gene, CG15630 (a predicted cell surface receptor), is likely to be directly involved in the functioning of the CPG generating the pulse song pattern. Future studies should ascertain its functional role in the neurons that constitute the song CPG. Other genes (Sps2, CG34460), whose CNS-specific knockdown resulted in IPI reduction, are also worthy of detailed examination. PMID:25494872

  7. Knockdown of embryonic myosin heavy chain reveals an essential role in the morphology and function of the developing heart

    PubMed Central

    Rutland, Catrin Sian; Polo-Parada, Luis; Ehler, Elisabeth; Alibhai, Aziza; Thorpe, Aaran; Suren, Suganthi; Emes, Richard D.; Patel, Bhakti; Loughna, Siobhan

    2011-01-01

    The expression and function of embryonic myosin heavy chain (eMYH) has not been investigated within the early developing heart. This is despite the knowledge that other structural proteins, such as alpha and beta myosin heavy chains and cardiac alpha actin, play crucial roles in atrial septal development and cardiac function. Most cases of atrial septal defects and cardiomyopathy are not associated with a known causative gene, suggesting that further analysis into candidate genes is required. Expression studies localised eMYH in the developing chick heart. eMYH knockdown was achieved using morpholinos in a temporal manner and functional studies were carried out using electrical and calcium signalling methodologies. Knockdown in the early embryo led to abnormal atrial septal development and heart enlargement. Intriguingly, action potentials of the eMYH knockdown hearts were abnormal in comparison with the alpha and beta myosin heavy chain knockdowns and controls. Although myofibrillogenesis appeared normal, in knockdown hearts the tissue integrity was affected owing to apparent focal points of myocyte loss and an increase in cell death. An expression profile of human skeletal myosin heavy chain genes suggests that human myosin heavy chain 3 is the functional homologue of the chick eMYH gene. These data provide compelling evidence that eMYH plays a crucial role in important processes in the early developing heart and, hence, is a candidate causative gene for atrial septal defects and cardiomyopathy. PMID:21862559

  8. Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity.

    PubMed

    Chao, Pei-Ting; Yang, Liang; Aja, Susan; Moran, Timothy H; Bi, Sheng

    2011-05-04

    Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  9. Identification of Genetic Suppressors of the Sin3A Knockdown Wing Phenotype

    PubMed Central

    Fox, Stephanie; Gammouh, Sarah; Pile, Lori A.

    2012-01-01

    The role of the Sin3A transcriptional corepressor in regulating the cell cycle is established in various metazoans. Little is known, however, about the signaling pathways that trigger or are triggered by Sin3A function. To discover genes that work in similar or opposing pathways to Sin3A during development, we have performed an unbiased screen of deficiencies of the Drosophila third chromosome. Additionally, we have performed a targeted loss of function screen to identify cell cycle genes that genetically interact with Sin3A. We have identified genes that encode proteins involved in regulation of gene expression, signaling pathways and cell cycle that can suppress the curved wing phenotype caused by the knockdown of Sin3A. These data indicate that Sin3A function is quite diverse and impacts a wide variety of cellular processes. PMID:23166712

  10. Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice.

    PubMed

    Tasfaout, Hichem; Buono, Suzie; Guo, Shuling; Kretz, Christine; Messaddeq, Nadia; Booten, Sheri; Greenlee, Sarah; Monia, Brett P; Cowling, Belinda S; Laporte, Jocelyn

    2017-06-07

    Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease.

  11. Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

    PubMed

    Sheta, Razan; Roux-Dalvai, Florence; Woo, Christina M; Fournier, Frédéric; Bourassa, Sylvie; Bertozzi, Carolyn R; Droit, Arnaud; Bachvarov, Dimcho

    2016-09-01

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

  12. RNAi knockdown of redox signaling protein Ape1 in the differentiation of mouse embryonic stem cells.

    PubMed

    Zou, Gang-Ming; Lebron, Cynthia; Fu, Yumei

    2010-01-01

    Murine embryonic stem cells (ES) are pluripotent cells and have the potential to become a wide variety of specialized cell types. Mouse ES cell differentiation can be regarded as a valuable biological tool that has led to major advances in our understanding of cell and developmental biology. In vitro differentiation of mouse ES cells can be directed to a specific lineage formation, such as hematopoietic lineage, by appropriate cytokine and/or growth factor stimulation. To study specific gene function in early developmental events, gene knockout approaches have been traditionally used; however, this is a time-consuming and expensive approach. Recently, we have shown that siRNA is an effective strategy to knockdown target gene expression, such as Ape1, during ES cell differentiation, and consequently, one can alter cell fates in ES-derived differentiated cells. This approach will be applicable to test the function of a wide variety of gene products using the ES cell differentiation system.

  13. Ets2 knockdown inhibits tumorigenesis in esophageal squamous cell carcinoma in vivo and in vitro

    PubMed Central

    Zhu, Liqiang; Zhang, Yanting; Ma, Shanshan; Zhang, Kun; Yang, Bo; Guan, Fangxia

    2016-01-01

    Increased expression of Ets2 is reported upregulated in esophageal squamous cell carcinoma tissue. However, the function of Ets2 in carcinogenesis of ESCC is poorly understood. Here, the rise of Ets2 was confirmed in ESCC cells and Ets2 depletion by RNA interference promotes cell apoptosis, inhibits cell proliferation, attenuates cell invasion and induces cell cycle G0/G1 arrest in vitro. Moreover, in vivo, Xenograft mouse model studies showed Ets2 knockdown inhibits tumor formation and metastasis significantly. Furthermore, Ets2 depletion inactivates the mTOR/p70S6K signaling pathway both in vitro and in vivo. Taken together, these findings strongly suggest that a critical role of Ets2 in human ESCC pathogenesis via the inactivation of the mTOR/p70S6K signaling pathway. PMID:27556183

  14. Knockdown of Maternal Homeobox Transcription Factor SEBOX Gene Impaired Early Embryonic Development in Porcine Parthenotes

    PubMed Central

    ZHENG, Zhong; ZHAO, Ming-Hui; JIA, Jia-Lin; HEO, Young-Tae; CUI, Xiang-Shun; OH, Jeong Su; KIM, Nam-Hyung

    2013-01-01

    Abstract A number of germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, the role of these factors during early embryonic development has been poorly explored. In the present study, we investigated the role of SEBOX, a maternal homeobox transcription factor, during early embryonic development in porcine parthenotes. mRNA for SEBOX is preferentially expressed in oocytes, and expression persists until embryonic genome activation (EGA). Knockdown of SEBOX by siRNA disrupted early embryonic development, but not oocyte maturation. Many maternal genes essential for early embryonic development were upregulated in SEBOX-depleted embryos. Moreover, some pluripotency-associated genes, including SOX2 and NANOG, were upregulated when SEBOX was knocked down. Therefore, our data demonstrate that SEBOX is required for early embryonic development in pigs and appears to regulate the degradation of maternal transcripts and the expression of pluripotency genes. PMID:24018616

  15. Knockdown of maternal homeobox transcription factor SEBOX gene impaired early embryonic development in porcine parthenotes.

    PubMed

    Zheng, Zhong; Zhao, Ming-Hui; Jia, Jia-Lin; Heo, Young-Tae; Cui, Xiang-Shun; Oh, Jeong Su; Kim, Nam-Hyung

    2013-12-17

    A number of germ cell-specific transcription factors essential for ovarian formation and folliculogenesis have been identified and studied. However, the role of these factors during early embryonic development has been poorly explored. In the present study, we investigated the role of SEBOX, a maternal homeobox transcription factor, during early embryonic development in porcine parthenotes. mRNA for SEBOX is preferentially expressed in oocytes, and expression persists until embryonic genome activation (EGA). Knockdown of SEBOX by siRNA disrupted early embryonic development, but not oocyte maturation. Many maternal genes essential for early embryonic development were upregulated in SEBOX-depleted embryos. Moreover, some pluripotency-associated genes, including SOX2 and NANOG, were upregulated when SEBOX was knocked down. Therefore, our data demonstrate that SEBOX is required for early embryonic development in pigs and appears to regulate the degradation of maternal transcripts and the expression of pluripotency genes.

  16. Effect of {beta}4 integrin knockdown by RNA interference in anaplastic thyroid carcinoma.

    PubMed

    Noh, Tae Woong; Soung, Young Hwa; Kim, Hong Im; Gil, Hyea Jin; Kim, Jeong Mo; Lee, Eun Jig; Chung, Jun

    2010-11-01

    Integrin α6β4 is a known tumor antigen; however, its function in different subtypes of thyroid cancer is not known. This study reports that α6β4 expression is selectively up-regulated in anaplastic thyroid cancer (ATC) cells, the most malignant subtype of human thyroid cancer. To assess the contribution of α6β4 in ATC progression, cell proliferation, motility and soft agar assay were performed in vitro and a xenograft tumor growth assay was performed in vivo. Knockdown of β4 integrin subunit expression by shRNA in ATC cells reduced the proliferation, migration, and anchorage-independent growth of ATC cells in vitro and xenograft tumor growth in vivo. These data suggest that integrin α6β4 contributes to the development of aggressive forms of thyroid cancer with poor prognostic potential, such as ATC, and thus may be a novel therapeutic target for the treatment for this subtype of thyroid cancer.

  17. RIG-I knockdown impedes neurogenesis in a murine model of Japanese encephalitis.

    PubMed

    Mukherjee, Sriparna; Ghosh, Sourish; Nazmi, Arshed; Basu, Anirban

    2015-02-01

    Retinoic acid inducible gene I (RIG-I) is a well established pattern recognition receptor (PRR) in neurons infected with Japanese encephalitis virus (JEV) as reported previously from our laboratory. Japanese encephalitis (JE) virus infection in brain has been shown to decrease the proliferation of neural stem/progenitor cells (NSPCs) which has its implications in neurological sequelae in JE survivors. We have found that ablation of RIG-I both in vivo and in vitro models results in significant decrease in NSPC proliferation post JEV infection. We hypothesize that knockdown of RIG-I diminishes the expression of antiviral molecules resulting in an increase in viral replication, which in turn results in enhancement of the expression of cell cycle inhibitors, hence affecting the proliferation of NSPCs. © 2014 International Federation for Cell Biology.

  18. Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice

    PubMed Central

    Tasfaout, Hichem; Buono, Suzie; Guo, Shuling; Kretz, Christine; Messaddeq, Nadia; Booten, Sheri; Greenlee, Sarah; Monia, Brett P.; Cowling, Belinda S.; Laporte, Jocelyn

    2017-01-01

    Centronuclear myopathies (CNM) are non-dystrophic muscle diseases for which no effective therapy is currently available. The most severe form, X-linked CNM, is caused by myotubularin 1 (MTM1) loss-of-function mutations, while the main autosomal dominant form is due to dynamin2 (DNM2) mutations. We previously showed that genetic reduction of DNM2 expression in Mtm1 knockout (Mtm1KO) mice prevents development of muscle pathology. Here we show that systemic delivery of Dnm2 antisense oligonucleotides (ASOs) into Mtm1KO mice efficiently reduces DNM2 protein level in muscle and prevents the myopathy from developing. Moreover, systemic ASO injection into severely affected mice leads to reversal of muscle pathology within 2 weeks. Thus, ASO-mediated DNM2 knockdown can efficiently correct muscle defects due to loss of MTM1, providing an attractive therapeutic strategy for this disease. PMID:28589938

  19. Stable SET knockdown in breast cell carcinoma inhibits cell migration and invasion.

    PubMed

    Li, Jie; Yang, Xi-fei; Ren, Xiao-hu; Meng, Xiao-jing; Huang, Hai-yan; Zhao, Qiong-hui; Yuan, Jian-hui; Hong, Wen-xu; Xia, Bo; Huang, Xin-feng; Zhou, Li; Liu, Jian-jun; Zou, Fei

    2014-10-10

    Breast cancer is the most malignant tumor for women, however, the mechanisms underlying this devastating disease remain unclear. SET is an endogenous inhibitor of protein phosphatase 2A (PP2A) and involved in many physiological and pathological processes. SET could promote the occurrence of tumor through inhibiting PP2A. In this study, we explore the role of SET in the migration and invasion of breast cancer cells MDA-MB-231 and ZR-75-30. The stable suppression of SET expression through lentivirus-mediated RNA interference (RNAi) was shown to inhibit the growth, migration and invasion of breast cancer cells. Knockdown of SET increases the activity and expression of PP2Ac and decrease the expression of matrix metalloproteinase 9 (MMP-9). These data demonstrate that SET may be involved in the pathogenic processes of breast cancer, indicating that SET can serve as a potential therapeutic target for the treatment of breast cancer.

  20. Gene knockdown by morpholino-modified oligonucleotides in the zebrafish model: applications for developmental toxicology

    PubMed Central

    Timme-Laragy, Alicia R.; Karchner, Sibel I.; Hahn, Mark E.

    2014-01-01

    Summary The zebrafish (Danio rerio) has long been used as a model for developmental biology, making it an excellent model to use also in developmental toxicology. The many advantages of zebrafish include their small size, prolific spawning, rapid development, and transparent embryos. They can be easily manipulated genetically through the use of transgenic technology and gene knock-down via morpholino-modified antisense oligonucleotides (MOs). Knocking down specific genes to assess their role in the response to toxicant exposure provides a way to further our knowledge of how developmental toxicants work on a molecular and mechanistic level, while establishing a relationship between these molecular events and morphological, behavioral, and/or physiological effects (i.e. phenotypic anchoring). In this chapter we address important considerations for using MOs to study developmental toxicology in zebrafish embryos and provide a protocol for their use. PMID:22669659

  1. shRNA-Induced Gene Knockdown In Vivo to Investigate Neutrophil Function.

    PubMed

    Basit, Abdul; Tang, Wenwen; Wu, Dianqing

    2016-01-01

    To silence genes in neutrophils efficiently, we exploited the RNA interference and developed an shRNA-based gene knockdown technique. This method involves transfection of mouse bone marrow-derived hematopoietic stem cells with retroviral vector carrying shRNA directed at a specific gene. Transfected stem cells are then transplanted into irradiated wild-type mice. After engraftment of stem cells, the transplanted mice have two sets of circulating neutrophils. One set has a gene of interest knocked down while the other set has full complement of expressed genes. This efficient technique provides a unique way to directly compare the response of neutrophils with a knocked-down gene to that of neutrophils with the full complement of expressed genes in the same environment.

  2. Knockdown of transient receptor potential canonical-1 reduces the proliferation and migration of endothelial progenitor cells.

    PubMed

    Kuang, Chun-yan; Yu, Yang; Wang, Kui; Qian, De-hui; Den, Meng-yang; Huang, Lan

    2012-02-10

    Endothelial progenitor cells (EPCs) play an important role in accelerating endothelial repair after vascular injury. The proliferation and migration of EPCs is a critical first step in restoring endothelial. However, mechanisms for modulating EPC proliferation and migration are still being elucidated. Our previous study found that transient receptor potential canonical-1 (TRPC1) is involved in regulating store-operated Ca(2+) entry in EPCs through stromal interaction molecule 1. Therefore, in the present study, we sought to further investigate the regulation of proliferation and migration of EPCs by TRPC1. We found that the silencing of TRPC1 by 2 different RNA interference methods suppressed the proliferation and migration of EPCs. In addition, knockdown of TRPC1 significantly reduced of the amplitude of store-operated Ca(2+) entry and caused arrest of the EPC cell cycle in G1 phase. Analysis of the expression of 84 cell cycle genes by microarray showed that 9 genes were upregulated and 4 were downregulated by >2-fold in EPCs following TRPC1 silencing. The genes with expression changes were Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, Prm1, Bcl2, Mki67, Pmp22, and Ppp2r3a. Finally, we found that a Schlafen 1-blocking peptide partially reversed the abnormal cell cycle distribution and proliferation induced by TRPC1 knockdown, suggesting that Schlafen 1 is downstream of TRPC1 silencing in regulating EPC proliferation. In summary, these findings provide a new mechanism for modulating the biological properties of EPCs and suggest that TRPC1 may be a new target for inducing vascular repair by EPCs.

  3. Knockdown of Transient Receptor Potential Canonical-1 Reduces the Proliferation and Migration of Endothelial Progenitor Cells

    PubMed Central

    Kuang, Chun-yan; Yu, Yang; Wang, Kui; Qian, De-hui; Den, Meng-yang

    2012-01-01

    Endothelial progenitor cells (EPCs) play an important role in accelerating endothelial repair after vascular injury. The proliferation and migration of EPCs is a critical first step in restoring endothelial. However, mechanisms for modulating EPC proliferation and migration are still being elucidated. Our previous study found that transient receptor potential canonical-1 (TRPC1) is involved in regulating store-operated Ca2+ entry in EPCs through stromal interaction molecule 1. Therefore, in the present study, we sought to further investigate the regulation of proliferation and migration of EPCs by TRPC1. We found that the silencing of TRPC1 by 2 different RNA interference methods suppressed the proliferation and migration of EPCs. In addition, knockdown of TRPC1 significantly reduced of the amplitude of store-operated Ca2+ entry and caused arrest of the EPC cell cycle in G1 phase. Analysis of the expression of 84 cell cycle genes by microarray showed that 9 genes were upregulated and 4 were downregulated by >2-fold in EPCs following TRPC1 silencing. The genes with expression changes were Ak1, Brca2, Camk2b, p21, Ddit3, Inha, Slfn1, Mdm2, Prm1, Bcl2, Mki67, Pmp22, and Ppp2r3a. Finally, we found that a Schlafen 1-blocking peptide partially reversed the abnormal cell cycle distribution and proliferation induced by TRPC1 knockdown, suggesting that Schlafen 1 is downstream of TRPC1 silencing in regulating EPC proliferation. In summary, these findings provide a new mechanism for modulating the biological properties of EPCs and suggest that TRPC1 may be a new target for inducing vascular repair by EPCs. PMID:21361857

  4. Keap1 Knockdown increases markers of metabolic syndrome after long-term high fat diet feeding

    PubMed Central

    More, Vijay R; Xu, Jialin; Shimpi, Prajakta C; Belgrave, Clyde; Luyendyk, James P.; Yamamoto, Masayuki; Slitt, Angela L

    2013-01-01

    The Nuclear factor-E2 related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway upregulates antioxidant and biotransformation enzyme expression to counter cellular oxidative stress. The contribution of Nrf2 to other cellular functions, such as lipid homeostasis is emerging. The present study was conducted to determine how enhanced Nrf2 activity impacts progression of metabolic syndrome with long-term high fat diet (HFD) feeding. C57BL/6 and Keap1-Knockdown (Keap1-KD) mice, which exhibit enhanced Nrf2 activity, were fed a HFD for 24 weeks. Keap1-KD mice had higher body weight and white adipose tissue mass compared to C57BL/6 mice on HFD, along with increased inflammation and lipogenic gene expression. HFD feeding increased hepatic steatosis and inflammation to a greater extent in Keap1-KD mice compared to C57BL/6 mice, which was associated with increased liver Cd36, fatty acid binding protein 4 (Fabp4), and monocyte chemoattractant protein 1 (Mcp1) mRNA expression, as well as, increased acetyl CoA carboxylase 1 (Acc1) and Steroyl CoA desaturase 1 (Scd1) protein expression. The HFD altered short-term glucose homeostasis to a greater degree in Keap-KD mice compared to C57BL/6 mice, which was accompanied by down regulation of Insulin receptor substrate 1 mRNA expression in skeletal muscle. Together, the results indicate that Keap1 knockdown, on treatment with HFD, increases certain markers of metabolic syndrome. PMID:23507082

  5. RNAi knockdown of fatty acid elongase1 alters fatty acid composition in Brassica napus.

    PubMed

    Shi, Jianghua; Lang, Chunxiu; Wu, Xuelong; Liu, Renhu; Zheng, Tao; Zhang, Dongqing; Chen, Jinqing; Wu, Guanting

    2015-10-23

    The quality and end-use of oil from oilseed crops is determined by its fatty acid composition. In particular, the relative proportions of erucic and oleic acids are key selection traits for breeders. The goal of our research is to genetically improve the nutritional quality of Brassica napus cultivar CY2, the oil of which is high in erucic acid (about 40%) and low in oleic acid (about 20%). Here, we report the use of a seed-specific napin A promoter to drive the knockdown of BnFAE1 in transgenic CY2. Southern blotting results confirmed the presence of the transgene. RT-PCR analysis showed that the levels of BnFAE1 were greatly decreased in BnFAE1-Ri lines compared with the CY2 cultivar. Knockdown of BnFAE1 sharply decreased the levels of erucic acid (less than 3%), largely increased the contents of oleic acid (more than 60%) and slightly increased the polyunsaturated chain fatty acids. Compared with high erucic acid parents, expression of BnFAE1 was dramatically decreased in developing F1 seeds derived from reciprocally crossed BnFAE1-Ri lines and high erucic acid cultivars. In addition, F1 seeds derived from reciprocal crosses between BnFAE1-Ri lines and high erucic acid cultivars showed significantly increased oleic acid (more than 52%) and sharply decreased erucic acid (less than 4%), demonstrating that the RNAi construct of BnFAE1 can effectively interfere with the target gene in F1 seeds. Taken together, our results demonstrate that BnFAE1 is a reliable target for genetic improvement of rapeseed in seed oil quality promotion. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Chordin knockdown enhances the osteogenic differentiation of human mesenchymal stem cells

    PubMed Central

    Kwong, Francois NK; Richardson, Stephen M; Evans, Christopher H

    2008-01-01

    Introduction Bone morphogenetic proteins (BMPs) are critical growth factors in the osteogenic differentiation of progenitor cells during development in embryos and fracture repair in adults. Although recombinant BMPs are in use clinically, their clinical efficiency needs to be improved. The biological activities of BMPs are naturally regulated by extracellular binding proteins. The specific hypotheses tested in this study were as follows: the BMP inhibitor chordin is produced endogenously during the osteogenic differentiation of human mesenchymal stem cells (MSCs); and blockade of the activity of the BMP inhibitor increases the rate of osteogenic differentiation of human MSCs in vitro. Methods Human MSCs were derived from bone marrow from an iliac crest aspirate and from patients undergoing hip hemiarthroplasty. The MSCs were induced down the osteogenic pathway using standard osteogenic differentiation media, and expressions of BMP-2 and chordin were determined by gene expression analysis. During osteogenic differentiation, chordin knockdown was induced using RNA interference. Osteogenic differentiation was assessed by measuring the expression of alkaline phosphatase and calcium deposition. The differences in expression of osteogenic makers between groups were compared by analysis of variance, followed by Gabriel post hoc test. Results We demonstrate the expression of BMP-2 and chordin in human MSCs during osteogenic differentiation. Knockdown of chordin by RNA interference in vitro resulted in a significant increase in the expression of the osteogenic marker alkaline phosphatase and the deposition of extracellular mineral, in response to osteogenic stimulation. Conclusion We conclude that endogenously produced chordin constrains the osteogenic differentiation of human MSCs. The targeting of BMP inhibitors, such as chordin, may provide a novel strategy for enhancing bone regeneration. PMID:18533030

  7. RNAi-based targeted gene knockdown in the model oleaginous microalgae Nannochloropsis oceanica.

    PubMed

    Wei, Li; Xin, Yi; Wang, Qintao; Yang, Juan; Hu, Hanhua; Xu, Jian

    2017-03-01

    Microalgae are promising feedstock for renewable fuels such as biodiesel, yet development of industrial oleaginous strains has been hindered by the paucity and inefficiency of reverse genetics tools. Here we established an efficient RNAi-based targeted gene-knockdown method for Nannochloropsis spp., which are emerging model organisms for industrial microalgal oil production. The method achieved a 40-80% success rate in Nannochloropsis oceanica strain IMET1. When transcript level of one carbonic anhydrase (CA) was inhibited by 62-83% via RNAi, mutant cells exhibited photosynthetic oxygen evolution (POE) rates that were 68-100% higher than wild-type (WT) at pH 6.0, equivalent to WT at pH 8.2, yet 39-45% lower than WT at pH 9.0. Moreover, the mutant POE rates were negatively correlated with the increase of culture pH, an exact opposite of WT. Thus, a dynamic carbon concentration mechanism (CCM) that is highly sensitive to pH homeostasis was revealed, where the CA inhibition likely partially abrogated the mechanism that normally deactivates CCM under a high level of dissolved CO2 . Extension of the method to another sequenced N. oceanica strain of CCMP 1779 demonstrated comparable performance. Finally, McrBC-PCR followed by bisulfite sequencing revealed that the gene knockdown is mediated by the CG, CHG and CHH types of DNA methylation at the coding region of the targeted gene. The efficiency, robustness and general applicability of this reverse genetics approach suggested the possibility of large-scale RNAi-based gene function screening in industrial microalgae.

  8. AKI after Conditional and Kidney-Specific Knockdown of Stanniocalcin-1

    PubMed Central

    Huang, Luping; Belousova, Tatiana; Pan, Jenny Szu-Chin; Du, Jie; Ju, Huiming; Lu, Lianghao; Zhang, Pumin; Truong, Luan D.; Nuotio-Antar, Alli

    2014-01-01

    Stanniocalcin-1 is an intracrine protein; it binds to the cell surface, is internalized to the mitochondria, and diminishes superoxide generation through induction of uncoupling proteins. In vitro, stanniocalcin-1 inhibits macrophages and preserves endothelial barrier function, and transgenic overexpression of stanniocalcin-1 in mice protects against ischemia-reperfusion kidney injury. We sought to determine the kidney phenotype after kidney endothelium-specific expression of stanniocalcin-1 small hairpin RNA (shRNA). We generated transgenic mice that express stanniocalcin-1 shRNA or scrambled shRNA upon removal of a floxed reporter (phosphoglycerate kinase-driven enhanced green fluorescent protein) and used ultrasound microbubbles to deliver tyrosine kinase receptor-2 promoter-driven Cre to the kidney to permit kidney endothelium-specific shRNA expression. Stanniocalcin-1 mRNA and protein were expressed throughout the kidney in wild-type mice. Delivery of tyrosine kinase receptor-2 promoter-driven Cre to stanniocalcin-1 shRNA transgenic kidneys diminished the expression of stanniocalcin-1 mRNA and protein throughout the kidneys. Stanniocalcin-1 mRNA and protein expression did not change in similarly treated scrambled shRNA transgenic kidneys, and we observed no Cre protein expression in cultured and tyrosine kinase receptor-2 promoter-driven Cre–transfected proximal tubule cells, suggesting that knockdown of stanniocalcin-1 in epithelial cells in vivo may result from stanniocalcin-1 shRNA transfer from endothelial cells to epithelial cells. Kidney-specific knockdown of stanniocalcin-1 led to severe proximal tubule injury characterized by vacuolization, decreased uncoupling of protein-2 expression, greater generation of superoxide, activation of the unfolded protein response, initiation of autophagy, cell apoptosis, and kidney failure. Our observations suggest that stanniocalcin-1 is critical for tubular epithelial survival under physiologic conditions. PMID

  9. Transcriptome response and developmental implications of RNAi-mediated ODC knockdown in tobacco.

    PubMed

    Choubey, Ami; Rajam, M V

    2016-12-24

    Polyamines (PAs) are ubiquitously present polycationic compounds that play a critical role in various growth and developmental processes including stress responses in plants. Yet, their specific functions and mode of action remain largely unknown. In the present study, we have targeted tobacco ornithine decarboxylase gene (ODC) by RNA interference to modulate cellular PA levels and study the effects at different developmental time points. Down-regulation of ODC resulted in significant physiological and morphological anomalies including reduced leaf size, reduced chlorophyll and carotene content, decreased abiotic stress tolerance, early onset of senescence, delayed flowering, partial male and female sterility, reduced seed setting, delayed seed germination, reduced seed viability, and poor in vitro regeneration response from leaf explants. Also, for the first time, microarray analysis has been attempted to study genome-wide gene expression changes in response to lowered PA titers in an ODC knockdown line. A number of transcription factors, auxin- and ethylene-responsive genes, stress-induced genes, lignin-biosynthesis genes, photosynthesis-related genes, senescence-associated genes, membrane proteins, and protein kinases were found to be affected, suggesting a probable list of PA-responsive genes. Transcriptome analysis has also indicated many genes, which could directly or indirectly be responsible for regulating the PA metabolic pathway. Various phenotypic changes observed upon ODC knockdown along with the identification of a number of gene targets means it is a step forward in envisaging possible mechanisms of PA action and for assigning them with specific roles in various developmental processes they are known to be a part of.

  10. Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

    PubMed Central

    Reddy, B. K. Kishore; Landge, Sudhir; Ravishankar, Sudha; Patil, Vikas; Shinde, Vikas; Tantry, Subramanyam; Kale, Manoj; Raichurkar, Anandkumar; Menasinakai, Sreenivasaiah; Mudugal, Naina Vinay; Ambady, Anisha; Ghosh, Anirban; Tunduguru, Ragadeepthi; Kaur, Parvinder; Singh, Ragini; Kumar, Naveen; Bharath, Sowmya; Sundaram, Aishwarya; Bhat, Jyothi; Sambandamurthy, Vasan K.; Björkelid, Christofer; Jones, T. Alwyn; Das, Kaveri; Bandodkar, Balachandra; Malolanarasimhan, Krishnan; Mukherjee, Kakoli

    2014-01-01

    Pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by the coaA gene is an essential pantothenate kinase in Mycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-type M. tuberculosis strain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in a M. tuberculosis knockdown strain with reduced PanK expression levels. Additionally, in vitro and in vivo survival kinetic studies performed with a M. tuberculosis PanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK in M. tuberculosis. PMID:24687493

  11. Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype.

    PubMed

    Brendel, Christian; Guda, Swaroopa; Renella, Raffaele; Bauer, Daniel E; Canver, Matthew C; Kim, Young-Jo; Heeney, Matthew M; Klatt, Denise; Fogel, Jonathan; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2016-10-03

    Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%-70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease.

  12. siRNA-mediated knock-down of COX-2 in melanocytes suppresses melanogenesis.

    PubMed

    Kim, Ji Y; Shin, Jae Y; Kim, Miri R; Hann, Seung-Kyung; Oh, Sang H

    2012-06-01

    Cyclooxygenase-2 (COX-2) is an enzyme induced in response to multiple mitogenic and inflammatory stimuli, including UV light. UV-induced COX-2 expression induces production of prostaglandin E2 (PGE2) in keratinocytes, which mediates inflammation and cell proliferation. Until recently, studies regarding COX-2 and PGE2 in the skin have focused on keratinocytes and skin cancer and the effect of PGs produced by keratinocytes on melanocytes. However, the effects of COX-2 itself or COX-2 inhibitors on melanogenesis are not well known. Therefore, to establish the role of COX-2 in melanogenesis, we investigated the effects of knock-down of COX-2 in melanocytes on melanin production and the expression of melanogenic molecules through silencing of COX-2 expression with COX-2 short interfering RNA (siRNA). COX-2 knock-down in melanocytes decreased the expressions of tyrosinase, TRP-1, TRP-2, gp100 and MITF and also reduced tyrosinase enzyme activity. Furthermore, COX-2 siRNA-transfected melanocytes showed markedly reduced alpha-melanocyte stimulating hormone (α-MSH)-induced melanin production. In addition, α-MSH-induced COX-2 expression in both scrambled siRNA-transfected and COX-2 siRNA-transfected melanocytes was greater than α-MSH-untreated cells. Our results suggest that COX-2 might be a candidate target for the development of anti-melanogenic agents and α-MSH-induced pigmentation could be closely associated with COX-2 expression. COX-2 inhibitors might therefore be of particular use in whitening cosmetics for hyperpigmentation disorders such as melasma, postinflammatory hyperpigmentation and solar lentigo.

  13. Lineage-specific BCL11A knockdown circumvents toxicities and reverses sickle phenotype

    PubMed Central

    Brendel, Christian; Guda, Swaroopa; Renella, Raffaele; Bauer, Daniel E.; Canver, Matthew C.; Kim, Young-Jo; Heeney, Matthew M.; Klatt, Denise; Fogel, Jonathan; Milsom, Michael D.; Orkin, Stuart H.; Gregory, Richard I.

    2016-01-01

    Reducing expression of the fetal hemoglobin (HbF) repressor BCL11A leads to a simultaneous increase in γ-globin expression and reduction in β-globin expression. Thus, there is interest in targeting BCL11A as a treatment for β-hemoglobinopathies, including sickle cell disease (SCD) and β-thalassemia. Here, we found that using optimized shRNAs embedded within an miRNA (shRNAmiR) architecture to achieve ubiquitous knockdown of BCL11A profoundly impaired long-term engraftment of both human and mouse hematopoietic stem cells (HSCs) despite a reduction in nonspecific cellular toxicities. BCL11A knockdown was associated with a substantial increase in S/G2-phase human HSCs after engraftment into immunodeficient (NSG) mice, a phenotype that is associated with HSC exhaustion. Lineage-specific, shRNAmiR-mediated suppression of BCL11A in erythroid cells led to stable long-term engraftment of gene-modified cells. Transduced primary normal or SCD human HSCs expressing the lineage-specific BCL11A shRNAmiR gave rise to erythroid cells with up to 90% reduction of BCL11A protein. These erythrocytes demonstrated 60%–70% γ-chain expression (vs. < 10% for negative control) and a corresponding increase in HbF. Transplantation of gene-modified murine HSCs from Berkeley sickle cell mice led to a substantial improvement of sickle-associated hemolytic anemia and reticulocytosis, key pathophysiological biomarkers of SCD. These data form the basis for a clinical trial application for treating sickle cell disease. PMID:27599293

  14. Immobilization of the nematode Caenorhabditis elegans with addressable light-induced heat knockdown (ALINK).

    PubMed

    Chuang, Han-Sheng; Chen, Hsiang-Yu; Chen, Chang-Shi; Chiu, Wen-Tai

    2013-08-07

    Caenorhabditis (C.) elegans is a model animal used in genetics, neuroscience, and developmental biology. Researchers often immobilize squirming worms to obtain high-quality images for analysis. However, current methods usually require physical contact or anesthetics. This can cause injuries to worm bodies or neuron disturbances. This study presents an alternative technique, called addressable light-induced heat knockdown (ALINK), to effectively immobilize worms by using light-induced sublethal heat. A microchip composed of an indium-tin-oxide (ITO) glass plate and an ITO glass plate coated with a photoconductive layer (a-Si:H) was produced. Worms to be immobilized were immersed in a liquid medium and sandwiched between the two plates. When the worms were irradiated with a focused laser beam in the presence of electric fields (referred to as an optoelectric treatment), the optoelectric effect heated the liquid medium. The neural functions of the worms shut down temporarily when a critical temperature (>31 °C) was reached. Their neural functions resumed after the heat source was removed. A temperature above 37 °C killed all worms. Using short-wavelength light reduced the worms' recovery time. An equivalent circuit was modeled to predict the operating modes, and an optoelectric treatment with a high-concentration medium enhanced rapid heating. A safe operating range (20 Vpp (peak-to-peak voltage), 100 kHz to 10 MHz, 31 to 37 °C) to induce heat knockdown (KD) was also investigated. The results show that the heat KD was well controlled, autonomous, and reversible. This technique can be used for worm immobilization.

  15. DISC1 knockdown impairs the tangential migration of cortical interneurons by affecting the actin cytoskeleton

    PubMed Central

    Steinecke, André; Gampe, Christin; Nitzsche, Falk; Bolz, Jürgen

    2014-01-01

    Disrupted-in-Schizophrenia 1 (DISC1) is a risk gene for a spectrum of major mental disorders. It has been shown to regulate radial migration as well as dendritic arborization during neurodevelopment and corticogenesis. In a previous study we demonstrated through in vitro experiments that DISC1 also controls the tangential migration of cortical interneurons originating from the medial ganglionic eminence (MGE). Here we first show that DISC1 is necessary for the proper tangential migration of cortical interneurons in the intact brain. Expression of EGFP under the Lhx6 promotor allowed us to analyze exclusively interneurons transfected in the MGE after in utero electroporation. After 3 days in utero, DISC1 deficient interneurons displayed prolonged leading processes and, compared to control, fewer neurons reached the cortex. Time-lapse video microscopy of cortical feeder-layers revealed a decreased migration velocity due to a reduction of soma translocations. Immunostainings indicated that DISC1 is co-localized with F-actin in the growth cone-like structure of the leading process. DISC1 knockdown reduced F-actin levels whereas the overall actin level was not altered. Moreover, DISC1 knockdown also decreased levels of phosphorylated Girdin, which cross-links F-actin, as well as the Girdin-activator pAkt. In contrast, using time-lapse video microscopy of fluorescence-tagged tubulin and EB3 in fibroblasts, we found no effects on microtubule polymerization when DISC1 was reduced. However, DISC1 affected the acetylation of microtubules in the leading processes of MGE-derived cortical interneurons. Together, our results provide a mechanism how DISC1 might contribute to interneuron migration thereby explaining the reduced number of specific classes of cortical interneurons in some DISC1 mouse models. PMID:25071449

  16. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs

    PubMed Central

    Hines, John; Gough, Jonathan D.; Corson, Timothy W.; Crews, Craig M.

    2013-01-01

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects. PMID:23674677

  17. Posttranslational protein knockdown coupled to receptor tyrosine kinase activation with phosphoPROTACs.

    PubMed

    Hines, John; Gough, Jonathan D; Corson, Timothy W; Crews, Craig M

    2013-05-28

    Posttranslational knockdown of a specific protein is an attractive approach for examining its function within a system. Here we introduce phospho-dependent proteolysis targeting chimeras (phosphoPROTACs), a method to couple the conditional degradation of targeted proteins to the activation state of particular kinase-signaling pathways. We generated two phosphoPROTACs that couple the tyrosine phosphorylation sequences of either the nerve growth factor receptor, TrkA (tropomyosin receptor kinase A), or the neuregulin receptor, ErbB3 (erythroblastosis oncogene B3), with a peptide ligand for the E3 ubiquitin ligase von Hippel Lindau protein. These phosphoPROTACs recruit either the neurotrophic signaling effector fibroblast growth factor receptor substrate 2α or the survival-promoting phosphatidylinositol-3-kinase, respectively, to be ubiquitinated and degraded upon activation of specific receptor tyrosine kinases and phosphorylation of the phosphoPROTACs. We demonstrate the ability of these phosphoPROTACs to suppress the short- and long-term effects of their respective activating receptor tyrosine kinase pathways both in vitro and in vivo. In addition, we show that activation of phosphoPROTACs is entirely dependent on their kinase-mediated phosphorylation, as phenylalanine-containing null variants are inactive. Furthermore, stimulation of unrelated growth factor receptors does not induce target protein knockdown. Although comparable in efficiency to RNAi, this approach has the added advantage of providing a degree of temporal and dosing control as well as cell-type selectivity unavailable using nucleic acid-based strategies. By varying the autophosphorylation sequence of a phosphoPROTAC, it is conceivable that other receptor tyrosine kinase/effector pairings could be similarly exploited to achieve other biological effects.

  18. Knockdown of CSE1L Gene in Colorectal Cancer Reduces Tumorigenesis in Vitro.

    PubMed

    Pimiento, Jose M; Neill, Kevin G; Henderson-Jackson, Evita; Eschrich, Steven A; Chen, Dung-Tsa; Husain, Kazim; Shibata, David; Coppola, Domenico; Malafa, Mokenge P

    2016-10-01

    Human cellular apoptosis susceptibility (chromosomal segregation 1-like, CSE1L) gene plays a role in nuclear-to-cytoplasm transport and chromosome segregation during mitosis, cellular proliferation, and apoptosis. CSE1L is involved in colon carcinogenesis. CSE1L gene expression was assessed with three data sets using Affymetrix U133 + gene chips on normal human colonic mucosa (NR), adenomas (ADs), and colorectal carcinoma (CRC). CSE1L protein expression in CRC, AD, and NR from the same patients was measured by immunohistochemistry using a tissue microarray. We evaluated CSE1L expression in CRC cells (HCT116, SW480, and HT29) and its biological functions. CSE1L mRNA was significantly increased in all AD and CRC compared with NR (P < 0.001 and P = 0.02, respectivly). We observed a change in CSE1L staining intensity and cellular localization by immunohistochemistry. CSE1L was significantly increased during the transition from AD to CRC when compared with NR in a CRC tissue microarray (P = 0.01 and P < 0.001). HCT116, SW480, and HT29 cells also expressed CSE1L protein. CSE1L knockdown by shRNA inhibited protein, resulting in decreased cell proliferation, reduced colony formation in soft agar, and induction of apoptosis. CSE1L protein is expressed early and across all stages of CRC development. shRNA knockdown of CSE1L was associated with inhibition of tumorigenesis in CRC cells. CSE1L may represent a potential target for treatment of CRC.

  19. Knockdown of PKM2 Suppresses Tumor Growth and Invasion in Lung Adenocarcinoma

    PubMed Central

    Sun, Hong; Zhu, Anyou; Zhang, Lunjun; Zhang, Jie; Zhong, Zhengrong; Wang, Fengchao

    2015-01-01

    Accumulating evidence shows that activity of the pyruvate kinase M2 (PKM2) isoform is closely related to tumorigenesis. In this study, we investigated the relationship betweenPKM2 expression, tumor invasion, and the prognosis of patients with lung adenocarcinoma. We retrospectively analyzed 65 cases of patients with lung adenocarcinoma who were divided into low and a high expression groups based on PKM2immunohistochemical staining. High PKM2 expression was significantly associated with reduced patient survival. We used small interfering RNA (siRNA) technology to investigate the effect of targeted PKM2-knockout on tumor growth at the cellular level. In vitro, siRNA-mediated PKM2-knockdown significantly inhibited the proliferation, glucose uptake (25%), ATP generation (20%) and fatty acid synthesis of A549 cells, while the mitochondrial respiratory capacity of the cells increased (13%).Western blotting analysis showed that PKM2-knockout significantly inhibited the expression of the glucose transporter GLUT1 and ATP citrate lyase, which is critical for fatty acid synthesis. Further Western blotting analysis showed that PKM2-knockdown inhibited the expression of matrix metalloproteinase 2 (MMP-2) and vascular endothelial growth factor (VEGF), which are important in degradation of the extracellular matrix and angiogenesis, respectively. These observations show that PKM2 activates both glycolysis and lipid synthesis, thereby regulating cell proliferation and invasion. This information is important in elucidating the mechanisms by which PKM2 influences the growth and metastasis of lung adenocarcinoma at the cellular and molecular level, thereby providing the basic data required for the development of PKM2-targeted gene therapy. PMID:26501265

  20. Knockdown of ICB-1 gene enhanced estrogen responsiveness of ovarian and breast cancer cells.

    PubMed

    Konwisorz, Anna; Springwald, Anette; Haselberger, Martina; Goerse, Regina; Ortmann, Olaf; Treeck, Oliver

    2010-03-01

    ICB-1 chromosome 1 open reading frame 38 (C1orf38) is a human gene initially described by our group to be involved in differentiation processes of cancer cells. Recently, we have reported ICB-1 as a novel estrogen target gene and identified an estrogen response element in its promoter. In this study, we examined the role of ICB-1 in regulation of proliferation of breast and ovarian cancer cells. We knocked down its expression in estrogen-dependent MCF-7 breast cancer cells and hormone-unresponsive SK-OV-3 ovarian cancer cells by stable transfection with a specific shRNA plasmid followed by G-418 selection. Knockdown of ICB-1 enabled a considerable estrogen response of SK-OV-3 cells in terms of proliferation. This transformation of SK-OV-3 cells into an estrogen-responsive phenotype was accompanied by upregulation of estrogen receptor alpha (ERalpha) expression and a significant decrease of ERbeta expression on the mRNA level. Expression of ERalpha-dependent genes progesterone receptor, pS2, fibulin 1c, and c-fos was elevated in SK-OV-3 cells stably expressing ICB-1 shRNA. In MCF-7 cells, ICB-1 knockdown exerted similar effects on gene expression, supporting a general role of ICB-1 in estrogen responsiveness. Our data suggest that differentiation-associated gene ICB-1 might exert antagonistic actions on cellular estrogen response, which can result in inhibition of estradiol-triggered proliferation. The molecular mechanisms mediating this inhibitory effect of ICB-1 on estrogen signaling are suggested to be limitation of ERalpha transcript levels but sustaining high levels of ERbeta, reducing both activation of ERalpha target genes and cellular proliferation. The identification of ICB-1 as a new player in endocrine-related cancer encourages further studies on the significance of this gene in cancer development and therapy.

  1. Knockdown of Malic Enzyme 2 Suppresses Lung Tumor Growth, Induces Differentiation and Impacts PI3K/AKT Signaling

    PubMed Central

    Ren, Jian-Guo; Seth, Pankaj; Clish, Clary B.; Lorkiewicz, Pawel K.; Higashi, Richard M.; Lane, Andrew N.; Fan, Teresa W.-M.; Sukhatme, Vikas P.

    2014-01-01

    Mitochondrial malic enzyme 2 (ME2) catalyzes the oxidative decarboxylation of malate to yield CO2 and pyruvate, with concomitant reduction of dinucleotide cofactor NAD+ or NADP+. We find that ME2 is highly expressed in many solid tumors. In the A549 non-small cell lung cancer (NSCLC) cell line, ME2 depletion inhibits cell proliferation and induces cell death and differentiation, accompanied by increased reactive oxygen species (ROS) and NADP+/NADPH ratio, a drop in ATP, and increased sensitivity to cisplatin. ME2 knockdown impacts phosphoinositide-dependent protein kinase 1 (PDK1) and phosphatase and tensin homolog (PTEN) expression, leading to AKT inhibition. Depletion of ME2 leads to malate accumulation and pyruvate decrease, and exogenous cell permeable dimethyl-malate (DMM) mimics the ME2 knockdown phenotype. Both ME2 knockdown and DMM treatment reduce A549 cell growth in vivo. Collectively, our data suggest that ME2 is a potential target for cancer therapy. PMID:24957098

  2. Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

    PubMed Central

    Zemany, Laura; Kraus, Bettina J.; Norseen, Julie; Saito, Tsugumichi; Peroni, Odile D.; Johnson, Randy L.

    2014-01-01

    To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6−/− mice. Adipose-Stra6−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance. PMID:24421389

  3. Downregulation of STRA6 in adipocytes and adipose stromovascular fraction in obesity and effects of adipocyte-specific STRA6 knockdown in vivo.

    PubMed

    Zemany, Laura; Kraus, Bettina J; Norseen, Julie; Saito, Tsugumichi; Peroni, Odile D; Johnson, Randy L; Kahn, Barbara B

    2014-03-01

    To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6(-/-) mice. Adipose-Stra6(-/-) mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6(-/-) mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6(-/-) mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.

  4. shRNA-Mediated XRCC2 Gene Knockdown Efficiently Sensitizes Colon Tumor Cells to X-ray Irradiation in Vitro and in Vivo

    PubMed Central

    Wang, Qin; Wang, Yan; Du, Liqing; Xu, Chang; Sun, Yuanming; Yang, Bing; Sun, Zhijuan; Fu, Yue; Cai, Lu; Fan, Saijun; Fan, Feiyue; Liu, Qiang

    2014-01-01

    Colon cancer is one of the most common tumors of the digestive tract. Resistance to ionizing radiation (IR) decreased therapeutic efficiency in these patients’ radiotherapy. XRCC2 is the key protein of DNA homologous recombination repair, and its high expression is associated with enhanced resistance to DNA damage induced by IR. Here, we investigated the effect of XRCC2 silencing on colon tumor cells’ growth and sensitivity to X-radiation in vitro and in vivo. Colon tumor cells (T84 cell line) were cultivated in vitro and tumors originated from the cell line were propagated as xenografts in nude mice. The suppression of XRCC2 expression was achieved by using vector-based short hairpin RNA (shRNA) in T84 cells. We found that the knockdown of XRCC2 expression effectively decreased T84 cellular proliferation and colony formation, and led to cell apoptosis and cell cycle arrested in G2/M phase induced by X-radiation in vitro. In addition, tumor xenograft studies suggested that XRCC2 silencing inhibited tumorigenicity after radiation treatment in vivo. Our data suggest that the suppression of XRCC2 expression rendered colon tumor cells more sensitive to radiation therapy in vitro and in vivo, implying XRCC2 as a promising therapeutic target for the treatment of radioresistant human colon cancer. PMID:24481064

  5. Knockdown and mortality comparisons among spinosad-, imidacloprid-, and methomyl-containing baits against susceptible Musca domestica (Diptera: Muscidae) under laboratory conditions.

    PubMed

    White, W Hunter; McCoy, Christine M; Meyer, Jeffery A; Winkle, Joseph R; Plummer, Philip R; Kemper, Connie J; Starkey, Robert; Snyder, Daniel E

    2007-02-01

    The activity of spinosad, imidacloprid, and methomyl baits and technical actives were assessed against susceptible house flies, Musca domestica L. (Diptera: Muscidae). In a feeding assay, imidacloprid affected flies more rapidly than methomyl or spinosad, but spinosad was 2.7 times more potent than methomyl and 8 times more potent than imidacloprid. The profile of technical actives correlated with their respective fly bait formulations in laboratory assays. Although having the most rapid onset of activity in laboratory tests, up to 50% of flies remained alive after exposure to imidacloprid bait. In contrast, <5% of flies survived 24-h exposure to spinosad or methomyl baits. High temperature reduced the knockdown activity of imidacloprid bait and slowed the speed of kill for spinosad and methomyl baits over a 24-h exposure period. Spinosad and methomyl baits were also superior to imidacloprid when applied to the floors of environmentally controlled rooms at label recommended rates, providing good fly control for up to 21 d. The fact that a significant percentage of flies exposed to imidacloprid were rapidly knocked down but subsequently remained alive in all of the assays suggested that flies were recovering from initial exposure to this compound. Given its favorable safety profile, a high degree of initial and residual activity comparable with methomyl and lack of cross-resistance to other chemistries, spinosad bait may be a valuable component of house fly control programs to help control or delay the emergence of resistant populations.

  6. RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells.

    PubMed

    Dai, Chun-Hua; Li, Jian; Chen, Ping; Jiang, He-Guo; Wu, Ming; Chen, Yong-Chang

    2015-09-18

    Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance. In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin. Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage. Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.

  7. Efficient gene knockdown in Clostridium acetobutylicum by synthetic small regulatory RNAs.

    PubMed

    Cho, Changhee; Lee, Sang Yup

    2017-02-01

    Clostridium is considered a promising microbial host for the production of valuable industrial chemicals. However, Clostridium is notorious for the difficulty of genetic manipulations, and consequently metabolic engineering. Thus, much effort has been exerted to develop novel tools for genetic and metabolic engineering of Clostridium strains. Here, we report the development of a synthetic small regulatory RNA (sRNA)-based system for controlled gene expression in Clostridium acetobutylicum, consisting of a target recognition site, MicC sRNA scaffold, and an RNA chaperone Hfq. To examine the functional operation of sRNA system in C. acetobutylicum, expression control was first examined with the Evoglow fluorescent protein as a model protein. Initially, a C. acetobutylicum protein annotated as Hfq was combined with the synthetic sRNA based on the Escherichia coli MicC scaffold to knockdown Evoglow expression. However, C. acetobutylicum Hfq did not bind to E. coli MicC, while MicC scaffold-based synthetic sRNA itself was able to knockdown the expression of Evoglow. When E. coli hfq gene was introduced, the knockdown efficiency assessed by measuring fluorescence intensity, could be much enhanced. Then, this E. coli MicC scaffold-Hfq system was used to knock down adhE1 gene expression in C. acetobutylicum. Knocking down the adhE1 gene expression using the synthetic sRNA led to a 40% decrease in butanol production (2.5 g/L), compared to that (4.5 g/L) produced by the wild-type strain harboring an empty vector. The sRNA system was further extended to knock down the pta gene expression in the buk mutant C. acetobutylicum strain PJC4BK for enhanced butanol production. The PJC4BK (pPta-Hfq(Eco) ) strain, which has the pta gene expression knocked down, was able to produce 16.9 g/L of butanol, which is higher than that (14.9 g/L) produced by the PJC4BK strain, mainly due to reduced acetic acid production. Fed-batch culture of PJC4BK (pPta-Hfq(Eco) ) strain coupled with

  8. RNAi mediated gene knockdown and transgenesis by microinjection in the necromenic Nematode Pristionchus pacificus.

    PubMed

    Cinkornpumin, Jessica K; Hong, Ray L

    2011-10-16

    Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis and Pristionchus pacificus. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double-stranded RNA (ds

  9. Knockdown of Ice-Binding Proteins in Brachypodium distachyon Demonstrates Their Role in Freeze Protection

    PubMed Central

    Bredow, Melissa; Vanderbeld, Barbara; Walker, Virginia K.

    2016-01-01

    Sub-zero temperatures pose a major threat to the survival of cold-climate perennials. Some of these freeze-tolerant plants produce ice-binding proteins (IBPs) that offer frost protection by restricting ice crystal growth and preventing expansion-induced lysis of the plasma membranes. Despite the extensive in vitro characterization of such proteins, the importance of IBPs in the freezing stress response has not been investigated. Using the freeze-tolerant grass and model crop, Brachypodium distachyon, we characterized putative IBPs (BdIRIs) and generated the first ‘IBP-knockdowns’. Seven IBP sequences were identified and expressed in Escherichia coli, with all of the recombinant proteins demonstrating moderate to high levels of ice-recrystallization inhibition (IRI) activity, low levels of thermal hysteresis (TH) activity (0.03−0.09°C at 1 mg/mL) and apparent adsorption to ice primary prism planes. Following plant cold acclimation, IBPs purified from wild-type B. distachyon cell lysates similarly showed high levels of IRI activity, hexagonal ice-shaping, and low levels of TH activity (0.15°C at 0.5 mg/mL total protein). The transfer of a microRNA construct to wild-type plants resulted in the attenuation of IBP activity. The resulting knockdown mutant plants had reduced ability to restrict ice-crystal growth and a 63% reduction in TH activity. Additionally, all transgenic lines were significantly more vulnerable to electrolyte leakage after freezing to −10°C, showing a 13−22% increase in released ions compared to wild-type. IBP-knockdown lines also demonstrated a significant decrease in viability following freezing to −8°C, with some lines showing only two-thirds the survival seen in control lines. These results underscore the vital role IBPs play in the development of a freeze-tolerant phenotype and suggests that expression of these proteins in frost-susceptible plants could be valuable for the production of more winter-hardy crops. PMID:27959937

  10. RNAi Mediated Gene Knockdown and Transgenesis by Microinjection in the Necromenic Nematode Pristionchus pacificus

    PubMed Central

    Cinkornpumin, Jessica K.; Hong, Ray L.

    2011-01-01

    Although it is increasingly affordable for emerging model organisms to obtain completely sequenced genomes, further in-depth gene function and expression analyses by RNA interference and stable transgenesis remain limited in many species due to the particular anatomy and molecular cellular biology of the organism. For example, outside of the crown group Caenorhabditis that includes Caenorhabditis elegans3, stably transmitted transgenic lines in non-Caenorhabditis species have not been reported in this specious phylum (Nematoda), with the exception of Strongyloides stercoralis4 and Pristionchus pacificus5. To facilitate the expanding role of P. pacificus in the study of development, evolution, and behavior6-7, we describe here the current methods to use microinjection for making transgenic animals and gene knock down by RNAi. Like the gonads of C. elegans and most other nematodes, the gonads of P. pacificus is syncitial and capable of incorporating DNA and RNA into the oocytes when delivered by direct microinjection. Unlike C. elegans however, stable transgene inheritance and somatic expression in P. pacificus requires the addition of self genomic DNA digested with endonucleases complementary to the ends of target transgenes and coinjection markers5. The addition of carrier genomic DNA is similar to the requirement for transgene expression in Strongyloides stercoralis4 and in the germ cells of C. elegans. However, it is not clear if the specific requirement for the animals' own genomic DNA is because P. pacificus soma is very efficient at silencing non-complex multi-copy genes or that extrachromosomal arrays in P. pacificus require genomic sequences for proper kinetochore assembly during mitosis. The ventral migration of the two-armed (didelphic) gonads in hermaphrodites further complicates the ability to inject both gonads in individual worms8. We also demonstrate the use of microinjection to knockdown a dominant mutant (roller,tu92) by injecting double

  11. Impairment of HIV-1 cDNA synthesis by DBR1 knockdown.

    PubMed

    Galvis, Alvaro E; Fisher, Hugh E; Nitta, Takayuki; Fan, Hung; Camerini, David

    2014-06-01

    Previous studies showed that short hairpin RNA (shRNA) knockdown of the RNA lariat debranching enzyme (DBR1) led to a decrease in the production of HIV-1 cDNA. To further characterize this effect, DBR1 shRNA was introduced into GHOST-R5X4 cells, followed by infection at a multiplicity near unity with HIV-1 or an HIV-1-derived vector. DNA and RNA were isolated from whole cells and from cytoplasmic and nuclear fractions at different times postinfection. Inhibition of DBR1 had little or no effect on the formation of minus-strand strong-stop cDNA but caused a significant reduction in the formation of intermediate and full-length cDNA. Moreover, minus-strand strong-stop DNA rapidly accumulated in the cytoplasm in the first 2 h of infection but shifted to the nuclear fraction by 6 h postinfection. Regardless of DBR1 inhibition, greater than 95% of intermediate-length and full-length HIV-1 cDNA was found in the nuclear fraction at all time points. Thus, under these experimental conditions, HIV-1 cDNA synthesis was initiated in the cytoplasm and completed in the nucleus or perinuclear region of the infected cell. When nuclear import of the HIV-1 reverse transcription complex was blocked by expressing a truncated form of the mRNA cleavage and polyadenylation factor CPSF6, the completion of HIV-1 vector cDNA synthesis was detected in the cytoplasm, where it was not inhibited by DBR1 knockdown. Refinement of the cell fractionation procedure indicated that the completion of reverse transcription occurred both within nuclei and in the perinuclear region. Taken together the results indicate that in infections at a multiplicity near 1, HIV-1 reverse transcription is completed in the nucleus or perinuclear region of the infected cell, where it is dependent on DBR1. When nuclear transport is inhibited, reverse transcription is completed in the cytoplasm in a DBR1-independent manner. Thus, there are at least two mechanisms of HIV-1 reverse transcription that require different

  12. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing

    PubMed Central

    Bosch, Justin A.; Sumabat, Taryn M.; Hariharan, Iswar K.

    2016-01-01

    RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked “shadow RNAi” clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc. PMID:26984059

  13. Knockdown of HIF-1α and IL-8 induced apoptosis of hepatocellular carcinoma triggers apoptosis of vascular endothelial cells.

    PubMed

    Choi, Sung Hoon; Park, Jun Yong; Kang, Wonseok; Kim, Seung Up; Kim, Do Young; Ahn, Sang Hoon; Ro, Simon Wonsang; Han, Kwang-Hyub

    2016-01-01

    A local hypoxic microenvironment is one of the most important characteristics of solid tumors. Hypoxia inducible factor-1α (HIF-1α) and Interleukin-8 (IL-8) activate tumor survival from hypoxic-induced apoptosis in each pathway. This study aimed to evaluate whether knockdown of HIF-1α and IL-8 induced apoptosis of the hepatocellular carcinoma (HCC) and endothelial cell lines. HCC cell lines were infected with adenovirus-expressing shRNA for HIF-1α and IL-8 and maintained under hypoxic conditions (1% O2, 24 h). The expression levels of HIF-1α and both apoptotic and growth factors were examined by real-time quantitative PCR and western blot. We also investigated apoptosis by TUNEL assay (FACS and Immunofluorescence) and measured the concentration of cytochrome C. Inhibition of HIF-1α and IL-8 up-regulated the expression of apoptotic factors while downregulating anti-apoptotic factors simultaneously. Knockdown of HIF-1α and IL-8 increased the concentration of cytochrome C and enhanced DNA fragmentation in HCC cell lines. Moreover, culture supernatant collected from the knockdown of HIF-1α and IL-8 in HCC cell lines induced apoptosis in human umbilical vein endothelial cells under hypoxia, and the expression of variable apoptotic ligand increased from HCC cell lines, time-dependently. These data suggest that adenovirus-mediated knockdown of HIF-1α and IL-8 induced apoptosis in HCC cells and triggered apoptosis of vascular endothelial cells.

  14. Persistence of RNAi-Mediated Knockdown in Drosophila Complicates Mosaic Analysis Yet Enables Highly Sensitive Lineage Tracing.

    PubMed

    Bosch, Justin A; Sumabat, Taryn M; Hariharan, Iswar K

    2016-05-01

    RNA interference (RNAi) has emerged as a powerful way of reducing gene function in Drosophila melanogaster tissues. By expressing synthetic short hairpin RNAs (shRNAs) using the Gal4/UAS system, knockdown is efficiently achieved in specific tissues or in clones of marked cells. Here we show that knockdown by shRNAs is so potent and persistent that even transient exposure of cells to shRNAs can reduce gene function in their descendants. When using the FLP-out Gal4 method, in some instances we observed unmarked "shadow RNAi" clones adjacent to Gal4-expressing clones, which may have resulted from brief Gal4 expression following recombination but prior to cell division. Similarly, Gal4 driver lines with dynamic expression patterns can generate shadow RNAi cells after their activity has ceased in those cells. Importantly, these effects can lead to erroneous conclusions regarding the cell autonomy of knockdown phenotypes. We have investigated the basis of this phenomenon and suggested experimental designs for eliminating ambiguities in interpretation. We have also exploited the persistence of shRNA-mediated knockdown to design a sensitive lineage-tracing method, i-TRACE, which is capable of detecting even low levels of past reporter expression. Using i-TRACE, we demonstrate transient infidelities in the expression of some cell-identity markers near compartment boundaries in the wing imaginal disc.

  15. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

    DOE PAGES

    Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; ...

    2016-02-17

    Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs ormore » multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.« less

  16. Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana

    SciTech Connect

    Czarnecki, Olaf; Bryan, Anthony C.; Jawdy, Sara S.; Yang, Xiaohan; Cheng, Zong-Ming; Chen, Jin-Gui; Tuskan, Gerald A.

    2016-02-17

    Genetic engineering of plants that results in successful establishment of new biochemical or regulatory pathways requires stable introduction of one or more genes into the plant genome. It might also be necessary to down-regulate or turn off expression of endogenous genes in order to reduce activity of competing pathways. An established way to knockdown gene expression in plants is expressing a hairpin-RNAi construct, eventually leading to degradation of a specifically targeted mRNA. Knockdown of multiple genes that do not share homologous sequences is still challenging and involves either sophisticated cloning strategies to create vectors with different serial expression constructs or multiple transformation events that is often restricted by a lack of available transformation markers. Synthetic RNAi fragments were assembled in yeast carrying homologous sequences to six or seven non-family genes and introduced into pAGRIKOLA. Transformation of Arabidopsis thaliana and subsequent expression analysis of targeted genes proved efficient knockdown of all target genes. In conclusion, we present a simple and cost-effective method to create constructs to simultaneously knockdown multiple non-family genes or genes that do not share sequence homology. The presented method can be applied in plant and animal synthetic biology as well as traditional plant and animal genetic engineering.

  17. Selection on knockdown performance in Drosophila melanogaster impacts thermotolerance and heat-shock response differently in females and males.

    PubMed

    Folk, Donna G; Zwollo, Patty; Rand, David M; Gilchrist, George W

    2006-10-01

    We studied adaptive thermotolerance in replicate populations of Drosophila melanogaster artificially selected for high and low knockdown temperature (T(KD)), the upper temperature at which flies can no longer remain upright or locomote effectively. Responses to selection have generated High T(KD) populations capable of maintaining locomotor function at approximately 40 degrees C, and Low T(KD) populations with T(KD) of approximately 35 degrees C. We examined inducible knockdown thermotolerance, as well as inducible thermal survivorship, following a pretreatment heat-shock (known to induce heat-shock proteins) for males and females from the T(KD) selected lines. Both selection for knockdown and sex influenced inducible knockdown thermotolerance, whereas inducible thermal survivorship was influenced only by sex, and not by selection. Overall, our findings suggest that the relationships between basal and inducible thermotolerance are contingent upon the methods used to gauge thermotolerance, as well as the sex of the flies. Finally, we compared temporal profiles of the combined expression of two major heat-shock proteins, HSC70 and HSP70, during heat stress among the females and males from the selected T(KD) lines. The temporal profiles of the proteins differed between High and Low T(KD) females, suggesting divergence of the heat-shock response. We discuss a possible mechanism that may lead to the heat-shock protein patterns observed in the selected females.

  18. Rescue of impaired long-term facilitation at sensorimotor synapses of Aplysia following siRNA knockdown of CREB1.

    PubMed

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J; Byrne, John H

    2015-01-28

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches.

  19. Rescue of Impaired Long-Term Facilitation at Sensorimotor Synapses of Aplysia following siRNA Knockdown of CREB1

    PubMed Central

    Zhou, Lian; Zhang, Yili; Liu, Rong-Yu; Smolen, Paul; Cleary, Leonard J.

    2015-01-01

    Memory impairment is often associated with disrupted regulation of gene induction. For example, deficits in cAMP response element-binding protein (CREB) binding protein (CBP; an essential cofactor for activation of transcription by CREB) impair long-term synaptic plasticity and memory. Previously, we showed that small interfering RNA (siRNA)-induced knockdown of CBP in individual sensory neurons significantly reduced levels of CBP and impaired 5-HT-induced long-term facilitation (LTF) in sensorimotor cocultures from Aplysia. Moreover, computational simulations of the biochemical cascades underlying LTF successfully predicted training protocols that restored LTF following CBP knockdown. We examined whether simulations could also predict a training protocol that restores LTF impaired by siRNA-induced knockdown of the transcription factor CREB1. Simulations based on a previously described model predicted rescue protocols that were specific to CREB1 knockdown. Empirical studies demonstrated that one of these rescue protocols partially restored impaired LTF. In addition, the effectiveness of the rescue protocol was enhanced by pretreatment with rolipram, a selective cAMP phosphodiesterase inhibitor. These results provide further evidence that computational methods can help rescue disruptions in signaling cascades underlying memory formation. Moreover, the study demonstrates that the effectiveness of computationally designed training protocols can be enhanced with complementary pharmacological approaches. PMID:25632137

  20. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction

    PubMed Central

    Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2015-01-01

    RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNAmiR) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNAmiRs resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNAmiRs for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences. PMID:26080908

  1. RNAi-Mediated Knockdown of IKK1 in Transgenic Mice Using a Transgenic Construct Containing the Human H1 Promoter

    PubMed Central

    Moreno-Maldonado, Rodolfo; Murillas, Rodolfo; Page, Angustias; Suarez-Cabrera, Cristian; Alameda, Josefa P.; Bravo, Ana; Casanova, M. Llanos

    2014-01-01

    Inhibition of gene expression through siRNAs is a tool increasingly used for the study of gene function in model systems, including transgenic mice. To achieve perdurable effects, the stable expression of siRNAs by an integrated transgenic construct is necessary. For transgenic siRNA expression, promoters transcribed by either RNApol II or III (such as U6 or H1 promoters) can be used. Relatively large amounts of small RNAs synthesis are achieved when using RNApol III promoters, which can be advantageous in knockdown experiments. To study the feasibility of H1 promoter-driven RNAi-expressing constructs for protein knockdown in transgenic mice, we chose IKK1 as the target gene. Our results indicate that constructs containing the H1 promoter are sensitive to the presence of prokaryotic sequences and to transgene position effects, similar to RNApol II promoters-driven constructs. We observed variable expression levels of transgenic siRNA among different tissues and animals and a reduction of up to 80% in IKK1 expression. Furthermore, IKK1 knockdown led to hair follicle alterations. In summary, we show that constructs directed by the H1 promoter can be used for knockdown of genes of interest in different organs and for the generation of animal models complementary to knockout and overexpression models. PMID:24523631

  2. Effect of knockdown of ezrin, radixin, and moesin on P-glycoprotein function in HepG2 cells.

    PubMed

    Kano, Takashi; Wada, Sho; Morimoto, Kaori; Kato, Yukio; Ogihara, Takuo

    2011-12-01

    Ezrin, radixin, and moesin (ERM) proteins regulate functional expression of certain transporters, but little is known about their effect on P-glycoprotein (P-gp). Here, we investigated the influence of ERM proteins on the expression and activity of P-gp at the transcriptional, translational, and posttranslational levels, using HepG2 as a model cell line. Knockdown of ezrin with RNA interference decreased the level of P-gp messenger RNA. On the contrary, knockdown of radixin caused a decrease of the P-gp gene product at the cell surface, but not in whole cell lysate. Furthermore, a significant increase in accumulation of rhodamine123, a typical P-gp substrate, was observed in radixin knockdown cells, compared with control cells. Knockdown of moesin did not influence the expression or function of P-gp. These results indicate that ezrin influences the expression of P-gp at the translational level, whereas radixin is involved in membrane localization of P-gp in HepG2 cells.

  3. Knockdown of LMP1-induced miR-155 sensitizes nasopharyngeal carcinoma cells to radiotherapy in vitro

    PubMed Central

    WANG, YUSHENG; SUN, LE

    2016-01-01

    The present study aimed to confirm the promotion of microRNA (miR)-155 expression by latent membrane protein 1 (LMP1), and to recognize the oncogenic role of LMP1 and LMP1-promoted miR-155 in nasopharyngeal carcinoma (NPC), particularly the influence of miR-155 knockdown on the radiosensitivity of CNE-2 cells. Following the regulation of the levels of LMP1 or miR-155 and/or subsequent to radiation treatment, the proliferation ability of CNE-2 cells was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation and Cell Counting Kit-8 assays. The results demonstrated that miR-155 was upregulated by overexpression of LMP1 in CNE-2 cells, and LMP1 overexpression and miR-155 mimic transfection increased CNE-2 cell proliferation, whereas miR-155 knockdown attenuated the promotion of CNE-2 cell growth induced by LMP1 overexpression. Furthermore, knockdown of miR-155 enhanced the radiosensitivity of CNE-2 cells. In conclusion, the present study confirmed the oncogenic role of miR-155 in NPC, and demonstrated that knockdown of miR-155 inhibited the growth of NPC cells and sensitized NPC cells to radiotherapy. PMID:27123134

  4. ZFX knockdown inhibits growth and migration of non-small cell lung carcinoma cell line H1299.

    PubMed

    Li, Kui; Zhu, Zhi-Chuan; Liu, Yong-Jie; Liu, Ji-Wei; Wang, Hong-Tao; Xiong, Zhi-Qi; Shen, Xu; Hu, Ze-Lan; Zheng, Jing

    2013-01-01

    ZFX (zinc finger transcription factor, X chromosome-linked) contributes to the maintenance of different types of stem cells and the progression of various cancers. We have previously reported that ZFX knockdown inhibits proliferation of glioma in vitro and in vivo. Since overexpression of ZFX in lung cancer tissue correlates with lymph node metastasis, we hypothesized that ZFX may play a role in lung cancer. In this study, we identified ZFX as a promoter of lung cancer growth and migration in a NSCLC (non-small cell lung carcinoma) cell line H1299. ZFX knockdown caused proliferation inhibition determined by MTT assay and colony formation assay, G0/G1 arrest of cell cycle and slightly increased proportion of apoptotic cells assessed by flow cytometry assay, decreased population of migrating cells showed by wound-healing assay, increased cell senescence evidenced by senescence-associated β-galactosidase staining. ZFX knockdown also led to decreased proportion of tumor bearing mice and reduced mean tumor volume in a subcutaneous tumor model. In addition, western blot showed that ZFX knockdown down regulated a set of proteins involved in proliferation, survival and motility. Altogether, these results suggest that ZFX may be a potential therapeutic target for NSCLC.

  5. Efficacy of RNA interference knockdown using aerosolized short interfering RNAs bound to nanoparticles in three diverse aphid species.

    PubMed

    Thairu, M W; Skidmore, I H; Bansal, R; Nováková, E; Hansen, T E; Li-Byarlay, H; Wickline, S A; Hansen, A K

    2017-03-17

    RNA interference (RNAi) has emerged as a promising method for validating gene function; however, its utility in nonmodel insects has proven problematic, with delivery methods being one of the main obstacles. This study investigates a novel method of RNAi delivery in aphids, the aerosolization of short interfering RNA (siRNA)-nanoparticle complexes. By using nanoparticles as a siRNA carrier, the likelihood of cellular uptake is increased, when compared to methods previously used in insects. To determine the efficacy of this RNAi delivery system, siRNAs were aerosolized with and without nanoparticles in three aphid species: Acyrthosiphon pisum, Aphis glycines and Schizaphis graminum. The genes targeted for knockdown were carotene dehydrogenase (tor), which is important for pigmentation in Ac. pisum, and branched chain-amino acid transaminase (bcat), which is essential in the metabolism of branched-chain amino acids in all three aphid species. Overall, we observed modest gene knockdown of tor in Ac. pisum and moderate gene knockdown of bcat in Ap. glycines along with its associated phenotype. We also determined that the nanoparticle emulsion significantly increased the efficacy of gene knockdown. Overall, these results suggest that the aerosolized siRNA-nanoparticle delivery method is a promising new high-throughput and non-invasive RNAi delivery method in some aphid species.

  6. Long- and short-term CDK5 knockdown prevents spatial memory dysfunction and tau pathology of triple transgenic Alzheimer's mice.

    PubMed

    Castro-Alvarez, John F; Uribe-Arias, S Alejandro; Kosik, Kenneth S; Cardona-Gómez, Gloria P

    2014-01-01

    CDK5 is a member of the cyclin-dependent kinase family with diverse functions in both the developing and mature nervous system. The inappropriate activation of CDK5 due to the proteolytic release of the activator fragment p25 from the membrane contributes to the formation of neurofibrillary tangles and chronic neurodegeneration. At 18 months of age 3xTg-AD mice were sacrificed after 1 year (long term) or 3 weeks (short term) of CDK5 knockdown. In long-term animals CDK5 knockdown prevented insoluble Tau formation in the hippocampi and prevented spatial memory impairment. In short-term animals, CDK5 knockdown showed reduction of CDK5, reversed Tau aggregation, and improved spatial memory compared to scrambled treated old 3xTg-AD mice. Neither long-term nor short-term CDK5 knock-down had an effect on old littermates. These findings further validate CDK5 as a target for Alzheimer's disease both as a preventive measure and after the onset of symptoms.

  7. Proteome changes induced by knock-down of the deubiquitylating enzyme HAUSP/USP7.

    PubMed

    Kessler, Benedikt M; Fortunati, Elisabetta; Melis, Monique; Pals, Cornelieke E G M; Clevers, Hans; Maurice, Madelon M

    2007-11-01

    Modification of proteins by ubiquitin plays a major role in a broad array of biological processes. Reversal of this process through deubiquitylation likely represents an important regulatory step in the maintenance of cellular homeostasis. However, the biological functions of deubiquitylating enzymes still remain poorly characterized. To investigate the biological role of the herpes virus-associated ubiquitin-specific protease HAUSP/USP7, we have generated stably transfected cells carrying inducible shRNA expression plasmids. USP7 mRNA and protein were strongly down-regulated 48-72 h after shRNA induction. We used a selected clone to compare whole-cell proteomes by 2D-SDS-PAGE before and after knockdown of USP7. Alterations in 36 proteins were detected and their identities were revealed by mass spectrometry analysis. Components of the replication machinery, DNA/RNA binding proteins, enzymes involved in apoptosis and metabolism were found to be down-regulated upon USP7 removal, representing proteins that are either more rapidly turned over or synthesized less efficiently in the absence of USP7-mediated deubiquitylation. Alix/HP95, a protein implicated in endosomal organization and virus budding, was confirmed by immunoblotting to become down-regulated when USP7 levels were reduced. Our results extend the current list of USP7-dependent biological processes and suggest a role for this enzyme not only in transcriptional regulation but also in DNA replication, apoptosis, and possibly endosomal organization.

  8. Knockdown of poc1b causes abnormal photoreceptor sensory cilium and vision impairment in zebrafish.

    PubMed

    Zhang, Conghui; Zhang, Qi; Wang, Fang; Liu, Qin

    2015-10-02

    Proteomic analysis of the mouse photoreceptor sensory cilium identified a set of cilia proteins, including Poc1 centriolar protein b (Poc1b). Previous functional studies in human cells and zebrafish embryos implicated that Poc1b plays important roles in centriole duplication and length control, as well as ciliogenesis. To study the function of Poc1b in photoreceptor sensory cilia and other primary cilia, we expressed a tagged recombinant Poc1b protein in cultured renal epithelial cells and rat retina. Poc1b was localized to the centrioles and spindle bundles during cell cycle progression, and to the basal body of photoreceptor sensory cilia. A morpholino knockdown and complementation assay of poc1b in zebrafish showed that loss of poc1b led to a range of morphological anomalies of cilia commonly associated with human ciliopathies. In the retina, the development of retinal laminae was significantly delayed and the length of photoreceptor outer segments was shortened. Visual behavior studies revealed impaired visual function in the poc1b morphants. In addition, ciliopathy-associated developmental defects, such as small eyes, curved body axis, heart defects, and shortened cilia in Kupffer's vesicle, were observed as well. These data suggest that poc1b is required for normal development and ciliogenesis of retinal photoreceptor sensory cilia and other cilia. Furthermore, this conclusion is supported by recent findings that mutations in POC1B gene have been identified in patients with inherited retinal dystrophy and syndromic retinal ciliopathy.

  9. Osteopontin knockdown suppresses tumorigenicity of human metastatic breast carcinoma, MDA-MB-435

    PubMed Central

    Shevde, Lalita A.; Samant, Rajeev S.; Paik, Jason C.; Metge, Brandon J.; Chambers, Ann F.; Casey, Graham; Frost, Andra R.; Welch, Danny R.

    2006-01-01

    Elevated expression of osteopontin (OPN), a secreted phosphoglycoprotein, is frequently associated with many transformed cell lines. Various studies suggest that OPN may contribute to tumor progression as well as metastasis in multiple tumor types. High levels of OPN have been reported in patients with metastatic cancers, including breast. We found that the expression of OPN corroborates with the aggressive phenotype of the breast cancer cells i.e. the expression of OPN is acquired as the breast cancer cells become more aggressive. To assess the role(s) of OPN in breast carcinoma, expression of endogenous OPN was knocked down in metastatic MDA-MB-435 human breast carcinoma cells using RNA interference. We targeted multiple regions of the OPN transcript for RNA interference, along with ‘scrambled’ and ‘non-targeting siRNA pool’ controls to distinguish between target-specific and potential off-target effects including interferon-response gene (PeIF2-α) induction. The OPN knockdown by shRNA suppressed tumor take in immunocompromised mice. The ‘silenced’ cells also showed significantly lower invasion and migration in modified Boyden chamber assays and reduced ability to grow in soft agar. Thus, in addition to the widely reported roles of OPN in late stages of tumor progression, these results provide functional evidence that OPN contributes to breast tumor growth as well. PMID:16830223

  10. Knockdown of Lingo1b protein promotes myelination and oligodendrocyte differentiation in zebrafish.

    PubMed

    Yin, Wu; Hu, Bing

    2014-01-01

    Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the central nervous system (CNS), resulting in myelin sheath damage and axonal loss. Leucine-rich repeat and immunoglobulin domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1 (LINGO-1) have been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1 inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. In this study, we cloned the zebrafish homolog of the human LINGO-1 and found that lingo1b regulated myelination and oligodendrocyte differentiation. The expression of lingo1b started 1 (mRNA) and 2 (protein) days post-fertilization (dpf) in the CNS. Morpholino oligonucleotide knockdown of lingo1b resulted in developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord. The lack of lingo1b enhanced myelination and oligodendrocyte differentiation during embryogenesis. Furthermore, immunohistochemistry and movement analysis showed that lingo1b was involved in the axon development of primary motor neurons. These results suggested that Lingo1b protein functions as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development.

  11. Knockdown of proteins involved in iron metabolism limits tick reproduction and development

    PubMed Central

    Hajdusek, Ondrej; Sojka, Daniel; Kopacek, Petr; Buresova, Veronika; Franta, Zdenek; Sauman, Ivo; Winzerling, Joy; Grubhoffer, Libor

    2009-01-01

    Ticks are among the most important vectors of a wide range of human and animal diseases. During blood feeding, ticks are exposed to an enormous amount of free iron that must be appropriately used and detoxified. However, the mechanism of iron metabolism in ticks is poorly understood. Here, we show that ticks possess a complex system that efficiently utilizes, stores and transports non-heme iron within the tick body. We have characterized a new secreted ferritin (FER2) and an iron regulatory protein (IRP1) from the sheep tick, Ixodes ricinus, and have demonstrated their relationship to a previously described tick intracellular ferritin (FER1). By using RNA interference-mediated gene silencing in the tick, we show that synthesis of FER1, but not of FER2, is subject to IRP1-mediated translational control. Further, we find that depletion of FER2 from the tick plasma leads to a loss of FER1 expression in the salivary glands and ovaries that normally follows blood ingestion. We therefore suggest that secreted FER2 functions as the primary transporter of non-heme iron between the tick gut and the peripheral tissues. Silencing of the fer1, fer2, and irp1 genes by RNAi has an adverse impact on hatching rate and decreases postbloodmeal weight in tick females. Importantly, knockdown of fer2 dramatically impairs the ability of ticks to feed, thus making FER2 a promising candidate for development of an efficient anti-tick vaccine. PMID:19171899

  12. Cocaine self-administration in mice with forebrain knock-down of trpc5 ion channels.

    PubMed

    Pomrenze, Matthew B; Baratta, Michael V; Rasmus, Kristin C; Cadle, Brian A; Nakamura, Shinya; Birnbaumer, Lutz; Cooper, Donald C

    2013-01-01

    Canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation and dendritic growth. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain; b) are densely expressed in deep layer pyramidal neurons of the prefrontal cortex (PFC); and c) modulate neuronal persistent activity necessary for working memory and attention. In order to evaluate the causal role of TRPC5 in motivation/reward-related behaviors, conditional forebrain TRPC5 knock-down (trpc5-KD) mice were generated and trained to nose-poke for intravenous cocaine. Here we present a data set containing the first 6 days of saline or cocaine self-administration in wild type (WT) and trpc5-KD mice. In addition, we also present a data set showing the dose-response to cocaine after both groups had achieved similar levels of cocaine self-administration. Compared to WT mice, trpc5-KD mice exhibited an apparent increase in self-administration on the first day of cocaine testing without prior operant training. There were no apparent differences between WT and trpc5-KD mice for saline responding on the first day of training. Both groups showed similar dose-response sensitivity to cocaine after several days of achieving similar levels of cocaine intake.

  13. Cocaine self-administration in mice with forebrain knock-down of trpc5 ion channels

    PubMed Central

    Cooper, Donald C

    2013-01-01

    Canonical transient receptor potential (TRPC) channels are a family of non-selective cation channels that play a crucial role in modulating neuronal excitability due to their involvement in intracellular Ca2+ regulation and dendritic growth. TRPC5 channels a) are one of the two most prevalent TRPC channels in the adult rodent brain; b) are densely expressed in deep layer pyramidal neurons of the prefrontal cortex (PFC); and c) modulate neuronal persistent activity necessary for working memory and attention. In order to evaluate the causal role of TRPC5 in motivation/reward-related behaviors, conditional forebrain TRPC5 knock-down (trpc5-KD) mice were generated and trained to nose-poke for intravenous cocaine. Here we present a data set containing the first 6 days of saline or cocaine self-administration in wild type (WT) and trpc5-KD mice. In addition, we also present a data set showing the dose-response to cocaine after both groups had achieved similar levels of cocaine self-administration. Compared to WT mice, trpc5-KD mice exhibited an apparent increase in self-administration on the first day of cocaine testing without prior operant training. There were no apparent differences between WT and trpc5-KD mice for saline responding on the first day of training. Both groups showed similar dose-response sensitivity to cocaine after several days of achieving similar levels of cocaine intake. PMID:24358869

  14. Expression Analysis and Knockdown of Two Antennal Odorant-Binding Protein Genes in Aedes aegypti

    PubMed Central

    Sengul, Meryem S.; Tu, Zhijian

    2010-01-01

    The presence and expression of odorant-binding proteins (OBPs) in the olfactory organs suggest that they play an important role in mosquito olfaction. However, no direct evidence has been found for their involvement in the host-seeking behavior of mosquitoes. It is important to establish a method in which a loss-of-function test can be performed to determine the possible role of these genes in olfaction. In this study, a double subgenomic Sindbis virus expression system was used to reduce the expression of two Obp genes in Aedes aegypti L (Diptera: Culicidae), AaegObp1 and AaegObp2. Quantitative real-time PCR analysis showed predominant expression of both genes in the female antennae, the primary olfactory tissue of mosquitoes. Moreover, at 11 days post virus-inoculation, the mRNA levels of AaegObp1 and AaegObp2 were significantly reduced in olfactory tissues of recombinant virus-inoculated female mosquitoes compared to that of controls by approximately 8 and 100-fold, respectively. These data suggest that the double subgenomic Sindbis virus expression system can be efficiently used to knockdown Obp gene expression in olfactory tissues of mosquitoes. We discuss the potential for a systematic analysis of the molecular players involved in mosquito olfaction using this newly developed technique. Such analysis will provide an important step to interfere with the host-seeking behavior of mosquitoes to prevent the transmission of diseases. PMID:21062207

  15. Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes

    PubMed Central

    Place, Elsie S.; Smith, James C.

    2017-01-01

    Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos. PMID:28192479

  16. A PATO-compliant zebrafish screening database (MODB): management of morpholino knockdown screen information.

    PubMed

    Knowlton, Michelle N; Li, Tongbin; Ren, Yongliang; Bill, Brent R; Ellis, Lynda Bm; Ekker, Stephen C

    2008-01-07

    The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies. The MOrpholino DataBase is a MySQL relational database with an online, PHP interface. Multiple quality control levels allow differential access to data in raw and finished formats. MODBv1 includes sequence information relating to almost 800 morpholinos and their targets and phenotypic data regarding the dose effect of each morpholino (mortality, toxicity and defects). To improve the searchability of this database, we have incorporated a fixed-vocabulary defect ontology that allows for the organization of morpholino affects based on anatomical structure affected and defect produced. This also allows comparison between species utilizing Phenotypic Attribute Trait Ontology (PATO) designated terminology. MODB is also cross-linked with ZFIN, allowing full searches between the two databases. MODB offers users the ability to retrieve morpholino data by sequence of morpholino or target, name of target, anatomical structure affected and defect produced. MODB data can be used for functional genomic analysis of morpholino design to maximize efficacy and minimize toxicity. MODB also serves as a template for future sequence-based functional genetic screen databases, and it is currently being used as a model for the creation of a mutagenic insertional transposon database.

  17. Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice

    PubMed Central

    Carrell, Samuel T.; Carrell, Ellie M.; Auerbach, David; Pandey, Sanjay K.; Bennett, C. Frank; Dirksen, Robert T.; Thornton, Charles A.

    2016-01-01

    Myotonic dystrophy type 1 (DM1) is a genetic disorder in which dominant-active DM protein kinase (DMPK) transcripts accumulate in nuclear foci, leading to abnormal regulation of RNA processing. A leading approach to treat DM1 uses DMPK-targeting antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. However, basal levels of DMPK protein are reduced by half in DM1 patients. This raises concern that intolerance for further DMPK loss may limit ASO therapy, especially since mice with Dmpk gene deletion reportedly show cardiac defects and skeletal myopathy. We re-examined cardiac and muscle function in mice with Dmpk gene deletion, and studied post-maturity knockdown using Dmpk-targeting ASOs in mice with heterozygous deletion. Contrary to previous reports, we found no effect of Dmpk gene deletion on cardiac or muscle function, when studied on two genetic backgrounds. In heterozygous knockouts, the administration of ASOs reduced Dmpk expression in cardiac and skeletal muscle by > 90%, yet survival, electrocardiogram intervals, cardiac ejection fraction and muscle strength remained normal. The imposition of cardiac stress by pressure overload, or muscle stress by myotonia, did not unmask a requirement for DMPK. Our results support the feasibility and safety of using ASOs for post-transcriptional silencing of DMPK in muscle and heart. PMID:27522499

  18. Proteomics analysis of E-cadherin knockdown in epithelial breast cancer cells.

    PubMed

    Vergara, Daniele; Simeone, Pasquale; Latorre, Dominga; Cascione, Francesca; Leporatti, Stefano; Trerotola, Marco; Giudetti, Anna Maria; Capobianco, Loredana; Lunetti, Paola; Rizzello, Antonia; Rinaldi, Rosaria; Alberti, Saverio; Maffia, Michele

    2015-05-20

    E-cadherin is the core protein of the epithelial adherens junction. Through its cytoplasmic domain, E-cadherin interacts with several signaling proteins; among them, α- and β-catenins mediate the link of E-cadherin to the actin cytoskeleton. Loss of E-cadherin expression is a crucial step of epithelial-mesenchymal transition (EMT) and is involved in cancer invasion and metastatization. In human tumors, down-regulation of E-cadherin is frequently associated with poor prognosis. Despite the critical role of E-cadherin in cancer progression, little is known about proteome alterations linked with its down-regulation. To address this point, we investigated proteomics, biophysical and functional changes of epithelial breast cancer cell lines upon shRNA-mediated stable knockdown of E-cadherin expression (shEcad). shEcad cells showed a distinct proteomic signature including altered expression of enzymes and proteins involved in cytoskeletal dynamic and migration. Moreover, these results suggest that, besides their role in mechanical adhesion, loss of E-cadherin expression may contribute to cancer progression by modifying a complex network of pathways that tightly regulate fundamental processes as oxidative stress, immune evasion and cell metabolism. Altogether, these results extend our knowledge on the cellular modifications associated with E-cadherin down-regulation in breast cancer cells.

  19. Sex determination in beetles: Production of all male progeny by Parental RNAi knockdown of transformer

    PubMed Central

    Shukla, Jayendra Nath; Palli, Subba Reddy

    2012-01-01

    Sex in insects is determined by a cascade of regulators ultimately controlling sex-specific splicing of a transcription factor, Doublesex (Dsx). We recently identified homolog of dsx in the red flour beetle, Tribolium castaneum (Tcdsx). Here, we report on the identification and characterization of a regulator of Tcdsx splicing in T. castaneum. Two male-specific and one female-specific isoforms of T. castaneum transformer (Tctra) were identified. RNA interference-aided knockdown of Tctra in pupa or adults caused a change in sex from females to males by diverting the splicing of Tcdsx pre-mRNA to male-specific isoform. All the pupa and adults developed from Tctra dsRNA injected final instar larvae showed male-specific sexually dimorphic structures. Tctra parental RNAi caused an elimination of females from the progeny resulting in production of all male progeny. Transformer parental RNAi could be used to produce all male population for use in pest control though sterile male release methods. PMID:22924109

  20. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection.

    PubMed

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes.

  1. Knockdown of ELMO3 Suppresses Growth, Invasion and Metastasis of Colorectal Cancer

    PubMed Central

    Peng, Hui-Yun; Yu, Qiong-Fang; Shen, Wei; Guo, Cheng-Ming; Li, Zhen; Zhou, Xiao-Yan; Zhou, Nan-Jin; Min, Wei-Ping; Gao, Dian

    2016-01-01

    The engulfment and cell motility (ELMOs) family of proteins plays a crucial role in tumor cell migration and invasion. However, the function of ELMO3 is poorly defined. To elucidate its role in the development and progression of colorectal cancer (CRC), we examined the expression of ELMO3 in 45 cases of paired CRC tumor tissues and adjacent normal tissues. Furthermore, we assessed the effect of the knockdown of ELMO3 on cell proliferation, cell cycle, migration, invasion and F-actin polymerization in HCT116 cells. The result shows that the expression of ELMO3 in CRC tissues was significantly increased in comparison to the adjacent normal colorectal tissues. Moreover, this overexpression was associated with tumor size (p = 0.007), tumor differentiation (p = 0.001), depth of invasion (p = 0.009), lymph node metastasis (p = 0.003), distant metastasis (p = 0.013) and tumor, node, metastasis (TNM)-based classification (p = 0.000). In in vitro experiments, the silencing of ELMO3 inhibited cell proliferation, invasion, metastasis, and F-actin polymerization, and induced Gap 1 (G1) phase cell cycle arrest. Our study demonstrates that ELMO3 is involved in the processes of growth, invasion and metastasis of CRC, and could be used a potential molecular diagnostic tool or therapy target of CRC. PMID:27999268

  2. Knockdown of USP39 induces cell cycle arrest and apoptosis in melanoma.

    PubMed

    Zhao, Yuan; Zhang, Bo; Lei, Yu; Sun, Jingying; Zhang, Yaohua; Yang, Sen; Zhang, Xuejun

    2016-10-01

    The spliceosome machinery composed of multimeric protein complexes guides precursor messenger RNAs (mRNAs) (pre-mRNAs) splicing in eukaryotic cells. Spliceosome components have been shown to be downregulated in cancer and could be a promising molecular target for anticancer therapy. The ubiquitin-specific protease 39 (USP39) is essential for pre-mRNA splicing, and upregulated USP39 expression is noted in a variety of cancers. However, the role of USP39 in the development and progression of melanoma remains unclear. In the present study, USP39 expression was found to be increased in melanoma tissues compared with that in nevus tissues. USP39 silencing via lentivirus-mediated short hairpin RNA (shRNA) significantly suppressed melanoma cell proliferation, induced G0/G1 cell cycle phase arrest, and increased apoptosis in vitro. Moreover, USP39 knockdown suppressed melanoma tumor growth in a xenograft model. In addition, USP39 silencing was associated with the increased expressions of p21, p27, and Bax. Furthermore, the inhibition of USP39 expression decreased the phosphorylation of extracellular signal-regulated kinase (ERK)1/2, indicating that ERK signaling pathways might be involved in the regulation of melanoma cell proliferation by USP39. Our findings suggest that USP39 may play crucial roles in the development and pathogenesis of melanoma, and it may serve as a potential therapeutic target for melanoma.

  3. Versican knock-down compromises chondrogenesis in the embryonic chick limb.

    PubMed

    Shepard, John B; Gliga, Diana A; Morrow, Amanda P; Hoffman, Stanley; Capehart, Anthony A

    2008-01-01

    Mesenchymal cell aggregation is critical for cartilage formation in the vertebrate limb. The extracellular matrix (ECM) plays a critical role in governing cell behavior and cell phenotype in this tissue, and the hyalectin versican is highly expressed in the ECM of precartilage mesenchymal cells and developing synovial joints. Although several in vitro studies have been conducted in an attempt to address versican's role during limb mesenchymal condensation, factors such as differences in cell density in culture, variations between chondrogenic cell lines, and the inability to prolong the viability of limb explants have led to conflicting data, mandating an in vivo analysis. By using a morpholino directed strategy in ovo, we performed knock-down of versican expression in the presumptive ulnar region of the developing chick wing at time points critical to skeletogenesis. These data indicate that in ovo misexpression of versican compromised mesenchymal condensation with resulting ulnar cartilages reduced in length distally by an average of 53% relative to contralateral control limbs. In select versican morphants the olecranon process was also reduced in size proximally and failed to cup the humerus, likely impairing joint morphogenesis. This study represents the first report assessing the role of versican in the developing chick limb in ovo, further demonstrating the importance of versican proteoglycan expression during chondrogenesis and extending previous findings to suggest a role for versican during synovial joint development.

  4. The Knockdown of αkap Alters the Postsynaptic Apparatus of Neuromuscular Junctions in Living Mice

    PubMed Central

    Martinez-Pena y Valenzuela, Isabel; Aittaleb, Mohamed; Chen, Po-Ju

    2015-01-01

    A muscle-specific nonkinase anchoring protein (αkap), encoded within the calcium/calmodulin kinase II (camk2) α gene, was recently found to control the stability of acetylcholine receptor (AChR) clusters on the surface of cultured myotubes. However, it remains unknown whether this protein has any effect on receptor stability and the maintenance of the structural integrity of neuromuscular synapses in vivo. By knocking down the endogenous expression of αkap in mouse sternomastoid muscles with shRNA, we found that the postsynaptic receptor density was dramatically reduced, the turnover rate of receptors at synaptic sites was significantly increased, and the insertion rates of both newly synthesized and recycled receptors into the postsynaptic membrane were depressed. Moreover, we found that αkap shRNA knockdown impaired synaptic structure as postsynaptic AChR clusters and their associated postsynaptic scaffold proteins within the neuromuscular junction were completely eliminated. These results provide new mechanistic insight into the role of αkap in regulating the stability of the postsynaptic apparatus of neuromuscular synapses. PMID:25834039

  5. Duplication and Retention Biases of Essential and Non-Essential Genes Revealed by Systematic Knockdown Analyses

    PubMed Central

    Rivers, David; Warnecke, Tobias; Jeffries, Sean J.; Kwon, Taejoon; Rogers, Anthony; Hurst, Laurence D.; Ahringer, Julie

    2013-01-01

    When a duplicate gene has no apparent loss-of-function phenotype, it is commonly considered that the phenotype has been masked as a result of functional redundancy with the remaining paralog. This is supported by indirect evidence showing that multi-copy genes show loss-of-function phenotypes less often than single-copy genes and by direct tests of phenotype masking using select gene sets. Here we take a systematic genome-wide RNA interference approach to assess phenotype masking in paralog pairs in the Caenorhabditis elegans genome. Remarkably, in contrast to expectations, we find that phenotype masking makes only a minor contribution to the low knockdown phenotype rate for duplicate genes. Instead, we find that non-essential genes are highly over-represented among duplicates, leading to a low observed loss-of-function phenotype rate. We further find that duplicate pairs derived from essential and non-essential genes have contrasting evolutionary dynamics: whereas non-essential genes are both more often successfully duplicated (fixed) and lost, essential genes are less often duplicated but upon successful duplication are maintained over longer periods. We expect the fundamental evolutionary duplication dynamics presented here to be broadly applicable. PMID:23675306

  6. Conservation, expression, and knockdown of zebrafish plxnb2a and plxnb2b.

    PubMed

    Perälä, Nina; Peitsaro, Nina; Sundvik, Maria; Koivula, Henri; Sainio, Kirsi; Sariola, Hannu; Panula, Pertti; Immonen, Tiina

    2010-10-01

    In mice lacking Plexin B2, a receptor of the axon guidance molecules Semaphorin 4C and Semaphorin 4D, the closure of the neural tube and structural organization of the cerebellum are severely impaired. We cloned two Plexin B2 orthologs, plxnb2a and plxnb2b, in zebrafish, which is a widely used model for the development of the vertebrate central nervous system (CNS). The predicted proteins, Plexin B2a and Plexin B2b, contain all the conserved and functional domains of the plexin B-subfamily. During embryonic development, plxnb2a is expressed, e.g., in pharyngeal arches while plxnb2b expression is more confined to neuronal structures like the cerebellum. However, both plxnb2a and plxnb2b are expressed at the midbrain-hindbrain boundary, in the otic vesicles, facial ganglia, and pectoral fins. Knockdown of both plxnb2a and plxnb2b simultaneously (>95% and 45%, respectively) resulted in normal CNS structure, axon guidance and swimming performance of the morphants.

  7. Knockdown of Unconventional Myosin ID Expression Induced Morphological Change in Oligodendrocytes

    PubMed Central

    Yamazaki, Reiji; Ishibashi, Tomoko; Baba, Hiroko

    2016-01-01

    Myelin is a special multilamellar structure involved in various functions in the nervous system. In the central nervous system, the oligodendrocyte (OL) produces myelin and has a unique morphology. OLs have a dynamic membrane sorting system associated with cytoskeletal organization, which aids in the production of myelin. Recently, it was reported that the assembly and disassembly of actin filaments is crucial for myelination. However, the partner myosin molecule which associates with actin filaments during the myelination process has not yet been identified. One candidate myosin is unconventional myosin ID (Myo1d) which is distributed throughout central nervous system myelin; however, its function is still unclear. We report here that Myo1d is expressed during later stages of OL differentiation, together with myelin proteolipid protein (PLP). In addition, Myo1d is distributed at the leading edge of the myelin-like membrane in cultured OL, colocalizing mainly with actin filaments, 2′,3′-cyclic nucleotide phosphodiesterase and partially with PLP. Myo1d-knockdown with specific siRNA induces significant morphological changes such as the retraction of processes and degeneration of myelin-like membrane, and finally apoptosis. Furthermore, loss of Myo1d by siRNA results in the impairment of intracellular PLP transport. Together, these results suggest that Myo1d may contribute to membrane dynamics either in wrapping or transporting of myelin membrane proteins during formation and maintenance of myelin. PMID:27655972

  8. RNAi-based conditional gene knockdown in mice using a U6 promoter driven vector.

    PubMed

    Shukla, Vivek; Coumoul, Xavier; Deng, Chu-Xia

    2007-01-05

    RNA interference (RNAi) is a powerful tool widely used for studying gene function in a number of species. We have previously developed an approach that allows conditional expression of a polymerase III promoter based small hairpin RNA (shRNA) in mice using the Cre-LoxP system. This approach uses a U6 promoter, which is inactive due to the presence of a ploxPneo cassette in the promoter; this promoter can be activated after excision of the neo gene in transgenic mice that express a Cre recombinase transgene. As a proof of principle, we have previously knocked down over 95% of Fgfr2 transcripts in mouse germlines, leading to embryonic lethality, while restricting the knockdown to the progress zone of the limb results in live animals with malformation of digits of both the forelimbs and hindlimbs. We now provide a detailed protocol, including a simplified single-step cloning procedure for vector construction. This method provides a fast yet efficient way to decipher gene functions in vivo in a tissue specific manner.

  9. Knockdown of TCTN1 Strongly Decreases Growth of Human Colon Cancer Cells

    PubMed Central

    Dai, Xiaoyu; Dong, Mingjun; Yu, Hua; Xie, Yangyang; Yu, Yongming; Cao, Yisheng; Kong, Zhenfang; Zhou, Baofeng; Xu, Yidong; Yang, Tong; Li, Keqiang

    2017-01-01

    Background Tectonic family member 1 (TCTN1), a member of the tectonic family, is involved in several developmental processes and is aberrantly expressed in multiple solid tumors. However, the expression and regulation of TCTN1 in human colorectal cancer (CRC) is still not clear. Material/Methods The expression of TCTN1 mRNA was first explored by using Oncomine microarray datasets. TCTN1 expression was silenced in human CRC cell lines HCT116 and SW1116 via RNA interference (RNAi). Furthermore, we investigated the effect of TCTN1 depletion on CRC cell growth by MTT, colony formation, and flow cytometry in vitro. Results In this study, meta-analysis showed that the expressions of TCTN1 mRNA in CRC specimens were significantly higher than that in normal specimens. Knockdown of TCTN1 expression potently inhibited the abilities of cell proliferation and colony formation as determined. Flow cytometry analysis showed that depletion of TCTN1 could cause cell cycle arrest at the G2/M phase. In addition, Annexin V/7-AAD double-staining indicated that TCTN1 silencing promoted cell apoptosis through down-regulation of caspase 3 and Bcl-2 and upregulation of cleaved caspase 3 and PARP. Conclusions Our results indicate that TCTN1 may be crucial for CRC cell growth, providing a novel alternative to target therapies of CRC. Further research on this topic is warranted. PMID:28123172

  10. p27 Nuclear localization and growth arrest caused by perlecan knockdown in human endothelial cells

    SciTech Connect

    Sakai, Katsuya; Oka, Kiyomasa; Matsumoto, Kunio; Nakamura, Toshikazu

    2010-02-12

    Perlecan, a secreted heparan sulfate proteoglycan, is a major component of the vascular basement membrane and participates in angiogenesis. Here, we used small interference RNA-mediated knockdown of perlecan expression to investigate the regulatory function of perlecan in the growth of human vascular endothelial cells. Basic fibroblast growth factor (bFGF)-induced ERK phosphorylation and cyclin D1 expression were unchanged by perlecan deficiency in endothelial cells; however, perlecan deficiency inhibited the Rb protein phosphorylation and DNA synthesis induced by bFGF. By contrast to cytoplasmic localization of the cyclin-dependent kinase inhibitor p27 in control endothelial cells, p27 was localized in the nucleus and its expression increased in perlecan-deficient cells, which suggests that p27 mediates inhibition of Rb phosphorylation. In addition to the well-characterized function of perlecan as a co-receptor for heparin-binding growth factors such as bFGF, our results suggest that perlecan plays an indispensible role in endothelial cell proliferation and acts through a mechanism that involves subcellular localization of p27.

  11. SHMT1 knockdown induces apoptosis in lung cancer cells by causing uracil misincorporation

    PubMed Central

    Paone, A; Marani, M; Fiascarelli, A; Rinaldo, S; Giardina, G; Contestabile, R; Paiardini, A; Cutruzzolà, F

    2014-01-01

    Reprogramming of cellular metabolism towards de novo serine production fuels the growth of cancer cells, providing essential precursors such as amino acids and nucleotides and controlling the antioxidant and methylation capacities of the cell. The enzyme serine hydroxymethyltransferase (SHMT) has a key role in this metabolic shift, and directs serine carbons to one-carbon units metabolism and thymidilate synthesis. While the mitochondrial isoform of SHMT (SHMT2) has recently been identified as an important player in the control of cell proliferation in several cancer types and as a hot target for anticancer therapies, the role of the cytoplasmic isoform (SHMT1) in cancerogenesis is currently less defined. In this paper we show that SHMT1 is overexpressed in tissue samples from lung cancer patients and lung cancer cell lines, suggesting that, in this widespread type of tumor, SHMT1 plays a relevant role. We show that SHMT1 knockdown in lung cancer cells leads to cell cycle arrest and, more importantly, to p53-dependent apoptosis. Our data demonstrate that the induction of apoptosis does not depend on serine or glycine starvation, but is because of the increased uracil accumulation during DNA replication. PMID:25412303

  12. Focused ultrasound for targeted delivery of siRNA and efficient knockdown of Htt expression.

    PubMed

    Burgess, Alison; Huang, Yuexi; Querbes, William; Sah, Dinah W; Hynynen, Kullervo

    2012-10-28

    RNA interference is a promising strategy for the treatment of Huntington's disease (HD) as it can specifically decrease the expression of the mutant Huntingtin protein (Htt). However, siRNA does not cross the blood-brain barrier and therefore delivery to the brain is limited to direct CNS delivery. Non-invasive delivery of siRNA through the blood-brain barrier (BBB) would be a significant advantage for translating this therapy to HD patients. Focused ultrasound (FUS), combined with intravascular delivery of microbubble contrast agent, was used to locally and transiently disrupt the BBB in the right striatum of adult rats. 48h following treatment with siRNA, the right (treated) and the left (control) striatum were dissected and analyzed for Htt mRNA levels. We demonstrate that FUS can non-invasively deliver siRNA-Htt directly to the striatum leading to a significant reduction of Htt expression in a dose dependent manner. Furthermore, we show that reduction of Htt with siRNA-Htt was greater when the extent of BBB disruption was increased. This study demonstrates that siRNA treatment for knockdown of mutant Htt is feasible without the surgical intervention previously required for direct delivery to the brain.

  13. Efficient Gene Knockdown in Mouse Oocytes through Peptide Nanoparticle-Mediated SiRNA Transfection

    PubMed Central

    Jin, Zhen; Li, Ruichao; Zhou, Chunxiang; Shi, Liya; Zhang, Xiaolan; Yang, Zhixia; Zhang, Dong

    2016-01-01

    The use of mouse oocytes as a model for studying female meiosis is very important in reproductive medicine. Gene knockdown by specific small interfering RNA (siRNA) is usually the first step in the study of the function of a target gene in mouse oocytes during in vitro maturation. Traditionally, the only way to introduce siRNA into mouse oocytes is through microinjection, which is certainly less efficient and strenuous than siRNA transfection in somatic cells. Recently, in research using somatic cells, peptide nanoparticle-mediated siRNA transfection has been gaining popularity over liposome nanoparticle-mediated methods because of its high efficiency, low toxicity, good stability, and strong serum compatibility. However, no researchers have yet tried transfecting siRNA into mouse oocytes because of the existence of the protective zona pellucida surrounding the oocyte membrane (vitelline membrane). We therefore tested whether peptide nanoparticles can introduce siRNA into mouse oocytes. In the present study, we showed for the first time that our optimized program can efficiently knock down a target gene with high specificity. Furthermore, we achieved the expected meiotic phenotypes after we knocked down a test unknown target gene TRIM75. We propose that peptide nanoparticles may be superior for preliminary functional studies of unknown genes in mouse oocytes. PMID:26974323

  14. DJ-1 knock-down in astrocytes impairs astrocyte-mediated neuroprotection against rotenone.

    PubMed

    Mullett, Steven J; Hinkle, David A

    2009-01-01

    Mutations that eliminate DJ-1 expression cause a familial form of Parkinson's disease (PD). In sporadic PD, and many other neurodegenerative diseases, reactive astrocytes over-express DJ-1 whereas neurons maintain its expression at non-disease levels. Since DJ-1 has neuroprotective properties, and since astrocytes are known to support and protect neurons, DJ-1 over-expression in reactive astrocytes may reflect an attempt by these cells to protect themselves and surrounding neurons against disease progression. We used neuron-astrocyte contact and non-contact co-cultures to show that DJ-1 knock-down in astrocytes impaired their neuroprotective capacity, relative to wild-type astrocytes, against the neurotoxin rotenone. Conversely, DJ-1 over-expression in astrocytes augmented their neuroprotective capacity. Experiments using astrocyte conditioned media on neuron-only cultures suggested that astrocyte-released, soluble factors were involved in the DJ-1-dependent, astrocyte-mediated neuroprotective mechanism. Our findings support the developing view that astrocytic dysfunction, in addition to neuronal dysfunction, may contribute to the progression of a variety of neurodegenerative disorders.

  15. si-RNA-mediated knockdown of PDLIM5 suppresses gastric cancer cell proliferation in vitro.

    PubMed

    Li, Yanliang; Gao, Yongsheng; Xu, Yue; Sun, Xianjun; Song, Xilin; Ma, Heng; Yang, Mingshan

    2015-04-01

    Gastric cancer is the second most prominent cause of cancer mortality in the world. This study was designed to identify the possible use of si-RNA-mediated PDLIM5 gene silencing as a therapeutic tool for gastric cancer. Expression levels of PDLIM5 were detected in several gastric cancer cell lines using Western blot and qRT-PCR. We found PDLIM5 is highly expressed in all cultured gastric cancer cell lines. Small interfering RNA (si-RNA) was then employed to knock down PDLIM5 expression in MGC80-3 gastric cancer cells. Knockdown of PDLIM5 significantly inhibited cell proliferation and colony formation. Moreover, the absence of PDLIM5 in MGC80-3 cells led to S phase cell cycle arrest and apoptosis. This study highlights the critical role of PDLIM5 in gastric cancer cell growth and suggests that si-RNA-mediated silencing of PDLIM5 might serve as a potential therapeutic approach for the treatment of gastric cancer.

  16. Effective knockdown of Drosophila long non-coding RNAs by CRISPR interference

    PubMed Central

    Ghosh, Sanjay; Tibbit, Charlotte; Liu, Ji-Long

    2016-01-01

    Long non-coding RNAs (lncRNAs) have emerged as regulators of gene expression across metazoa. Interestingly, some lncRNAs function independently of their transcripts – the transcription of the lncRNA locus itself affects target genes. However, current methods of loss-of-function analysis are insufficient to address the role of lncRNA transcription from the transcript which has impeded analysis of their function. Using the minimal CRISPR interference (CRISPRi) system, we show that coexpression of the catalytically inactive Cas9 (dCas9) and guide RNAs targeting the endogenous roX locus in the Drosophila cells results in a robust and specific knockdown of roX1 and roX2 RNAs, thus eliminating the need for recruiting chromatin modifying proteins for effective gene silencing. Additionally, we find that the human and Drosophila codon optimized dCas9 genes are functional and show similar transcription repressive activity. Finally, we demonstrate that the minimal CRISPRi system suppresses roX transcription efficiently in vivo resulting in loss-of-function phenotype, thus validating the method for the first time in a multicelluar organism. Our analysis expands the genetic toolkit available for interrogating lncRNA function in situ and is adaptable for targeting multiple genes across model organisms. PMID:26850642

  17. Rb knockdown accelerates bladder cancer progression through E2F3 activation.

    PubMed

    Wang, Jiang-Ping; Jiao, Yong; Wang, Cheng-Yuan; Xu, Zhi-Bin; Zhang, Bo

    2017-01-01

    Bladder cancer is one of the most common cancers diagnosed in the world and leads to significant mortality and morbidity among affected patients. The retinoblastoma (Rb) protein is a main tumor suppressor, controlling cellular responses to potentially oncogenic stimulation. E2F3 was invariably disrupted in different human cancers for its central role in the control of cellular proliferation. Here, we investigated how Rb is integrated to control bladder cancer progression through E2F3 and p53 regulation. The results exhibit that Rb expression is lower in patients with bladder tumor, while E2F3 level is high. Rb knockdown enhanced bladder tumor cell proliferation and migration, aggravated with p53 silence. Interestingly, Rb silence results in E2F3, Myc and mTOR signaling pathway activation, contributing to bladder cancer cell proliferation and apoptosis suppression mainly through caspase-3 inhibition in vitro and in vivo. Immunohistochemical analysis revealed that Rb is highly expressed in normal bladder cells, but was repressed in tumor tissues of the bladder completely, suggesting a possible role of Rb as a tumor suppressor.

  18. Targeted knockdown of polo-like kinase 1 alters metabolic regulation in melanoma.

    PubMed

    Gutteridge, Rosie Elizabeth Ann; Singh, Chandra K; Ndiaye, Mary Ann; Ahmad, Nihal

    2017-05-28

    A limited number of studies have indicated an association of the mitotic kinase polo-like kinase 1 (PLK1) and cellular metabolism. Here, employing an inducible RNA interference approach in A375 melanoma cells coupled with a PCR array and multiple validation approaches, we demonstrated that PLK1 alters a number of genes associated with cellular metabolism. PLK1 knockdown resulted in a significant downregulation of IDH1, PDP2 and PCK1 and upregulation of FBP1. Ingenuity Pathway Analysis (IPA) identified that 1) glycolysis and the pentose phosphate pathway are major canonical pathways associated with PLK1, and 2) PLK1 inhibition-modulated genes were largely associated with cellular proliferation, with FBP1 being the key modulator. Further, BI 6727-mediated inhibition of PLK1 caused a decrease in PCK1 and increase in FBP1 in A375 melanoma cell implanted xenografts in vivo. Furthermore, an inverse correlation between PLK1 and FBP1 was found in melanoma cells, with FBP1 expression significantly downregulated in a panel of melanoma cells. In addition, BI 6727 treatment resulted in an upregulation in FBP1 in A375, Hs294T and G361 melanoma cells. Overall, our study suggests that PLK1 may be an important regulator of metabolism maintenance in melanoma cells. Published by Elsevier B.V.

  19. Multi-gene engineering: simultaneous expression and knockdown of six genes off a single platform.

    PubMed

    Greber, David; Fussenegger, Martin

    2007-04-01

    Increases in our understanding of gene function have greatly expanded the repertoire of possible genetic interventions at our disposal with the consequence that many genetic engineering applications require multiple manipulations in which target genes can be both overexpressed and silenced in a simple and co-ordinated manner. Using synthetic introns as a source of encoding short-interfering RNA (siRNA), we demonstrate that it is possible to simultaneously express both a transgene and siRNA from a single polymerase (Pol) II promoter. By encoding siRNA as an intron between two protein domains requiring successful splicing for functionality, it was possible to demonstrate that splicing was occurring, that the coding genes (exonic transgenes) resulted in functional protein, and that the spliced siRNA-containing lariat was capable of modulating expression of a separate target gene. We subsequently extended this concept to develop pTRIDENT-based multi-cistronic vectors that were capable of co-ordinated expression of up to three siRNAs and three transgenes off a single genetic platform. Such multi-gene engineering technology, enabling concomitant transgene overexpression and target gene knockdown, should be useful for therapeutic, biopharmaceutical production, and basic research applications.

  20. Genetic Architecture of a Hormonal Response to Gene Knockdown in Honey Bees

    PubMed Central

    Rueppell, Olav; Huang, Zachary Y.; Wang, Ying; Fondrk, M. Kim; Page, Robert E.; Amdam, Gro V.

    2015-01-01

    Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general. PMID:25596612

  1. Comparison of Zebrafish tmem88a mutant and morpholino knockdown phenotypes.

    PubMed

    Eve, Alexander M J; Place, Elsie S; Smith, James C

    2017-01-01

    Tmem88a is a transmembrane protein that is thought to be a negative regulator of the Wnt signalling pathway. Several groups have used antisense morpholino oligonucleotides in an effort to characterise the role of tmem88a in zebrafish cardiovascular development, but they have not obtained consistent results. Here, we generate an 8 bp deletion in the coding region of the tmem88a locus using TALENs, and we have gone on to establish a viable homozygous tmem88aΔ8 mutant line. Although tmem88aΔ8 mutants have reduced expression of some key haematopoietic genes, differentiation of erythrocytes and neutrophils is unaffected, contradicting our previous study using antisense morpholino oligonucleotides. We find that expression of the tmem88a paralogue tmem88b is not significantly changed in tmem88aΔ8 mutants and injection of the tmem88a splice-blocking morpholino oligonucleotide into tmem88aΔ8 mutants recapitulates the reduction of erythrocytes observed in morphants using o-Dianisidine. This suggests that there is a partial, but inessential, requirement for tmem88a during haematopoiesis and that morpholino injection exacerbates this phenotype in tmem88a morpholino knockdown embryos.

  2. Adipose tissue insulin receptor knockdown via a new primate-derived hybrid recombinant AAV serotype

    PubMed Central

    Liu, Xianglan; Magee, Daniel; Wang, Chuansong; McMurphy, Travis; Slater, Andrew; During, Matthew; Cao, Lei

    2014-01-01

    Adipose tissue plays an essential role in metabolic homeostasis and holds promise as an alternative depot organ in gene therapy. However, efficient methods of gene transfer into adipose tissue in vivo have yet to be established. Here, we assessed the transduction efficiency to fat depots by a family of novel engineered hybrid capsid serotypes (Rec1~4) recombinant adeno-associated viral (AAV) vectors in comparison with natural serotypes AAV1, AAV8, and AAV9. Rec2 serotype led to widespread transduction in both brown fat and white fat with the highest efficiency among the seven serotypes tested. As a proof-of-efficacy, Rec2 serotype was used to deliver Cre recombinase to adipose tissues of insulin receptor floxed animals. Insulin receptor knockdown led to decreased fat pad mass and morphological and molecular changes in the targeted depot. These novel hybrid AAV vectors can serve as powerful tools to genetically manipulate adipose tissue and provide valuable vehicles to gene therapy targeting adipose tissue. PMID:25383359

  3. Induction of fibrillin-2 and periostin expression in Osterix-knockdown MC3T3-E1 cells.

    PubMed

    Lee, So-Jeong; Lee, Eun-Hye; Park, Seung-Yoon; Kim, Jung-Eun

    2017-01-05

    Osteoporosis is the most common age-related bone disease that is characterized by an imbalance between osteoblasts for bone formation and osteoclasts for bone resorption. Anti-catabolic drugs have been developed to inhibit osteoclast activity and to prevent bone loss in osteoporosis. However, because it is difficult to increase bone mass in osteoporotic bone, it would be beneficial to simultaneously enhance osteoblast function and thus form bone. Osterix (Osx) is an essential transcription factor for osteoblast differentiation. To date, many studies have focused on discovering Osx target genes and on increasing osteoblast differentiation. However, Osx targets and the mechanisms controlling osteoblast differentiation, are not well known. Here, we generated stable Osx-knockdown cell lines by employing shRNA in MC3T3-E1 osteoblastic cells. Stable Osx-knockdown osteoblasts exhibited a significant reduction in cell differentiation and nodule formation, which was similar to the reduced osteoblast activity observed in an Osx-deficient mouse model. Using an Affymetrix GeneChip microarray, we determined the differential gene expression profile in response to Osx knockdown, which provided insight into molecular mechanisms underlying osteoblast differentiation. Of 2743 genes with roles in cell differentiation, 15 were upregulated and 2 were downregulated in Osx-knockdown osteoblasts. In particular, the expression of fibrillin-2 and periostin was significantly increased in Osx-knockdown osteoblasts compared to that in control cells, as validated by RT-PCR and quantitative real-time PCR. Finally, this study showed differential gene expression profiles for Osx-mediated osteoblast differentiation, suggesting that fibrillin-2 and periostin will be target candidates of Osx in osteoblast differentiation.

  4. Downregulation of E-cadherin is an essential event in activating beta-catenin/Tcf-dependent transcription and expression of its target genes in Pdcd4 knockdown cells.

    PubMed

    Wang, Q; Sun, Z-X; Allgayer, H; Yang, H-S

    2010-01-07

    We reported earlier that knockdown of tumor suppressor Pdcd4 (programed cell death 4) downregulates E-cadherin expression and activates beta-catenin/Tcf (T-cell factor)-dependent transcription in colon tumor cells. However, the underlying mechanism of these observations remains unknown. In this study, we showed that knockdown of Pdcd4 downregulates E-cadherin expression through elevated protein level of Snail. Over-expression of Pdcd4 upregulates E-cadherin expression and inhibits beta-catenin/Tcf-dependent transcription. We then showed that knockdown of E-cadherin activates beta-catenin/Tcf-dependent transcription. Conversely, over-expression of E-cadherin in Pdcd4 knockdown cells inhibits beta-catenin/Tcf-dependent transcription. In addition, Pdcd4 knockdown stimulates urokinase-type plasminogen activator receptor (u-PAR) and c-Myc expression, whereas u-PAR and c-Myc expression can be reversed by over-expressing E-cadherin in Pdcd4 knockdown cells. Using chromatin immunoprecipitation, we showed that beta-catenin/Tcf4 directly binds to the promoters of u-PAR and c-myc in Pdcd4 knockdown cells. Futhermore, knockdown of u-PAR or c-Myc inhibits invasion in Pdcd4 knockdown cells, suggesting that both u-PAR and c-Myc contribute to invasion induced by Pdcd4 knockdown. Taken together, our data showed that elevated Snail expression by Pdcd4 knockdown leads to downregulation of E-cadherin resulting in activating beta-catenin/Tcf-dependent transcription and stimulating the expression of c-Myc and u-PAR, thus providing molecular explanation of how Pdcd4 suppresses tumor invasion.

  5. Insecticide resistance and resistance mechanisms in bed bugs, Cimex spp. (Hemiptera: Cimicidae).

    PubMed

    Dang, Kai; Doggett, Stephen L; Veera Singham, G; Lee, Chow-Yang

    2017-06-29

    The worldwide resurgence of bed bugs [both Cimex lectularius L. and Cimex hemipterus (F.)] over the past two decades is believed in large part to be due to the development of insecticide resistance. The transcriptomic and genomic studies since 2010, as well as morphological, biochemical and behavioral studies, have helped insecticide resistance research on bed bugs. Multiple resistance mechanisms, including penetration resistance through thickening or remodelling of the cuticle, metabolic resistance by increased activities of detoxification enzymes (e.g. cytochrome P450 monooxygenases and esterases), and knockdown resistance by kdr mutations, have been experimentally identified as conferring insecticide resistance in bed bugs. Other candidate resistance mechanisms, including behavioral resistance, some types of physiological resistance (e.g. increasing activities of esterases by point mutations, glutathione S-transferase, target site insensitivity including altered AChEs, GABA receptor insensitivity and altered nAChRs), symbiont-mediated resistance and other potential, yet undiscovered mechanisms may exist. This article reviews recent studies of resistance mechanisms and the genes governing insecticide resistance, potential candidate resistance mechanisms, and methods of monitoring insecticide resistance in bed bugs. This article provides an insight into the knowledge essential for the development of both insecticide resistance management (IRM) and integrated pest management (IPM) strategies for successful bed bug management.

  6. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    NASA Astrophysics Data System (ADS)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  7. Knockdown of L calcium channel subtypes: differential effects in neuropathic pain.

    PubMed

    Fossat, Pascal; Dobremez, Eric; Bouali-Benazzouz, Rabia; Favereaux, Alexandre; Bertrand, Sandrine S; Kilk, Kalle; Léger, Claire; Cazalets, Jean-René; Langel, Ulo; Landry, Marc; Nagy, Frédéric

    2010-01-20

    The maintenance of chronic pain states requires the regulation of gene expression, which relies on an influx of calcium. Calcium influx through neuronal L-type voltage-gated calcium channels (LTCs) plays a pivotal role in excitation-transcription coupling, but the involvement of LTCs in chronic pain remains unclear. We used a peptide nucleic acid (transportan 10-PNA conjugates)-based antisense strategy to investigate the role of the LTC subtypes Ca(V)1.2 and Ca(V)1.3 in long-term pain sensitization in a rat model of neuropathy (spinal nerve ligation). Our results demonstrate that specific knockdown of Ca(V)1.2 in the spinal dorsal horn reversed the neuropathy-associated mechanical hypersensitivity and the hyperexcitability and increased responsiveness of dorsal horn neurons. Intrathecal application of anti-Ca(V)1.2 siRNAs confirmed the preceding results. We also demonstrated an upregulation of Ca(V)1.2 mRNA and protein in neuropathic animals concomitant to specific Ca(V)1.2-dependent phosphorylation of the cAMP response element (CRE)-binding protein (CREB) transcription factor. Moreover, spinal nerve ligation animals showed enhanced transcription of the CREB/CRE-dependent gene COX-2 (cyclooxygenase 2), which also depends strictly on Ca(V)1.2 activation. We propose that L-type calcium channels in the spinal dorsal horn play an important role in pain processing, and that the maintenance of chronic neuropathic pain depends specifically on channels comprising Ca(V)1.2.

  8. Knockdown of orexin type 2 receptor in the lateral pontomesencephalic tegmentum of rats increases REM sleep

    PubMed Central

    Chen, Lichao; McKenna, James T.; Bolortuya, Yunren; Brown, Ritchie E.

    2012-01-01

    Dysfunction of the orexin/hypocretin neurotransmitter system causes the sleep disorder narcolepsy, characterized by intrusion of rapid-eye-movement (REM) sleep-like events into normal wakefulness. The sites where orexins act to suppress REM sleep are incompletely understood. Previous studies suggested that the lateral pontomesencephalic tegmentum (lPMT) contains an important REM sleep inhibitory area, and proposed that orexins inhibit REM sleep via orexin type 2 receptors (OxR2) in this region. However, this hypothesis has heretofore not been tested. We thus performed bilateral injection of small interfering RNAs (siRNAs) targeting Ox2R into the lPMT on two consecutive days. This led to a ~30 % increase of time spent in REM sleep in both the dark and light periods for the first two days after injection, with a return to baseline over the next two post-injection days. This increase was mainly due to more longer (>120 s) REM episodes. Cataplexy-like episodes were not observed. The percentage of time spent in wakefulness and NREM sleep, as well as the power spectral profile of NREM and REM sleep, were unaffected. Control animals injected with scrambled siRNA had no sleep changes post-injection. Quantification of the knockdown revealed that unilateral microinjection of siRNAs targeting OxR2 into the lPMT induced a ~40% reduction of OxR2 mRNA two days following the injections when compared to the contralateral side receiving control (scrambled) siRNA. Orexin type 1 receptor (OxR1) mRNA level was unaffected. Our results indicate that removal of OxR2 neurotransmission in the lPMT enhances REM sleep by increasing the duration of REM episodes. PMID:23282008

  9. Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells

    PubMed Central

    Doig, Craig L.; Battaglia, Sebastiano; Khanim, Farhat L.; Bunce, Christopher M.; Campbell, Moray J.

    2017-01-01

    Background The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. Methods In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. Results These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. Conclusions These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer. PMID:26429394

  10. Pheophytinase Knockdown Impacts Carbon Metabolism and Nutraceutical Content Under Normal Growth Conditions in Tomato.

    PubMed

    Lira, Bruno Silvestre; Rosado, Daniele; Almeida, Juliana; de Souza, Amanda Pereira; Buckeridge, Marcos Silveira; Purgatto, Eduardo; Guyer, Luzia; Hörtensteiner, Stefan; Freschi, Luciano; Rossi, Magdalena

    2016-03-01

    Although chlorophyll (Chl) degradation is an essential biochemical pathway for plant physiology, our knowledge regarding this process still has unfilled gaps. Pheophytinase (PPH) was shown to be essential for Chl breakdown in dark-induced senescent leaves. However, the catalyzing enzymes involved in pigment turnover and fruit ripening-associated degreening are still controversial. Chl metabolism is closely linked to the biosynthesis of other isoprenoid-derived compounds, such as carotenoids and tocopherols, which are also components of the photosynthetic machinery. Chls, carotenoids and tocopherols share a common precursor, geranylgeranyl diphosphate, produced by the plastidial methylerythritol 4-phosphate (MEP) pathway. Additionally, the Chl degradation-derived phytol can be incorporated into tocopherol biosynthesis. In this context, tomato turns out to be an interesting model to address isoprenoid-metabolic cross-talk since fruit ripening combines degreening and an intensely active MEP leading to carotenoid accumulation. Here, we investigate the impact of PPH deficiency beyond senescence by the comprehensive phenotyping of SlPPH-knockdown tomato plants. In leaves, photosynthetic parameters indicate altered energy usage of excited Chl. As a mitigatory effect, photosynthesis-associated carotenoids increased while tocopherol content remained constant. Additionally, starch and soluble sugar profiles revealed a distinct pattern of carbon allocation in leaves that suggests enhanced sucrose exportation. The higher levels of carbohydrates in sink organs down-regulated carotenoid biosynthesis. Additionally, the reduction in Chl-derived phytol recycling resulted in decreased tocopherol content in transgenic ripe fruits. Summing up, tocopherol and carotenoid metabolism, together with the antioxidant capacity of the hydrophilic and hydrophobic fractions, were differentially affected in leaves and fruits of the transgenic plants. Thus, in tomato, PPH plays a role beyond

  11. Knockdown of LI-cadherin alters expression of matrix metalloproteinase-2 and -9 and galectin-3.

    PubMed

    Yu, Qiongfang; Shen, Wei; Zhou, Huangyan; Dong, Weiguo; Gao, Dian

    2016-05-01

    Liver-intestine cadherin (LI-cadherin), a novel member of the cadherin family, has been associated with the ability of a tumor to acquire an aggressive phenotype in several types of cancer. However, the exact function of LI-cadherin in the process of tumor invasion and metastasis remains predominantly unknown. To explore the effect of LI-cadherin on the regulation of matrix metalloproteinase-2 (MMP-2), MMP-9 and galectin-3 in LoVo human colorectal cancer cells, a RNA interference technique was applied to suppress the expression of LI‑cadherin. Subsequently, the mRNA levels and activities of MMP-2 and -9 were analyzed by semi-quantitative reverse transcription-polymerase chain reaction and gelatin zymography, respectively. Additionally, the protein expression level of galectin-3 was determined by western blot analysis. The results of the present study demonstrated that short hairpin RNA (shRNA)-silencing of LI-cadherin significantly increased the mRNA levels and activities of MMP‑2 and ‑9, and significantly reduced the protein levels of galectin‑3 in LoVo cells compared with control shRNA (P<0.05). These data indicate that knockdown of LI‑cadherin facilitates the invasion of cancer cells by degrading extracellular matrix components via activation of MMP‑2 and ‑9, and increases cancer cell adhesion and migration via altered expression of galectin‑3. This suggests that LI‑cadherin serves an important role in the invasion and metastasis of colorectal cancer, and may be used as a potential therapeutic target.

  12. Rip3 knockdown rescues photoreceptor cell death in blind pde6c zebrafish

    PubMed Central

    Viringipurampeer, I A; Shan, X; Gregory-Evans, K; Zhang, J P; Mohammadi, Z; Gregory-Evans, C Y

    2014-01-01

    Achromatopsia is a progressive autosomal recessive retinal disease characterized by early loss of cone photoreceptors and later rod photoreceptor loss. In most cases, mutations have been identified in CNGA3, CNGB3, GNAT2, PDE6C or PDE6H genes. Owing to this genetic heterogeneity, mutation-independent therapeutic schemes aimed at preventing cone cell death are very attractive treatment strategies. In pde6cw59 mutant zebrafish, cone photoreceptors expressed high levels of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3) kinases, key regulators of necroptotic cell death. In contrast, rod photoreceptor cells were alternatively immunopositive for caspase-3 indicating activation of caspase-dependent apoptosis in these cells. Morpholino gene knockdown of rip3 in pde6cw59 embryos rescued the dying cone photoreceptors by inhibiting the formation of reactive oxygen species and by inhibiting second-order neuron remodelling in the inner retina. In rip3 morphant larvae, visual function was restored in the cones by upregulation of the rod phosphodiesterase genes (pde6a and pde6b), compensating for the lack of cone pde6c suggesting that cones are able to adapt to their local environment. Furthermore, we demonstrated through pharmacological inhibition of RIP1 and RIP3 activity that cone cell death was also delayed. Collectively, these results demonstrate that the underlying mechanism of cone cell death in the pde6cw59 mutant retina is through necroptosis, whereas rod photoreceptor bystander death occurs through a caspase-dependent mechanism. This suggests that targeting the RIP kinase signalling pathway could be an effective therapeutic intervention in retinal degeneration patients. As bystander cell death is an important feature of many retinal diseases, combinatorial approaches targeting different cell death pathways may evolve as an important general principle in treatment. PMID:24413151

  13. Knockdown and Mortality of Five Stored Product Beetle Species After Short Exposures of Thiamethoxam.

    PubMed

    Tsaganou, Fotoula C; Vassilakos, Thomas N; Athanassiou, Christos G

    2014-12-01

    Laboratory bioassays were conducted to evaluate the effectiveness of thiamethoxam, against five major stored-grain beetle species, the lesser grain borer, Rhyzopertha dominica (F.), the rice weevil, Sitophilus oryzae (L.), the confused flour beetle, Tribolium confusum Jacquelin du Val, the larger grain borer, Prostephanus truncatus (Horn), and the sawtoothed grain beetle, Oryzaephilus surinamensis (L.). Adults of the above species were exposed on wheat (or maize in the case of P. truncatus) treated with thiamethoxam at 0.1, 1, and 10 ppm for 0, 2, 4, 6, 8, 16, 40, 72, and 96 h. After each of these intervals, mortality was recorded (immediate mortality) and the surviving individuals were transferred in untreated wheat (or maize), where mortality was recorded again 7 d later (delayed mortality). During both immediate and delayed mortality counts, the number of adults that were knocked down was also recorded. Immediate mortality was low in all exposures, with the exception of the highest dose rate and after 72-96 h. At these conditions, during this interval, most of the surviving individuals were knocked down. Delayed mortality was further increased with the increase of dose and the initial exposure, but knockdown was extremely low, with the exception of P. truncatus. The results of the present work show that O. surinamensis was the least susceptible species, while P. truncatus was the most susceptible. These findings show that, despite the increased mortality, recovery after short exposures is likely for all species tested here. In this regard, partially treated areas on which the insects are exposed only for short intervals may reduce thiamethoxam efficacy.

  14. Single domain antibodies for the knockdown of cytosolic and nuclear proteins.

    PubMed

    Böldicke, Thomas

    2017-03-08

    Single domain antibodies (sdAbs) from camels or sharks comprise only the variable heavy chain domain. Human sdAbs comprise the variable domain of the heavy chain (VH) or light chain (VL) and can be selected from human antibodies. SdAbs are stable, non aggregating molecules in vitro and in vivo compared to complete antibodies and scFv fragments. They are excellent novel inhibitors of cytosolic/nuclear proteins because they are correctly folded inside the cytosol in contrast to scFv fragments. SdAbs are unique because of their excellent specificity and possibility to target posttranslational modifications such as phosphorylation sites, conformers or interaction regions of proteins that cannot be targeted with genetic knockout techniques and are impossible to knockdown with RNAi. The number of inhibiting cytosolic/nuclear sdAbs is increasing and usage of synthetic single pot single domain libraries will boost the generation of these fascinating molecules without the need of immunization. The most frequently selected antigenic epitopes belong to viral and oncogenic proteins, followed by toxins, proteins of the nervous system as well as plant- and drosophila proteins. It is now possible to select functional sdAbs against virtually every cytosolic/nuclear protein and desired epitope. The development of new endosomal escape protein domains and cell-penetrating peptides for efficient transfection broaden the application of inhibiting sdAbs. Last but not least, the generation of relatively new cell-specific nanoparticles such as polymersomes and polyplexes carrying cytosolic/nuclear sdAb-DNA or -protein will pave the way to apply cytosolic/nuclear sdAbs for inhibition of viral infection and cancer in the clinic. This article is protected by copyright. All rights reserved.

  15. Effect of lamin A/C knockdown on osteoblast differentiation and function.

    PubMed

    Akter, Rahima; Rivas, Daniel; Geneau, Graziello; Drissi, Hicham; Duque, Gustavo

    2009-02-01

    Recent studies have associated mutations in lamin A/C, a component of the nuclear lamina, with premature aging and severe bone loss. In this study, we hypothesized that reduced expression of lamin A/C has a negative impact on osteoblastogenesis and bone formation in vitro. We inhibited lamin A/C using increasing doses of lamin A/C siRNA in normal human osteoblasts and differentiating mesenchymal stem cells (MSCs). Untreated cells and cells treated with vehicle but without the siRNA-oligo were used as control. The level of effectiveness of siRNA was determined by RT-PCR, Western blot, and immunofluorescence. Nuclear blebbing, a typical finding of lamin A/C inhibition, was quantified using propidium iodine staining, and its effect on cell survival was determined using MTS-formazan. Furthermore, alizarin red and alkaline phosphatase staining were correlated with osteocalcin secretion and levels of expression of osteocalcin, osterix, bone sialoprotein, and Runx2. Finally, the nuclear binding activity of Runx2, an essential transcription factor for osteoblast differentiation, was assessed using ELISA and EMSA. A successful inhibitory effect on the lamin A/C gene at doses of 400-800 nM oligo was obtained without affecting cell survival. Whereas osteoblast function was significantly affected by lamin A/C inhibition, siRNA-treated MSC showed a higher incidence of nuclear changes, lower osteoblast differentiation, and enhanced adipocyte differentiation. Finally, lamin A/C knockdown reduced Runx2 nuclear binding activity without affecting Runx2 expression. In summary, our results indicate that lamin A/C is a new factor needed for osteoblast differentiation that plays an important role in the cellular mechanisms of age-related bone loss.

  16. Knockdown of LjIPT3 influences nodule development in Lotus japonicus.

    PubMed

    Chen, Yaping; Chen, Wei; Li, Xueliu; Jiang, Huawu; Wu, Pingzhi; Xia, Kuaifei; Yang, Yali; Wu, Guojiang

    2014-01-01

    Cytokinins play important roles in legume-rhizobia symbiosis. Here we report isolation of six genes encoding isopentenyl transferase (IPT) from Lotus japonicus, which catalyze the rate-limiting step of cytokinin biosynthesis. The LjIPT3 gene was found to be up-regulated in infected roots and mature nodules. Histochemical analysis demonstrated expression of Pro(LjIPT3):GUS (β-glucuronidase) in vegetative and reproductive organs, and was especially high in the vascular bundles of roots. When inoculated with Mesorhizobium loti MAFF303099, LjIPT3 was undetectable in the nodule primordia and developing nodules, and later it was expressed only in the vascular bundles of mature nodules. In addition, knockdown of LjIPT3 (LjIPT3i) by RNA interference reduced levels of endogenous cytokinins, affected plant development and accelerated Chl degradation during dark-induced leaf senescence. Compared with the wild type, LjIPT3i plants produced fewer infection threads and nodules. In addition, expression of downstream nodulation-related transcription factor genes LjNSP1, LjNSP2 and LjNIN decreased dramatically in LjIPT3i plants. These results suggest that LjIPT3 regulates the CRE1-dependent cytokinin pathway, affecting nodule initiation and thereby influencing the number of infection threads and nodules. Detection of nitrogenase activity and observation of nodule structure showed that endogenous cytokinins are required for full development of the infected cells in mature nodules by preventing early senescence. Therefore, our results indicate that the LjIPT3 gene product is required for nodule initiation and development, and does not appear to be involved in early infection events.

  17. Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells

    PubMed Central

    Jovanovic, Katarina; Veale, Rob B.; Weiss, Stefan F. T.

    2015-01-01

    Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment. PMID:26427016

  18. Knockdown of ROS1 gene sensitizes breast tumor growth to doxorubicin in a syngeneic mouse model.

    PubMed

    Tiash, Snigdha; Chua, Ming Jang; Chowdhury, Ezharul Hoque

    2016-06-01

    Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.

  19. Mitofusin-2 knockdown increases ER-mitochondria contact and decreases amyloid β-peptide production.

    PubMed

    Leal, Nuno Santos; Schreiner, Bernadette; Pinho, Catarina Moreira; Filadi, Riccardo; Wiehager, Birgitta; Karlström, Helena; Pizzo, Paola; Ankarcrona, Maria

    2016-09-01

    Mitochondria are physically and biochemically in contact with other organelles including the endoplasmic reticulum (ER). Such contacts are formed between mitochondria-associated ER membranes (MAM), specialized subregions of ER, and the outer mitochondrial membrane (OMM). We have previously shown increased expression of MAM-associated proteins and enhanced ER to mitochondria Ca(2+) transfer from ER to mitochondria in Alzheimer's disease (AD) and amyloid β-peptide (Aβ)-related neuronal models. Here, we report that siRNA knockdown of mitofusin-2 (Mfn2), a protein that is involved in the tethering of ER and mitochondria, leads to increased contact between the two organelles. Cells depleted in Mfn2 showed increased Ca(2+) transfer from ER to mitchondria and longer stretches of ER forming contacts with OMM. Interestingly, increased contact resulted in decreased concentrations of intra- and extracellular Aβ40 and Aβ42 . Analysis of γ-secretase protein expression, maturation and activity revealed that the low Aβ concentrations were a result of impaired γ-secretase complex function. Amyloid-β precursor protein (APP), β-site APP-cleaving enzyme 1 and neprilysin expression as well as neprilysin activity were not affected by Mfn2 siRNA treatment. In summary, our data shows that modulation of ER-mitochondria contact affects γ-secretase activity and Aβ generation. Increased ER-mitochondria contact results in lower γ-secretase activity suggesting a new mechanism by which Aβ generation can be controlled. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  20. Knockdown of LRP/LR Induces Apoptosis in Breast and Oesophageal Cancer Cells.

    PubMed

    Khumalo, Thandokuhle; Ferreira, Eloise; Jovanovic, Katarina; Veale, Rob B; Weiss, Stefan F T

    2015-01-01

    Cancer is a global burden due to high incidence and mortality rates and is ranked the second most diagnosed disease amongst non-communicable diseases in South Africa. A high expression level of the 37kDa/67kDa laminin receptor (LRP/LR) is one characteristic of cancer cells. This receptor is implicated in the pathogenesis of cancer cells by supporting tumor angiogenesis, metastasis and especially for this study, the evasion of apoptosis. In the current study, the role of LRP/LR on cellular viability of breast MCF-7, MDA-MB 231 and WHCO1 oesophageal cancer cells was investigated. Western blot analysis revealed that total LRP expression levels of MCF-7, MDA-MB 231 and WHCO1 were significantly downregulated by targeting LRP mRNA using siRNA-LAMR1. This knockdown of LRP/LR resulted in a significant decrease of viability in the breast and oesophageal cancer cells as determined by an MTT assay. Transfection of MDA-MB 231 cells with esiRNA-RPSA directed against a different region of the LRP mRNA had similar effects on LRP/LR expression and cell viability compared to siRNA-LAMR1, excluding an off-target effect of siRNA-LAMR1. This reduction in cellular viability is as a consequence of apoptosis induction as indicated by the exposure of the phosphatidylserine protein on the surface of breast MCF-7, MDA-MB 231 and oesophageal WHCO1 cancer cells, respectively, detected by an Annexin-V/FITC assay as well as nuclear morphological changes observed post-staining with Hoechst. These observations indicate that LRP/LR is crucial for the maintenance of cellular viability of breast and oesophageal cancer cells and recommend siRNA technology targeting LRP expression as a possible novel alternative technique for breast and oesophageal cancer treatment.

  1. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity.

    PubMed

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S; Zhang, Lin; Rodgers, Joseph T; Pirinen, Eija; Pulinilkunnil, Thomas C; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A; Asara, John M; Peroni, Odile D; Monia, Brett P; Bhanot, Sanjay; Alhonen, Leena; Puigserver, Pere; Kahn, Barbara B

    2014-04-10

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor. Nicotinamide is a precursor of NAD(+), an important cofactor linking cellular redox states with energy metabolism. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine-spermine N(1)-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD(+) levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD(+)-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes.

  2. STAT3 Knockdown Reduces Pancreatic Cancer Cell Invasiveness and Matrix Metalloproteinase-7 Expression in Nude Mice

    PubMed Central

    Huang, Ke jian; Wu, Wei dong; Jiang, Tao; Cao, Jun; Feng, Zhen zhong; Qiu, Zheng jun

    2011-01-01

    Aims Transducer and activator of transcription-3 (STAT3) plays an important role in tumor cell invasion and metastasis. The aim of the present study was to investigate the effects of STAT3 knockdown in nude mouse xenografts of pancreatic cancer cells and underlying gene expression. Methods A STAT3 shRNA lentiviral vector was constructed and infected into SW1990 cells. qRT-PCR and western immunoblot were performed to detect gene expression. Nude mouse xenograft assays were used to assess changes in phenotypes of these stable cells in vivo. HE staining was utilized to evaluate tumor cell invasion and immunohistochemistry was performed to analyze gene expression. Results STAT3 shRNA successfully silenced expression of STAT3 mRNA and protein in SW1990 cells compared to control cells. Growth rate of the STAT3-silenced tumor cells in nude mice was significantly reduced compared to in the control vector tumors and parental cells-generated tumors. Tumor invasion into the vessel and muscle were also suppressed in the STAT3-silenced tumors compared to controls. Collagen IV expression was complete and continuous surrounding the tumors of STAT3-silenced SW1990 cells, whereas collagen IV expression was incomplete and discontinuous surrounding the control tumors. Moreover, microvessel density was significantly lower in STAT3-silenced tumors than parental or control tumors of SW1990 cells. In addition, MMP-7 expression was reduced in STAT3-silenced tumors compared to parental SW1990 xenografts and controls. In contrast, expression of IL-1β and IgT7α was not altered. Conclusion These data clearly demonstrate that STAT3 plays an important role in regulation of tumor growth, invasion, and angiogenesis, which could be act by reducing MMP-7 expression in pancreatic cancer cells. PMID:21991388

  3. Knockdown of AKR1C3 exposes a potential epigenetic susceptibility in prostate cancer cells.

    PubMed

    Doig, Craig L; Battaglia, Sebastiano; Khanim, Farhat L; Bunce, Christopher M; Campbell, Moray J

    2016-01-01

    The aldo-keto reductase 1C3 (AKR1C3) has been heavily implicated in the propagation of prostate malignancy. AKR1C3 protein is elevated within prostate cancer tissue, it contributes to the formation of androgens and downstream stimulation of the androgen receptor (AR). Elevated expression of AKR1C3 is also reported in acute myeloid leukemia but the target nuclear receptors have been identified as members of the peroxisome-proliferator activated receptor (PPARs) subfamily. Thus, AKR1C3 cancer biology is likely to be tissue dependent and hormonally linked to the availability of ligands for both the steroidogenic and non-steroidogenic nuclear receptors. In the current study we investigated the potential for AKR1C3 to regulate the availability of prostaglandin-derived ligands for PPARg mainly, prostaglandin J2 (PGJ2). Using prostate cancer cell lines with stably reduced AKR1C3 levels we examined the impact of AKR1C3 upon proliferation mediated by PPAR ligands. These studies revealed knockdown of AKR1C3 had no effect upon the sensitivity of androgen receptor independent prostate cancer cells towards PPAR ligands. However, the reduction of levels of AKR1C3 was accompanied by a significantly reduced mRNA expression of a range of HDACs, transcriptional co-regulators, and increased sensitivity towards SAHA, a clinically approved histone deacetylase inhibitor. These results suggest a hitherto unidentified link between AKR1C3 levels and the epigenetic status in prostate cancer cells. This raises an interesting possibility of a novel rational to target AKR1C3, the utilization of AKRIC3 selective inhibitors in combination with HDAC inhibition as part of novel epigenetic therapies in androgen deprivation therapy recurrent prostate cancer. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Nicotinamide N-methyltransferase knockdown protects against diet-induced obesity

    PubMed Central

    Kraus, Daniel; Yang, Qin; Kong, Dong; Banks, Alexander S.; Zhang, Lin; Rodgers, Joseph T.; Pirinen, Eija; Pulinilkunnil, Thomas C.; Gong, Fengying; Wang, Ya-chin; Cen, Yana; Sauve, Anthony A.; Asara, John M.; Peroni, Odile D.; Monia, Brett P.; Bhanot, Sanjay; Alhonen, Leena; Puigserver, Pere; Kahn, Barbara B.

    2014-01-01

    In obesity and type 2 diabetes, Glut4 glucose transporter expression is decreased selectively in adipocytes1. Adipose-specific knockout or overexpression of Glut4 alters systemic insulin sensitivity2. Here we show, using DNA array analyses, that nicotinamide N-methyltransferase (Nnmt) is the most strongly reciprocally regulated gene when comparing gene expression in white adipose tissue (WAT) from adipose-specific Glut4-knockout or adipose-specific Glut4-overexpressing mice with their respective controls. NNMT methylates nicotinamide (vitamin B3) using S-adenosylmethionine (SAM) as a methyl donor3,4. Nicotinamide is a precursor of NAD+, an important cofactor linking cellular redox states with energy metabolism5. SAM provides propylamine for polyamine biosynthesis and donates a methyl group for histone methylation6. Polyamine flux including synthesis, catabolism and excretion, is controlled by the rate-limiting enzymes ornithine decarboxylase (ODC) and spermidine–spermine N1-acetyltransferase (SSAT; encoded by Sat1) and by polyamine oxidase (PAO), and has a major role in energy metabolism7,8. We report that NNMT expression is increased in WAT and liver of obese and diabetic mice. Nnmt knockdown in WAT and liver protects against diet-induced obesity by augmenting cellular energy expenditure. NNMT inhibition increases adipose SAM and NAD+ levels and upregulates ODC and SSAT activity as well as expression, owing to the effects of NNMT on histone H3 lysine 4 methylation in adipose tissue. Direct evidence for increased polyamine flux resulting from NNMT inhibition includes elevated urinary excretion and adipocyte secretion of diacetylspermine, a product of polyamine metabolism. NNMT inhibition in adipocytes increases oxygen consumption in an ODC-, SSAT- and PAO-dependent manner. Thus, NNMT is a novel regulator of histone methylation, polyamine flux and NAD+-dependent SIRT1 signalling, and is a unique and attractive target for treating obesity and type 2 diabetes. PMID

  5. Effect of TRPC6 knockdown on puromycin aminonucleoside-induced podocyte injury.

    PubMed

    Sun, Xifeng; Chu, Yongli; Zhang, Chun; Du, Xiyun; He, Fangfang; Chen, Shan; Gao, Pan; Liu, Jianshe; Zhu, Zhonghua; Meng, Xianfang

    2012-06-01

    This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

  6. An experimental approach to evaluate the impact of impaired transport function on hepatobiliary drug disposition using Mrp2-deficient TR- rat sandwich-cultured hepatocytes in combination with Bcrp knockdown.

    PubMed

    Yang, Kyunghee; Pfeifer, Nathan D; Hardwick, Rhiannon N; Yue, Wei; Stewart, Paul W; Brouwer, Kim L R

    2014-03-03

    Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR(-)) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1-10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR(-) status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR(-) rat SCH decreased by 28.6 (95% CI: 5.8-51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5-9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR(-) rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR(-) rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0-12.8) and 7.5 (3.7-11.3), respectively, relative to noninfected SCH. These

  7. Knockdown of Decoy Receptor 3 Impairs Growth and Invasiveness of Hepatocellular Carcinoma Cell Line of HepG2

    PubMed Central

    Zhou, Xiao-Na; Li, Guang-Ming; Xu, Ying-Chen; Zhao, Tuan-Jie; Wu, Ji-Xiang

    2016-01-01

    Background: Decoy receptor 3 (DcR3) binds to Fas ligand (FasL) and inhibits FasL-induced apoptosis. The receptor is overexpressed in hepatocellular carcinoma (HCC), and it is associated with the growth and metastatic spread of tumors. DcR3 holds promises as a new target for the treatment of HCC, but little is known regarding the molecular mechanisms underlying the oncogenic properties of DcR3. The present work, therefore, examined the role of DcR3 in regulating the growth and invasive property of liver cancer cell HepG2. Methods: HepG2 cells were stably transfected with lentivirus-based short hairpin RNA vector targeting DcR3. After the knockdown of DcR3 was confirmed, cell proliferation, clone formation, ability of migrating across transwell membrane, and wound healing were assessed in vitro. Matrix metalloproteinase-9 (MMP 9) and vascular epithelial growth factor (VEGF)-C and D expressions of the DcR3 knockdown were also studied. Comparisons between multiple groups were done using one-way analysis of variance (ANOVA), while pairwise comparisons were performed using Student's t test. P < 0.05 was regarded statistically significant. Results: DcR3 was overexpressed in HepG2 compared to other HCC cell lines and normal hepatocyte Lo-2. Stable knockdown of DcR3 slowed down the growth of HepG2 (P < 0.05) and reduced the number of clones formed by 50% compared to those without DcR3 knockdown (P < 0.05). The knockdown also reduced the migration of HepG2 across transwell matrix membrane by five folds compared to the control (P < 0.05) and suppressed the closure of scratch wound (P < 0.05). In addition, the messenger RNA levels of MMP 9, VEGF-C, and VEGF-D were significantly suppressed by DcR3 knockdown by 90% when compared with the mock control (P < 0.05). Conclusions: Loss of DcR3 impaired the growth and invasive property of HCC cell line of HepG2. Targeting DcR3 may be a potential therapeutic approach for the treatment of HCC. PMID:27779171

  8. Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

    PubMed

    Dong, Zhiwei; Chen, Yajie; Peng, Yuan; Wang, Fan; Yang, Zichen; Huang, Guangtao; Chen, Yu; Yuan, Zhiqiang; Cao, Tongtong; Peng, Yizhi

    2017-01-01

    Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection. © 2017 The Author(s). Published by S. Karger AG, Basel.

  9. Knockdown of stem cell regulator Oct4A in ovarian cancer reveals cellular reprogramming associated with key regulators of cytoskeleton-extracellular matrix remodelling

    PubMed Central

    Samardzija, Chantel; Greening, David W.; Escalona, Ruth; Chen, Maoshan; Bilandzic, Maree; Luwor, Rodney; Kannourakis, George; Findlay, Jock K.; Ahmed, Nuzhat

    2017-01-01

    Oct4A is a master regulator of self-renewal and pluripotency in embryonic stem cells. It is a well-established marker for cancer stem cell (CSC) in malignancies. Recently, using a loss of function studies, we have demonstrated key roles for Oct4A in tumor cell survival, metastasis and chemoresistance in in vitro and in vivo models of ovarian cancer. In an effort to understand the regulatory role of Oct4A in tumor biology, we employed the use of an ovarian cancer shRNA Oct4A knockdown cell line (HEY Oct4A KD) and a global mass spectrometry (MS)-based proteomic analysis to investigate novel biological targets of Oct4A in HEY samples (cell lysates, secretomes and mouse tumor xenografts). Based on significant differential expression, pathway and protein network analyses, and comprehensive literature search we identified key proteins involved with biologically relevant functions of Oct4A in tumor biology. Across all preparations of HEY Oct4A KD samples significant alterations in protein networks associated with cytoskeleton, extracellular matrix (ECM), proliferation, adhesion, metabolism, epithelial-mesenchymal transition (EMT), cancer stem cells (CSCs) and drug resistance was observed. This comprehensive proteomics study for the first time presents the Oct4A associated proteome and expands our understanding on the biological role of this stem cell regulator in carcinomas. PMID:28406185

  10. Knockdown of a cellulose synthase gene BoiCesA affects the leaf anatomy, cellulose content and salt tolerance in broccoli

    PubMed Central

    Li, Shuangtao; Zhang, Lei; Wang, Ying; Xu, Fengfeng; Liu, Mengyun; Lin, Peng; Ren, Shuxin; Ma, Rui; Guo, Yang-Dong

    2017-01-01

    Cellulose is the major component of cell wall materials. A 300 bp specific fragment from the cDNA fragment was chosen to insert into vector pFGC1008 at forward and reverse orientations to construct the recombinant RNAi vector. Knockdown of BoiCesA caused “dwarf” phenotype with smaller leaves and a loss of the content of cellulose. Moreover, RT-PCR analysis confirmed that the expression of the RNAi apparatus could repress expression of the CesA gene. Meanwhile, examination of the leaves from the T3 of RNAi transformants indicated reduction of cell expansion in vascular bundles, particularly on their abaxial surface. The proline and soluble sugar content increased contrarily. Under the salt stress, the T3 of RNAi plants showed significant higher resistance. The expression levels of some salt tolerance related genes (BoiProH, BoiPIP2;2, BoiPIP2;3) were significantly changed in T3 of RNAi plants. The results showed that the hairpin structure of CesA specific fragment inhibited the endogenous gene expression and it was proved that the cDNA fragment was relevant to the cellulose biosynthesis. Moreover, modulation cellulose synthesis probably was an important influencing factor in polysaccharide metabolism and adaptations of plants to stresses. This will provide technological possibilities for the further study of modulation of the cellulose content of crops. PMID:28169290

  11. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    DOE PAGES

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; ...

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstratemore » reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.« less

  12. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila

    SciTech Connect

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E.; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Cantu Gutierrez, Manuel; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L.; Booth, Benjamin W.; Evans-Holm, Martha; Venken, Koen J.T.; Levis, Robert W.; Spradling, Allan C.; Hoskins, Roger A.; Bellen, Hugo J.

    2015-03-31

    Here, we document a collection of ~7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  13. A library of MiMICs allows tagging of genes and reversible, spatial and temporal knockdown of proteins in Drosophila.

    PubMed

    Nagarkar-Jaiswal, Sonal; Lee, Pei-Tseng; Campbell, Megan E; Chen, Kuchuan; Anguiano-Zarate, Stephanie; Gutierrez, Manuel Cantu; Busby, Theodore; Lin, Wen-Wen; He, Yuchun; Schulze, Karen L; Booth, Benjamin W; Evans-Holm, Martha; Venken, Koen J T; Levis, Robert W; Spradling, Allan C; Hoskins, Roger A; Bellen, Hugo J

    2015-03-31

    Here, we document a collection of ∼7434 MiMIC (Minos Mediated Integration Cassette) insertions of which 2854 are inserted in coding introns. They allowed us to create a library of 400 GFP-tagged genes. We show that 72% of internally tagged proteins are functional, and that more than 90% can be imaged in unfixed tissues. Moreover, the tagged mRNAs can be knocked down by RNAi against GFP (iGFPi), and the tagged proteins can be efficiently knocked down by deGradFP technology. The phenotypes associated with RNA and protein knockdown typically correspond to severe loss of function or null mutant phenotypes. Finally, we demonstrate reversible, spatial, and temporal knockdown of tagged proteins in larvae and adult flies. This new strategy and collection of strains allows unprecedented in vivo manipulations in flies for many genes. These strategies will likely extend to vertebrates.

  14. Knockdown of Aurora-B inhibits the growth of non-small cell lung cancer A549 cells.

    PubMed

    Yu, Jing Jing; Zhou, Long Dian; Zhao, Tian Tian; Bai, Wei; Zhou, Jing; Zhang, Wei

    2015-09-01

    Elevated expression of Aurora-B affects cell apoptosis and proliferation in a variety of solid tumors. However, the role of Aurora-B has been poorly evaluated in non-small cell lung cancer (NSCLC). In the present study, it was found that Aurora-B was overexpressed in tissue specimens obtained from 174 patients with lung cancer. It was also demonstrated that knockdown of Aurora-B induces apoptosis and inhibits the growth of lung cancer A549 cells in vitro and in vivo. Furthermore, it was found that silencing Aurora-B decreased the activity of the phosphoinositide 3-kinase (PI3K)/AKT pathway. Therefore, it was concluded that knockdown of Aurora-B induces apoptosis and inhibits growth in NSCLC A549 cells, in addition to inhibiting the activity of the PI3K/AKT signaling pathway. Targeting Aurora-B may provide a novel target for lung cancer therapy.

  15. GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

    PubMed Central

    Wang, Yong; Song, Wei; Li, Shuchun; Guan, Xin; Miao, Shiying; Zong, Shudong; Koide, SS; Wang, Linfang

    2009-01-01

    Background Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules. Results Stable mRHBDD1 knockdown GC-1 cells were sensitive to apoptotic stimuli, PS341 and UV irradiation. In vitro, they survived and proliferated normally. However, they lost the ability to survive and differentiate in mouse seminiferous tubules. Conclusion Our findings suggest that mRHBDD1 may be associated with mammalian spermatogenesis. PMID:19358743

  16. Gucy2f zebrafish knockdown – a model for Gucy2d-related leber congenital amaurosis

    PubMed Central

    Stiebel-Kalish, Hadas; Reich, Ehud; Rainy, Nir; Vatine, Gad; Nisgav, Yael; Tovar, Anna; Gothilf, Yoav; Bach, Michael

    2012-01-01

    Mutations in retinal-specific guanylate cyclase (Gucy2d) are associated with Leber congenital amaurosis-1 (LCA1). Zebrafish offer unique advantages relative to rodents, including their excellent color vision, precocious retinal development, robust visual testing strategies, low cost, relatively easy transgenesis and shortened experimental times. In this study we will demonstrate the feasibility of using gene-targeting in the zebrafish as a model for the photoreceptor-specific GUCY2D-related LCA1, by reporting the visual phenotype and retinal histology resulting from Gucy2f knockdown. Gucy2f zebrafish LCA-orthologous cDNA was identified and isolated by PCR amplification. Its expression pattern was determined by whole-mount in-situ hybridization and its function was studied by gene knockdown using two different morpholino-modified oligos (MO), one that blocks translation of Gucy2f and one that blocks splicing of Gucy2f. Visual function was assessed with an optomotor assay on 6-days-post-fertilization larvae, and by analyzing changes in retinal histology. Gucy2f knockdown resulted in significantly lower vision as measured by the optomotor response compared with uninjected and control MO-injected zebrafish larvae. Histological changes in the Gucy2f-knockdown larvae included loss and shortening of cone and rod outer segments. A zebrafish model of Gucy2f-related LCA1 displays early visual dysfunction and photoreceptor layer dystrophy. This study serves as proof of concept for the use of zebrafish as a simple, inexpensive model with excellent vision on which further study of LCA-related genes is possible. PMID:22378290

  17. [Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

    PubMed

    Liu, Yanhua; Li, Bingong; Wang, Yuqin; Wang, Delong; Zou, Jin; Ke, Xuan; Hao, Yanqin

    2016-12-01

    Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

  18. Knockdown of ILK inhibits glioma development via upregulation of E-cadherin and downregulation of cyclin D1.

    PubMed

    Zheng, Kebin; Wang, Guangyi; Li, Chunhui; Shan, Xiaosong; Liu, Haipeng

    2015-07-01

    Integrin-linked kinase (ILK) is a highly conserved serine-threonine protein kinase that interacts with cytoplasmic domains of integrin subunits in tumor tissues. However, the relationship between gliomas and ILK is elusive. The present study aimed to investigate the role of ILK in a human glioma cell line (U251). ILK stable expressing vector, U251ILK-PGFP-V-RS-shRNA, was established and named as U251-si. The empty-PGFP-V-RS-shRNA (U251-N) was employed as the control. Quantitative real-time PCR and western blot analysis were used to detect ILK and E-cadherin mRNA and protein expression, respectively. Cell cycle analysis was employed to examine the cell cycle distribution. Cell migration was detected using a wound healing assay, and cell invasion was detected using a Transwell invasion assay. Tumor size and weight were also examined. The results indicated that ILK was expressed at a lower level at both the mRNA and protein levels in the U251-si group compared with the U251-N group (p<0.01). ILK knockdown suppressed cell proliferation of the glioma cells. Knockdown of ILK reduced the migratory and invasive potentials of the glioma cells. Inhibition of ILK expression upregulated E-cadherin and downregulated cyclin D1 in the glioma cells compared to the U251-N group (p<0.05). Knockdown of ILK in the U251 cells attenuated the ability of U251 cells to form tumors in nude mice and impaired glioma cell in vivo tumorigenicity. In conclusion, knockdown of ILK inhibits glioma cell migration, invasion and proliferation through upregulation of E-cadherin and downregulation of cyclin D1. Our results suggest that ILK may serve as a promising therapeutic target for glioma.

  19. Signal Transducer and Activator of Transcription 1 (STAT1) Knock-down Induces Apoptosis in Malignant Pleural Mesothelioma.

    PubMed

    Arzt, Lisa; Halbwedl, Iris; Gogg-Kamerer, Margit; Popper, Helmut H

    2017-07-01

    Malignant pleural mesothelioma (MPM) is the most common primary tumor of the pleura. Its incidence is still increasing in Europe and the prognosis remains poor. We investigated the oncogenic function of signal transducer and activator of transcription 1 (STAT1) in MPM in more detail. A miRNA profiling was performed on 52 MPM tissue samples. Upregulated miRNAs (targeting SOCS1/3) were knocked-down using miRNA inhibitors. mRNA expression levels of STAT1/3, SOCS1/3 were detected in MPM cell lines. STAT1 has been knocked-down using siRNA and qPCR was used to detect mRNA expression levels of all JAK/STAT family members and genes that regulate them. An immunohistochemical staining was performed to detect the expression of caspases. STAT1 was upregulated and STAT3 was downregulated, SOCS1/3 protein was not detected but it was possible to detect SOCS1/3 mRNA in MPM cell lines. The upregulated miRNAs were successfully knocked-down, however the expected effect on SOCS1 expression was not detected. STAT1 knock-down had different effects on STAT3/5 expression. Caspase 3a and 8 expression was found to be increased after STAT1 knock-down. The physiologic regulation of STAT1 via SOCS1 is completely lost in MPM and it does not seem that the miRNAs identified by now, do inhibit the expression of SOCS1. MPM cell lines compensate STAT1 knock-down by increasing the expression of STAT3 or STAT5a, two genes which are generally considered to be oncogenes. And much more important, STAT1 knock-down induces apoptosis in MPM cell lines and STAT1 might therefore be a target for therapeutic intervention.

  20. miRNA-embedded shRNAs for Lineage-specific BCL11A Knockdown and Hemoglobin F Induction.

    PubMed

    Guda, Swaroopa; Brendel, Christian; Renella, Raffaele; Du, Peng; Bauer, Daniel E; Canver, Matthew C; Grenier, Jennifer K; Grimson, Andrew W; Kamran, Sophia C; Thornton, James; de Boer, Helen; Root, David E; Milsom, Michael D; Orkin, Stuart H; Gregory, Richard I; Williams, David A

    2015-09-01

    RNA interference (RNAi) technology using short hairpin RNAs (shRNAs) expressed via RNA polymerase (pol) III promoters has been widely exploited to modulate gene expression in a variety of mammalian cell types. For certain applications, such as lineage-specific knockdown, embedding targeting sequences into pol II-driven microRNA (miRNA) architecture is required. Here, using the potential therapeutic target BCL11A, we demonstrate that pol III-driven shRNAs lead to significantly increased knockdown but also increased cytotoxcity in comparison to pol II-driven miRNA adapted shRNAs (shRNA(miR)) in multiple hematopoietic cell lines. We show that the two expression systems yield mature guide strand sequences that differ by a 4 bp shift. This results in alternate seed sequences and consequently influences the efficacy of target gene knockdown. Incorporating a corresponding 4 bp shift into the guide strand of shRNA(miR)s resulted in improved knockdown efficiency of BCL11A. This was associated with a significant de-repression of the hemoglobin target of BCL11A, human γ-globin or the murine homolog Hbb-y. Our results suggest the requirement for optimization of shRNA sequences upon incorporation into a miRNA backbone. These findings have important implications in future design of shRNA(miR)s for RNAi-based therapy in hemoglobinopathies and other diseases requiring lineage-specific expression of gene silencing sequences.

  1. Effects of hnRNP A2/B1 Knockdown on Inhibition of Glioblastoma Cell Invasion, Growth and Survival.

    PubMed

    Deng, Jinmu; Chen, Song; Wang, Feng; Zhao, Hongxin; Xie, Zongyi; Xu, Zhongye; Zhang, Qingtao; Liang, Ping; Zhai, Xuan; Cheng, Yuan

    2016-03-01

    Heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNP A2/B1) plays an important role in influence of pre-messenger RNA (pre-mRNA) processing and mRNA metabolism and transportation in cells. Increasing evidence indicates that hnRNP A2/B1 played an important role in development and progression of various human cancers. Forty cases of normal and human glioma tissue samples were analyzed using immunohistochemistry to reveal the expression of hnRNP A2/B1 protein in the samples. Then, knockdown of hnRNP A2/B1 expression induced by RNA interference (RNAi) method was used to analyze the role of hnRNP A2/B1 in glioblastoma cell viability, adhesion, migration, invasion, and chemoresistance for temozolomide (TMZ). The data showed that hnRNP A2/B1 protein was overexpressed in glioma tissue specimens and associated with advanced glioma grades. Knockdown of hnRNP A2/B1 could reduce glioblastoma cell viability, adhesion, migration, invasion, and chemoresistance for TMZ capacity, but induced tumor cells to apoptosis and reactive oxygen species (ROS) generation in glioma U251 and SHG44 cells. Molecularly, hnRNP A2/B1 knockdown reduced expression of phospho-STAT3 and MMP-2. Detection of hnRNP A2/B1 expression may be useful as a biomarker for prediction of glioma progression and knockdown of hnRNP A2/B1 expression as a novel strategy in future control of glioblastoma in clinic.

  2. Knockdown of IQGAP1 inhibits proliferation and epithelial–mesenchymal transition by Wnt/β-catenin pathway in thyroid cancer

    PubMed Central

    Su, Dongyue; Liu, Yang; Song, Tao

    2017-01-01

    Background Thyroid cancer is the most common endocrine malignant disease with a high incidence rate. The expression of IQGAP1 is upregulated in various cancers, including thyroid cancer. However, the role and underlying mechanism of IQGAP1 in thyroid cancer are still not clear. Materials and methods The expression of IQGAP1 in thyroid cancer tissues and cells was determined by reverse transcription polymerase chain reaction and Western blot analysis. Cells were transfected with different siRNAs using Lipofectamine 2000 or were treated with various concentrations of XAV939. The effects of IQGAP1 knockdown on proliferation and epithelial–mesenchymal transition (EMT) of thyroid cancer cells were determined by MTT assay and Western blot analysis. Animal experiments were performed to investigate the effects of IQGAP1 knockdown on the growth of tumors in vivo. Results High IQGAP1 expression is found in thyroid cancer tissues and cells. Knockdown of IQGAP1 had inhibitory effects on cell proliferation and EMT, as well as on the Wnt/β-catenin pathway. Additionally, inactivation of the Wnt/β-catenin pathway by XAV939 or si-β-catenin suppressed cell proliferation and EMT. Furthermore, suppression of the Wnt/β-catenin pathway reversed the positive effects of pcDNA-IQGAP1 on cell proliferation and EMT in vitro. Moreover, downregulation of IQGAP1 suppressed tumor growth and EMT in SW579 tumor xenografts through the Wnt/β-catenin pathway in vivo. Conclusion Our study demonstrated that knockdown of IQGAP1 inhibited cell proliferation and EMT through blocking the Wnt/β-catenin pathway in thyroid cancer. PMID:28352188

  3. Knockdown of kinesin KIF11 abrogates directed migration in response to epidermal growth factor-mediated chemotaxis.

    PubMed

    Wang, Fang; Lin, Stanley Li

    2014-09-26

    Establishment of microtubule polarity is critical for directional cell migration involved in morphogenesis, differentiation, cell division, and metastasis. Current models, involving iterative microtubule capture and inactivation of microtubule depolymerizing mechanisms at the leading edge, cannot account for the biased migration exhibited by cells in culture in the absence of directional cues, suggesting central mechanisms governing microtubule polarity remain unknown. We engineered two human MDA-MB-231/IMP1 breast carcinoma cell lines, denoted kdKIF11-1 and kdKIF11-2, in which the kinesin KIF11 (also known as Eg5) was stably knocked down by two different shRNAs. Western blot analysis showed knockdown by each shRNA decreased KIF11 expression by 58% and 79% for kdKIF11-1 and kdKIF11-2, respectively, whereas Rac1 expression was unaffected. All cell lines retained a well-defined microtubule structure. Compared to cells infected with the control viral vector, both KIF11 knockdown cell lines displayed a 14-45% increase in cell motility in a scratch wound healing assay. In contrast, KIF11 knockdown decreased invasion by 70%, compared to the control, as measured by invasion through Matrigel-coated transwells. To determine whether the reduction in invasion was due to reduced chemotaxis, we substituted collagen for Matrigel in the transwell assay and similarly observed a 44-54% reduction in migration, using EGF as the chemoattractant. However, when including EGF in both the upper and lower chambers of the transwell to stimulate migration but eliminate chemotaxis, transwell migration decreased for the control cell line only, indicating that KIF11 knockdown did not impair migration, but severely impaired chemotaxis. We conclude KIF11 is a key downstream molecule that responds to directional cues in chemotaxis to govern the direction of migration.

  4. Knockdown of a Zebrafish Aryl Hydrocarbon Receptor Repressor (AHRRa) Affects Expression of Genes Related to Photoreceptor Development and Hematopoiesis

    PubMed Central

    Aluru, Neelakanteswar; Jenny, Matthew J.; Hahn, Mark E.

    2014-01-01

    The aryl hydrocarbon receptor repressor (AHRR) is a transcriptional repressor of aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF) and is regulated by an AHR-dependent mechanism. Zebrafish (Danio rerio) possess two AHRR paralogs; AHRRa regulates constitutive AHR signaling during development, whereas AHRRb regulates polyaromatic hydrocarbon-induced gene expression. However, little is known about the endogenous roles and targets of AHRRs. The objective of this study was to elucidate the role of AHRRs during zebrafish development using a loss-of-function approach followed by gene expression analysis. Zebrafish embryos were microinjected with morpholino oligonucleotides against AHRRa or AHRRb to knockdown AHRR protein expression. At 72 h postfertilization (hpf), microarray analysis revealed that the expression of 279 and 116 genes was altered by knockdown of AHRRa and AHRRb, respectively. In AHRRa-morphant embryos, 97 genes were up-regulated and 182 genes were down-regulated. Among the down-regulated genes were several related to photoreceptor function, including cone-specific genes such as several opsins (opn1sw1, opn1sw2, opn1mw1, and opn1lw2), phosphodiesterases (pde6H and pde6C), retinol binding protein (rbp4l), phosducin, and arrestins. Down-regulation was confirmed by RT-PCR and with samples from an independent experiment. The four genes tested (opn1sw1, pde6H, pde6C, and arr3b) were not inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin. AHRRa knockdown also caused up-regulation of embryonic hemoglobin (hbbe3), suggesting a role for AHRR in regulating hematopoiesis. Knockdown of AHRRb caused up-regulation of 31 genes and down-regulation of 85 genes, without enrichment for any specific biological process. Overall, these results suggest that AHRRs may have important roles in development, in addition to their roles in regulating xenobiotic signaling. PMID:24675095

  5. Small interfering RNA mediated knockdown of irisin suppresses food intake and modulates appetite regulatory peptides in zebrafish.

    PubMed

    Sundarrajan, Lakshminarasimhan; Unniappan, Suraj

    2017-10-01

    Irisin is a myokine encoded in fibronectin type III domain containing 5 (FNDC5). FNDC5 forms an integral part of the muscle post-exercise, and causes an increase in energy expenditure in mammals. Irisin is abundantly expressed in cardiac and skeletal muscles and is secreted upon activation of peroxisome proliferator-activated receptor gamma coactivator-1 (PGC-1 alpha). Irisin regulates feeding behaviour and cardiovascular function in mammals. More recently, irisin has gained importance as a potential biomarker for myocardial infarction due to its abundance in cardiac muscle. The goal of this research was to determine whether irisin influences feeding, and regulates appetite regulatory peptides in zebrafish. Intraperitoneal injection of irisin [0.1, 1, 10 and 100ng/g body weight (BW)] did not affect feeding, but its knockdown using siRNA (10ng/g BW) caused a significant reduction in food intake. Knockdown of irisin reduced ghrelin and orexin-A mRNA expression, and increased cocaine and amphetamine regulated transcript mRNA expression in zebrafish brain and gut. siRNA mediated knockdown of irisin also downregulated brain derived neurotrophic factor mRNA in zebrafish. The role of endogenous irisin on food intake is likely mediated by its actions on other metabolic peptides. Collectively, these results indicate that unaltered endogenous irisin is required to maintain food intake in zebrafish. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Knockdown of heme oxygenase-1 promotes apoptosis and autophagy and enhances the cytotoxicity of doxorubicin in breast cancer cells

    PubMed Central

    ZHU, XIAO-FENG; LI, WEN; MA, JIE-YI; SHAO, NAN; ZHANG, YUN-JIAN; LIU, RUI-MING; WU, WEI-BIN; LIN, YING; WANG, SHEN-MING

    2015-01-01

    Heme oxygenase-1 (HMOX-1) is a microsomal enzyme that exerts anti-apoptotic and cytoprotective effects. In the present study, HMOX-1 was demonstrated to be overexpressed and able to be induced by doxorubicin in breast cancer cell lines. Knockdown of HMOX-1 using short interfering (si)RNA enhanced the cytotoxicity of doxorubicin in MDA-MB-231 and BT549 cells. Knockdown of HMOX-1 downregulated B cell lymphoma (Bcl)-2 and Bcl-extra large expression, and significantly enhanced doxorubicin-induced apoptosis in MDA-MB-231 and BT549 cells. Additionally, knockdown of HMOX-1 upregulated light chain 3B expression and markedly increased the accumulation of autophagic vacuoles in MDA-MB-231 and BT549 cells treated with doxorubicin. These results indicated that HMOX-1 may be involved in conferring the chemoresistance of breast cancer cells, by preventing apoptosis and autophagy. Therefore, HMOX-1 may represent a potential therapeutic target for enhancing the cytotoxicity and efficacy of doxorubicin during the treatment of breast cancer. PMID:26722274

  7. Functional analysis of ESM1 by siRNA knockdown in primary and metastatic head and neck cancer cells.

    PubMed

    Bender, Onur; Gunduz, Mehmet; Cigdem, Sadik; Hatipoglu, Omer Faruk; Acar, Muradiye; Kaya, Mesut; Grenman, Reidar; Gunduz, Esra; Ugur, Kadriye Serife

    2017-10-11

    Genetic factors play a large role in cancer and thus there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities are of utmost importance. The aim of our study is to illuminate the role of ESM1 (endothelial cell specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers. ESM1 expression was shown with immunofluorescence assay by using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real time RT-PCR and western blot. Cell proliferation and migration assays were performed by xCELLigence real time cell analysis system. Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells. These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  8. RNAi-mediated knock-down of Dab and Numb attenuate Aβ levels via γ-secretase mediated APP processing

    PubMed Central

    2012-01-01

    Amyloid-β-protein (Aβ), the key component of senile plaques in Alzheimer's disease (AD) brain, is produced from amyloid precursor protein (APP) by cleavage of β-secretase and then γ-secretase. APP adaptor proteins with phosphotyrosine-binding (PTB) domains, including Dab (gene: DAB) and Numb (gene: NUMB), can bind to and interact with the conserved YENPTY-motif in the APP C-terminus. Here we describe, for the first time, the effects of RNAi knock-down of Dab and Numb expression on APP processing and Aβ production. RNAi knock-down of Dab and Numb in H4 human neuroglioma cells stably transfected to express either FL-APP (H4-FL-APP cells) or APP-C99 (H4-APP-C99 cells) increased levels of APP-C-terminal fragments (APP-CTFs) and lowered Aβ levels in both cell lines by inhibiting γ-secretase cleavage of APP. Finally, RNAi knock-down of APP also reduced levels of Numb in H4-APP cells. These findings suggest that pharmacologically blocking interaction of APP with Dab and Numb may provide novel therapeutic strategies of AD. The notion of attenuating γ-secretase cleavage of APP via the APP adaptor proteins, Dab and Numb, is particularly attractive with regard to therapeutic potential, given that side effects of γ-secretase inhibition owing to impaired proteolysis of other γ-secretase substrates, e.g. Notch, might be avoided. PMID:23211096

  9. A multicolor panel of TALE-KRAB based transcriptional repressor vectors enabling knockdown of multiple gene targets

    PubMed Central

    Zhang, Zhonghui; Wu, Elise; Qian, Zhijian; Wu, Wen-Shu

    2014-01-01

    Stable and efficient knockdown of multiple gene targets is highly desirable for dissection of molecular pathways. Because it allows sequence-specific DNA binding, transcription activator-like effector (TALE) offers a new genetic perturbation technique that allows for gene-specific repression. Here, we constructed a multicolor lentiviral TALE-Kruppel-associated box (KRAB) expression vector platform that enables knockdown of multiple gene targets. This platform is fully compatible with the Golden Gate TALEN and TAL Effector Kit 2.0, a widely used and efficient method for TALE assembly. We showed that this multicolor TALE-KRAB vector system when combined together with bone marrow transplantation could quickly knock down c-kit and PU.1 genes in hematopoietic stem and progenitor cells of recipient mice. Furthermore, our data demonstrated that this platform simultaneously knocked down both c-Kit and PU.1 genes in the same primary cell populations. Together, our results suggest that this multicolor TALE-KRAB vector platform is a promising and versatile tool for knockdown of multiple gene targets and could greatly facilitate dissection of molecular pathways. PMID:25475013

  10. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum.

    PubMed

    Abd El Halim, Hesham M; Alshukri, Baida M H; Ahmad, Munawar S; Nakasu, Erich Y T; Awwad, Mohammed H; Salama, Elham M; Gatehouse, Angharad M R; Edwards, Martin G

    2016-07-14

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30-60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control.

  11. A Glyoxalase-1 Knockdown Does Not Have Major Short Term Effects on Energy Expenditure and Atherosclerosis in Mice.

    PubMed

    Wortmann, Markus; Hakimi, Maani; Fleming, Thomas; Peters, Andreas S; Sijmonsma, Tjeerd P; Herzig, Stephan; Nawroth, Peter P; Böckler, Dittmar; Dihlmann, Susanne

    2016-01-01

    Objective. Glyoxalase-1 is an enzyme detoxifying methylglyoxal (MG). MG is a potent precursor of advanced glycation endproducts which are regarded to be a key player in micro- and macrovascular damage. Yet, the role of Glo1 in atherosclerosis remains unclear. In this study, the effect of Glo1 on mouse metabolism and atherosclerosis is evaluated. Methods. Glo1 knockdown mice were fed a high fat or a standard diet for 10 weeks. Body weight and composition were investigated by Echo MRI. The PhenoMaster system was used to measure the energy expenditure. To evaluate the impact of Glo1 on atherosclerosis, Glo1(KD) mice were crossed with ApoE-knockout mice and fed a high fat diet for 14 weeks. Results. Glo1 activity was significantly reduced in heart, liver, and kidney lysates derived from Glo1(KD) mice. Yet, there was no increase in methylglyoxal-derived AGEs in all organs analyzed. The Glo1 knockdown did not affect body weight or body composition. Metabolic studies via indirect calorimetry did not show significant effects on energy expenditure. Glo1(KD) mice crossed to ApoE(-/-) mice did not show enhanced formation of atherosclerosis. Conclusion. A Glo1 knockdown does not have major short term effects on the energy expenditure or the formation of atherosclerotic plaques.

  12. RNAi-mediated knockdown of the voltage gated sodium ion channel TcNav causes mortality in Tribolium castaneum

    PubMed Central

    Abd El Halim, Hesham M.; Alshukri, Baida M. H.; Ahmad, Munawar S.; Nakasu, Erich Y. T.; Awwad, Mohammed H.; Salama, Elham M.; Gatehouse, Angharad M. R.; Edwards, Martin G.

    2016-01-01

    The voltage-gated sodium ion channel (VGSC) belongs to the largest superfamily of ion channels. Since VGSCs play key roles in physiological processes they are major targets for effective insecticides. RNA interference (RNAi) is widely used to analyse gene function, but recently, it has shown potential to contribute to novel strategies for selectively controlling agricultural insect pests. The current study evaluates the delivery of dsRNA targeted to the sodium ion channel paralytic A (TcNav) gene in Tribolium castaneum as a viable means of controlling this insect pest. Delivery of TcNav dsRNA caused severe developmental arrest with larval mortalities up to 73% post injection of dsRNA. Injected larvae showed significant (p < 0.05) knockdown in gene expression between 30–60%. Expression was also significantly (p < 0.05) reduced in pupae following injection causing 30% and 42% knockdown for early and late pupal stages, respectively. Oral delivery of dsRNA caused dose-dependant mortalities of between 19 and 51.34%; this was accompanied by significant (p < 0.05) knockdown in gene expression following 3 days of continuous feeding. The majority of larvae injected with, or fed, dsRNA died during the final larval stage prior to pupation. This work provides evidence of a viable RNAi-based strategy for insect control. PMID:27411529

  13. AMPK knockdown in Placental Labyrinthine Progenitor Cells Results in Restriction of Critical Energy Resources and Terminal Differentiation Failure.

    PubMed

    Waker, Christopher A; Albers, Renee E; Pye, Richard L; Doliboa, Savannah R; Wyatt, Christopher N; Brown, Thomas L; Mayes, Debra Ann

    2017-03-23

    Placental abnormalities can cause Pregnancy-Associated Disorders including preeclampsia, intrauterine growth restriction, and placental insufficiency that result in complications for both the mother and fetus. Trophoblast cells within the labyrinthine layer of the placenta facilitate the exchange of nutrients, gases, and waste between mother and fetus; therefore, the development of this cell layer is critical for fetal development. As trophoblast cells differentiate, it is assumed their metabolism changes with their energy requirements. We hypothesize that proper regulation of trophoblast metabolism is a key component of normal placental development; therefore, we examined the role of AMP-activated kinase (AMPK, PRKAA1/2), a sensor of cellular energy status. Our previous studies have shown that AMPK knockdown alters both trophoblast differentiation and nutrient transport. In this study, AMPKα1/2 shRNA was used to investigate the metabolic effects of AMPK knockdown on SM10 placental labyrinthine progenitor cells before and after differentiation. Extracellular flux analysis confirmed that AMPK knockdown was sufficient to reduce trophoblast glycolysis, mitochondrial respiration, and ATP coupling efficiency. A reduction in AMPK in differentiated trophoblasts also resulted in increased mitochondrial volume. These data indicate that a reduction in AMPK disrupts cellular metabolism in both progenitors and differentiated placental trophoblasts. This disruption correlates to abortive trophoblast differentiation that may contribute to the development of Pregnancy-Associated Disorders.

  14. Functional VEGFA knockdown with artificial 3′-tailed mirtrons defined by 5′ splice site and branch point

    PubMed Central

    Kock, Kian Hong; Kong, Kiat Whye; Hoon, Shawn; Seow, Yiqi

    2015-01-01

    Mirtrons are introns that form pre-miRNA hairpins after splicing to produce RNA interference (RNAi) effectors distinct from Drosha-dependent intronic miRNAs, and will be especially useful for co-delivery of coding genes and RNAi. A specific family of mirtrons – 3′-tailed mirtrons – has hairpins precisely defined on the 5′ end by the 5′ splice site and 3′ end by the branch point. Here, we present design principles for artificial 3′-tailed mirtrons and demonstrate, for the first time, efficient gene knockdown with tailed mirtrons within eGFP coding region. These artificial tailed mirtrons, unlike canonical mirtrons, have very few sequence design restrictions. Tailed mirtrons targeted against VEGFA mRNA, the misregulation of which is causative of several disorders including cancer, achieved significant levels of gene knockdown. Tailed mirtron-mediated knockdown was further shown to be splicing-dependent, and at least as effective as equivalent artificial intronic miRNAs, with the added advantage of very defined cleavage sites for generation of mature miRNA guide strands. Further development and exploitation of this unique mirtron biogenesis pathway for therapeutic RNAi coupled into protein-expressing genes can potentially enable the development of precisely controlled combinatorial gene therapy. PMID:26089392

  15. N-Myc knockdown and apigenin treatment controlled growth of malignant neuroblastoma cells having N-Myc amplification

    PubMed Central

    Hossain, Md. Motarab; Banik, Naren L.; Ray, Swapan K.

    2013-01-01

    Malignant neuroblastomas mostly occur in children and are frequently associated with N-Myc amplification. Oncogene amplification, which is selective increase in copy number of the oncogene, provides survival advantages in solid tumors including malignant neuroblastoma. We have decreased expression of N-Myc oncogene using short hairpin RNA (shRNA) plasmid to increase anti-tumor efficacy of the isoflavonoid apigenin (APG) in human malignant neuroblastoma SK-N-DZ and SK-N-BE2 cell lines that harbor N-Myc amplification. N-Myc knockdown induced morphological and biochemical features of neuronal differentiation. Combination of N-Myc knockdown and APG most effectively induced morphological and biochemical features of apoptotic death. This combination therapy also prevented cell migration and decreased N-Myc driven survival, angiogenic, and invasive factors. Collectively, N-Myc knockdown and APG treatment is a promising strategy for controlling the growth of human malignant neuroblastoma cell lines that harbor N-Myc amplification. PMID:23941992

  16. Resistance-resistant antibiotics.

    PubMed

    Oldfield, Eric; Feng, Xinxin

    2014-12-01

    New antibiotics are needed because drug resistance is increasing while the introduction of new antibiotics is decreasing. We discuss here six possible approaches to develop 'resistance-resistant' antibiotics. First, multitarget inhibitors in which a single compound inhibits more than one target may be easier to develop than conventional combination therapies with two new drugs. Second, inhibiting multiple targets in the same metabolic pathway is expected to be an effective strategy owing to synergy. Third, discovering multiple-target inhibitors should be possible by using sequential virtual screening. Fourth, repurposing existing drugs can lead to combinations of multitarget therapeutics. Fifth, targets need not be proteins. Sixth, inhibiting virulence factor formation and boosting innate immunity may also lead to decreased susceptibility to resistance. Although it is not possible to eliminate resistance, the approaches reviewed here offer several possibilities for reducing the effects of mutations and, in some cases, suggest that sensitivity to existing antibiotics may be restored in otherwise drug-resistant organisms.

  17. Knockdown of Merm1/Wbscr22 attenuates sensitivity of H460 non-small cell lung cancer cells to SN-38 and 5-FU without alteration to p53 expression levels.

    PubMed

    Yan, Dongmei; Zheng, Xiaoliang; Tu, Linglan; Jia, Jing; Li, Qin; Cheng, Liyan; Wang, Xiaoju

    2015-01-01

    Merm1/Wbscr22 is a novel metastasis promoter that has been shown to be involved in tumor metastasis, viability and apoptosis. To the best of our knowledge, there are currently no studies suggesting the possible correlation between the expression of Merm1/Wbscr22 in tumor cells and chemosensitivity to antitumor agents. In the present study, two human non-small cell lung cancer cell lines, H1299 and H460, were used to investigate whether Merm1/Wbscr22 affects chemosensitivity to antitumor agents, including cisplatin (CDDP), doxorubicin (ADM), paclitaxel (PTX), mitomycin (MMC), 7-Ethyl-10-hydroxycamptothecin (SN-38; the active metabolite of camptothecin) and 5-fluorouracil (5-FU). Merm1/Wbscr22 knockdown cell lines (H1299-shRNA and H460-shRNA) and negative control cell lines (H1299-NC and H460-NC) were established by stable transfection, and the efficiency of Merm1/Wbscr22 knockdown was confirmed by western blotting, immunofluorescence microscopy and quantitative polymerase chain reaction. The results demonstrated that shRNA-mediated knockdown of Merm1/Wbscr22 did not affect cell proliferation in vitro and in vivo. The H460 cells harboring wild type p53 were markedly more sensitive to all six antitumor agents as compared with the p53-null H1299 cells. Downregulation of Merm1/Wbscr22 did not affect H1299 sensitivity to any of the six antitumor agents, whereas attenuated H460 sensitivity to SN-38 and 5-FU, without significant alteration in p53 at both mRNA and protein levels, was identified. The reduced H460 sensitivity to SN-38 was further confirmed in vivo. SN-38 demonstrated significant tumor growth inhibitory activity in both H460 and H460‑NC tumor xenograft models, but only marginally suppressed the H460-shRNA xenograft tumor growth. Furthermore, CDDP (4, 10, 15 µg/ml)-resistant human non-small lung cancer cells A549 (A549-CDDPr-4, 10, 15) expressed significant amounts of Merm1/Wbscr22 protein, as compared with the parental A549 cells. In conclusion, sh

  18. Antimicrobial Resistance

    MedlinePlus

    ... spread of antimicrobial resistance. Present situation Resistance in bacteria Antibiotic resistance is present in every country. Patients with infections caused by drug-resistant bacteria are at increased risk of worse clinical outcomes ...

  19. Bax-inhibitor-1 knockdown phenotypes are suppressed by Buffy and exacerbate degeneration in a Drosophila model of Parkinson disease

    PubMed Central

    2017-01-01

    Background Bax inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective transmembrane protein that acts as a suppressor of Bax-induced apoptosis by regulation of endoplasmic reticulum stress-induced cell death. We knocked down BI-1 in the sensitive dopa decarboxylase (Ddc) expressing neurons of Drosophila melanogaster to investigate its neuroprotective functions. We additionally sought to rescue the BI-1-induced phenotypes by co-expression with the pro-survival Buffy and determined the effect of BI-1 knockdown on the neurodegenerative α-synuclein-induced Parkinson disease (PD) model. Methods We used organismal assays to assess longevity of the flies to determine the effect of the altered expression of BI-1 in the Ddc-Gal4-expressing neurons by employing two RNAi transgenic fly lines. We measured the locomotor ability of these RNAi lines by computing the climbing indices of the climbing ability and compared them to a control line that expresses the lacZ transgene. Finally, we performed biometric analysis of the developing eye, where we counted the number of ommatidia and calculated the area of ommatidial disruption. Results The knockdown of BI-1 in these neurons was achieved under the direction of the Ddc-Gal4 transgene and resulted in shortened lifespan and precocious loss of locomotor ability. The co-expression of Buffy, the Drosophila anti-apoptotic Bcl-2 homologue, with BI-1-RNAi resulted in suppression of the reduced lifespan and impaired climbing ability. Expression of human α-synuclein in Drosophila dopaminergic neurons results in neuronal degeneration, accompanied by the age-dependent loss in climbing ability. We exploited this neurotoxic system to investigate possible BI-1 neuroprotective function. The co-expression of α-synuclein with BI-1-RNAi results in a slight decrease in lifespan coupled with an impairment in climbing ability. In supportive experiments, we employed the neuron-rich Drosophila compound eye to investigate subtle phenotypes

  20. Clusterin knockdown sensitizes prostate cancer cells to taxane by modulating mitosis.

    PubMed

    Al Nakouzi, Nader; Wang, Chris Kedong; Beraldi, Eliana; Jager, Wolfgang; Ettinger, Susan; Fazli, Ladan; Nappi, Lucia; Bishop, Jennifer; Zhang, Fan; Chauchereau, Anne; Loriot, Yohann; Gleave, Martin

    2016-07-01

    Clusterin (CLU) is a stress-activated molecular chaperone that confers treatment resistance to taxanes when highly expressed. While CLU inhibition potentiates activity of taxanes and other anti-cancer therapies in preclinical models, progression to treatment-resistant disease still occurs implicating additional compensatory survival mechanisms. Taxanes are believed to selectively target cells in mitosis, a complex mechanism controlled in part by balancing antagonistic roles of Cdc25C and Wee1 in mitosis progression. Our data indicate that CLU silencing induces a constitutive activation of Cdc25C, which delays mitotic exit and hence sensitizes cancer cells to mitotic-targeting agents such as taxanes. Unchecked Cdc25C activation leads to mitotic catastrophe and cell death unless cells up-regulate protective mechanisms mediated through the cell cycle regulators Wee1 and Cdk1. In this study, we show that CLU silencing induces a constitutive activation of Cdc25C via the phosphatase PP2A leading to relief of negative feedback inhibition and activation of Wee1-Cdk1 to promote survival and limit therapeutic efficacy. Simultaneous inhibition of CLU-regulated cell cycle effector Wee1 may improve synergistic responses of biologically rational combinatorial regimens using taxanes and CLU inhibitors.

  1. [Knockdown of Larp4b in Lin(-) cells does not affect the colony forming ability of mouse hematopoietic cells].

    PubMed

    Wang, Xiao-Juan; Pang, Ya-Kun; Cheng, Hui; Dong, Fang; Liang, Hao-Yue; Zhang, Ying-Chi; Wang, Xiao-Min; Xu, Jing; Cheng, Tao; Yuan, Wei-Ping

    2013-06-01

    Larp4b is a member of the LARP family, which can interact with RNA and generally stimulate the translation of mRNA. Abnormal expression of Larp4b can be found in leukemia patients in our previous study. This study was purposed to detect the relative expression of Larp4b mRNA in different subpopulations of mouse hematopoietic cells, to construct lentivirus vector containing shLarp4b targeting mouse gene Larp4b and to explore its effects on mouse Lin(-) cells infected with shLarp4b by lentivirus. SF-LV-shLarP4b-EGFP and control vectors were constructed and two-plasmid lentivirus packing system was used to transfect 293T cells. After 48 h and 72 h, lentivirus SF-LV-shLarp4b-EGFP was harvested and was used to infect Lin(-) cells. After 48 h, EGFP(+) cells was sorted by flow cytometry (FCM). Meanwhile, semi-quantitative real time-PCR, AnnexinV-PE/7-AAD staining, PI staining and colony forming cell assay (CFC) were performed to determine the expression of Larp4b and its effect on the proliferation of hematopoietic progenitor cells. The results showed that Larp4b was highly expressed in myeloid cells. SF-LV-shLarp4b-EGFP was successfully constructed according to the restriction endonuclease digestion assay. RT-PCR confirmed that Larp4b was efficiently knockdown in mouse Lin(-) cells. The low expression of Larp4b did not affect the colony forming number, the apoptosis and cell cycle of Lin(-) cells. It is concluded that knockdown of Larp4b in mouse Lin(-) cells do not contribute to the colony forming ability and the growth of Lin(-) cells in vitro. This useful knockdown system will be used to study in vivo Larp4b in future.

  2. shRNA mediated knockdown of Nav1.7 in rat dorsal root ganglion attenuates pain following burn injury.

    PubMed

    Cai, Weihua; Cao, Jing; Ren, Xiuhua; Qiao, Liang; Chen, Xuemei; Li, Ming; Zang, Weidong

    2016-08-11

    Abnormal acute pain after burn injury still torments patients severely. In this study, we investigated that one voltage gated sodium channel Nav1.7 plays a vital role in lowering heat pain threshold after burn injury, and the hypothesis that knockdown of Nav1.7 attenuates pain following burn injury. Sixty eight adult male Sprague-Dawley rats were divided into 4 treatment groups: (1) sham, which hind paw was put on the room temperature metal plate for 15 s (2) burn model, which hind paw was put on the 85 °C metal plate for 15 s. (3) Burn injury + lentiviral vector -SCN9AsiRNA-GFP (LV- SCN9AsiRNA-GFP group, n = 18), which receive the DRG microinjection of LV- SCN9AsiRNA-GFP on the zero day. (4) Burn injury + lentiviral vector negative control (LV-NC-GFP group, n = 18), which receive the DRG microinjection of empty lentiviral vector on the zero day. Both mechanical and heat threshold were measured from day 1 to 21. Meanwhile, expression of sodium channels Nav1.7 in injured dorsal root ganglia were measured on post-operative days 7(POD 7). Rats exhibited decreased thresholds on both mechanical allodynia and thermal withdrawl latency, accompanied by increased Nav1.7 and c-fos expression in dorsal root ganglion (DRG). And knockdown of Nav1.7 in L5DRG led to the attenuation of burn injury-induced mechanical allodynia and thermal hyperalgesia in the rats. We provide evidence that shRNA mediated knockdown of Nav1.7 attenuates burn induced pain in rats as well as decreased the activiation of c-fos protein.

  3. Knockdown of MicroRNA-122 Protects H9c2 Cardiomyocytes from Hypoxia-Induced Apoptosis and Promotes Autophagy

    PubMed Central

    Zhang, Zaiwei; Li, Hu; Chen, Shasha; Li, Ying; Cui, Zhiyuan; Ma, Jie

    2017-01-01

    Background Acute myocardial infarction (AMI) is a severe disease causing heart failure and sudden death. Studies indicate that microRNAs (miRNAs) are involved in the pathophysiology of AMI. In the present study, we carefully explored the effects of miR-122 on myocardial hypoxia injury and its possible underlying mechanism. Material/Methods miR-122 expression was analyzed in H9c2 cardiomyocytes after being transfected with miR-122 mimic, ASO-miR-122, or negative control. Cell viability and apoptosis were investigated by CCK-8 assays and flow cytometry analysis, respectively. Cell migration was analyzed using wound-healing assays. Western blotting was performed to analyze the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/phosphatidylinositol 3-hydroxy kinase (PI3K)/AKT and LC3-II/LC3-I. Result Hypoxia exposure significantly inhibited H9c2 cell viability (P<0.01). miR-122 overexpression promoted the hypoxia-induced H9c2 cell proliferation and migration loss (P<0.05), and cell apoptosis was increased (P<0.05). miR-122 knockdown enhanced cell viability and decreased cell apoptosis (P<0.05). Knockdown of miR-122 enhanced PTEN/PI3K/AKT activation and cell autophagy. Overexpression of miR-122 inhibited the PTEN/PI3K/AKT pathway and cell autophagy pathway. Conclusions The expression of miR-122 is involved in hypoxia-induced H9c2 cardiomyocyte injury. Knockdown of miR-122 protects H9c2 cells from hypoxia-induced apoptosis and enhances cell viability. PMID:28871076

  4. Knockdown of SVCT2 impairs in-vitro cell attachment, migration and wound healing in bone marrow stromal cells.

    PubMed

    Sangani, Rajnikumar; Pandya, Chirayu D; Bhattacharyya, Maryka H; Periyasamy-Thandavan, Sudharsan; Chutkan, Norman; Markand, Shanu; Hill, William D; Hamrick, Mark; Isales, Carlos; Fulzele, Sadanand

    2014-03-01

    Bone marrow stromal cell (BMSC) adhesion and migration are fundamental to a number of pathophysiologic processes, including fracture and wound healing. Vitamin C is beneficial for bone formation, fracture repair and wound healing. However, the role of the vitamin C transporter in BMSC adhesion, migration and wound healing is not known. In this study, we knocked-down the sodium-dependent vitamin C transporter, SVCT2, the only known transporter of vitamin C in BMSCs, and performed cell adhesion, migration, in-vitro scratch wound healing and F-actin re-arrangement studies. We also investigated the role of oxidative stress on the above processes. Our results demonstrate that both oxidative stress and down-regulation of SVCT2 decreased cell attachment and spreading. A trans-well cell migration assay showed that vitamin C helped in BMSC migration and that knockdown of SVCT2 decreased cell migration. In the in-vitro scratch wound healing studies, we established that oxidative stress dose-dependently impairs wound healing. Furthermore, the supplementation of vitamin C significantly rescued the BMSCs from oxidative stress and increased wound closing. The knockdown of SVCT2 in BMSCs strikingly decreased wound healing, and supplementing with vitamin C failed to rescue cells efficiently. The knockdown of SVCT2 and induction of oxidative stress in cells produced an alteration in cytoskeletal dynamics. Signaling studies showed that oxidative stress phosphorylated members of the MAP kinase family (p38) and that vitamin C inhibited their phosphorylation. Taken together, these results indicate that both the SVCT2 transporter and oxidative stress play a vital role in BMSC attachment, migration and cytoskeletal re-arrangement. BMSC-based cell therapy and modulation of SVCT2 could lead to a novel therapeutic approach that enhances bone remodeling, fracture repair and wound healing in chronic disease conditions.

  5. CBP knockdown inhibits angiotensin II-induced vascular smooth muscle cells proliferation through downregulating NF-kB transcriptional activity.

    PubMed

    Yang, Jian; Jiang, Hong; Chen, Si-si; Chen, Jing; Xu, Sheng-kai; Li, Wan-qiang; Wang, Ji-chun

    2010-07-01

    CREB binding protein (CBP), a powerful transcriptional co-activator for various transcriptional factors, regulates cell behavior in many cell types. Angiotensin II (Ang II) contributes to vascular lesion by promoting vascular smooth muscle cells (VSMCs) proliferation and migration. Therefore, we examined whether CBP knockdown could suppress Ang II-induced VSMCs proliferation, and elucidated its underlying molecular mechanism. We constructed lentiviral vector expressing CBP-specific short hairpin RNAs (shRNAs) that efficiently silenced CBP. VSMCs proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation assay. Protein and mRNA expression of CBP and relevant cytokines were examined by Western blot, ELISA, and real-time PCR, respectively. We also used luciferase reporter gene and electrophoretic mobility shift assay (EMSA) to detect Nuclear factor kappaB (NF-kB) transcriptional activity and DNA binding. Meanwhile, NF-kB p65 subunit nuclear translocation was confirmed by immunoblotting. Lentiviral-mediated CBP-shRNAs at different multiplicities of infection (MOI = 100, 150) both significantly suppressed Ang II-induced CBP expression. Knockdown of CBP markedly inhibited Ang II-stimulated VSMCs proliferation and cytokines (TNF-alpha and IL-6) production. However, this inhibitory effect was not enhanced at MOI of 150 compared with MOI of 100 (P > 0.05). CBP siRNA showed the potent inhibition on Ang II-induced NF-kB transcriptional activity. Similarly, no significant difference was found between CBP siRNA lentivirus treatment groups. Furthermore, CBP gene silencing had no effect on NF-kB nuclear translocation and DNA binding. These findings suggest that CBP knockdown inhibits Ang II-induced VSMCs proliferation and the mechanism is involved with downregulation of NF-kB transcriptional activity, not through reduction in NF-kB nuclear translocation or DNA binding. Maintaining proper CBP level may be a potential therapeutic target for Ang II-induced cardiovascular

  6. Label-free mass spectrometry exploits dozens of detected peptides to quantify lamins in wildtype and knockdown cells.

    PubMed

    Swift, Joe; Harada, Takamasa; Buxboim, Amnon; Shin, Jae-Won; Tang, Hsin-Yao; Speicher, David W; Discher, Dennis E

    2013-01-01

    Label-free quantitation and characterization of proteins by mass spectrometry (MS) is now feasible, especially for moderately expressed structural proteins such as lamins that typically yield dozens of tryptic peptides from tissue cells. Using standard cell culture samples, we describe general algorithms for quantitative analysis of peptides identified in liquid chromatography tandem mass spectrometry (LC-MS/MS). The algorithms were foundational to the discovery that the absolute stoichiometry of A-type to B-type lamins scales with tissue stiffness (Swift et al., Science 2013). Isoform dominance helps make sense of why mutations and changes with age of mechanosensitive lamin-A,C only affect "stiff" tissues such as heart, muscle, bone, or even fat, but not brain. A Peak Ratio Fingerprinting (PRF) algorithm is elaborated here through its application to lamin-A,C knockdown. After demonstrating the large dynamic range of PRF using calibrated mixtures of human and mouse lysates, we validate measurements of partial knockdown with standard cell biology analyses using quantitative immunofluorescence and immunoblotting. Optimal sets of MS-detected peptides as determined by PRF demonstrate that the strongest peptide signals are not necessarily the most reliable for quantitation. After lamin-A,C knockdown, PRF computes an invariant set of "housekeeping" proteins as part of a broader proteomic analysis that also shows the proteome of mesenchymal stem cells (MSCs) is more broadly perturbed than that of a human epithelial cancer line (A549s), with particular variation in nuclear and cytoskeletal proteins. These methods offer exciting prospects for basic and clinical studies of lamin-A,C as well as other MS-detectable proteins.

  7. Knockdown of the Drosophila fused in sarcoma (FUS) homologue causes deficient locomotive behavior and shortening of motoneuron terminal branches.

    PubMed

    Sasayama, Hiroshi; Shimamura, Mai; Tokuda, Takahiko; Azuma, Yumiko; Yoshida, Tomokatsu; Mizuno, Toshiki; Nakagawa, Masanori; Fujikake, Nobuhiro; Nagai, Yoshitaka; Yamaguchi, Masamitsu

    2012-01-01

    Mutations in the fused in sarcoma/translated in liposarcoma gene (FUS/TLS, FUS) have been identified in sporadic and familial forms of amyotrophic lateral sclerosis (ALS). FUS is an RNA-binding protein that is normally localized in the nucleus, but is mislocalized to the cytoplasm in ALS, and comprises cytoplasmic inclusions in ALS-affected areas. However, it is still unknown whether the neurodegeneration that occurs in ALS is caused by the loss of FUS nuclear function, or by the gain of toxic function due to cytoplasmic FUS aggregation. Cabeza (Caz) is a Drosophila orthologue of human FUS. Here, we generated Drosophila models with Caz knockdown, and investigated their phenotypes. In wild-type Drosophila, Caz was strongly expressed in the central nervous system of larvae and adults. Caz did not colocalize with a presynaptic marker, suggesting that Caz physiologically functions in neuronal cell bodies and/or their axons. Fly models with neuron-specific Caz knockdown exhibited reduced climbing ability in adulthood and anatomical defects in presynaptic terminals of motoneurons in third instar larvae. Our results demonstrated that decreased expression of Drosophila Caz is sufficient to cause degeneration of motoneurons and locomotive disability in the absence of abnormal cytoplasmic Caz aggregates, suggesting that the pathogenic mechanism underlying FUS-related ALS should be ascribed more to the loss of physiological FUS functions in the nucleus than to the toxicity of cytoplasmic FUS aggregates. Since the Caz-knockdown Drosophila model we presented recapitulates key features of human ALS, it would be a suitable animal model for the screening of genes and chemicals that might modify the pathogenic processes that lead to the degeneration of motoneurons in ALS.

  8. Overexpression or knockdown of rat tryptophan hyroxylase-2 has opposing effects on anxiety behavior in an estrogen-dependent manner

    PubMed Central

    Hiroi, Ryoko; McDevitt, Ross A.; Morcos, Paul A.; Clark, Michael S.; Neumaier, John F.

    2010-01-01

    Previous studies showed that chronic estrogen treatment increases tryptophan hydroxylase-2 (TpH2) mRNA in the caudal dorsal raphe nucleus (DRN), and this increase was associated with decreased anxiety. The present study explored the interaction of estrogen and targeted, bidirectional manipulation of TpH2 expression in the caudal DRN by knockdown or viral overexpression, to decrease or increase tryptophan hydroxylase expression respectively, on anxiety behavior. Rats were ovariectomized and replaced with empty or estradiol capsules (OVX, OVX/E, respectively). Animals received microinfusions of either antisense TpH2 or control morpholino oligonucleotides into caudal DRN and were later tested in the open field test. A separate group of animals were microinfused with TpH2-GFP or GFP-only herpes simplex viral vectors into caudal DRN and tested in the open field. The bidirectional impact of manipulations on TpH2 expression was confirmed using a combination of quantitative protein and mRNA measurements; TpH2 expression changes were limited to discrete subregions of DRN that were targeted by the manipulations. Estradiol decreased anxiety in all behavioral measures. In the OVX/E group, TpH2 knockdown significantly decreased time spent in the center of the open field, but not in the OVX group, suggesting that TpH2 knockdown reduced the anxiolytic effects of estrogen. Conversely, TpH2 overexpression in the OVX group mimicked the effects of estrogen, as measured by increased time spent in the center of the open field. These results suggest that estrogen and TpH2 in the caudal DRN have a critical interaction in regulating anxiety-like behavior. PMID:21182901

  9. Knockdown of the fat mass and obesity gene disrupts cellular energy balance in a cell-type specific manner.

    PubMed

    Pitman, Ryan T; Fong, Jason T; Billman, Penny; Puri, Neelu

    2012-01-01

    Recent studies suggest that FTO variants strongly correlate with obesity and mainly influence energy intake with little effect on the basal metabolic rate. We suggest that FTO influences eating behavior by modulating intracellular energy levels and downstream signaling mechanisms which control energy intake and metabolism. Since FTO plays a particularly important role in adipocytes and in hypothalamic neurons, SH-SY5Y neuronal cells and 3T3-L1 adipocytes were used to understand how siRNA mediated knockdown of FTO expression alters cellular energy homeostasis. Cellular energy status was evaluated by measuring ATP levels using a luminescence assay and uptake of fluorescent glucose. FTO siRNA in SH-SY5Y cells mediated mRNA knockdown (-82%), increased ATP concentrations by up to 46% (P = 0.013) compared to controls, and decreased phosphorylation of AMPk and Akt in SH-SY5Y by -52% and -46% respectively as seen by immunoblotting. In contrast, FTO siRNA in 3T3-L1 cells decreased ATP concentration by -93% (p<0.0005), and increased AMPk and Akt phosphorylation by 204% and 70%, respectively suggesting that FTO mediates control of energy levels in a cell-type specific manner. Furthermore, glucose uptake was decreased in both SH-SY5Y (-51% p = 0.015) and 3T3-L1 cells (-30%, p = 0.0002). We also show that FTO knockdown decreases NPY mRNA expression in SH-SY5Y cells (-21%) through upregulation of pSTAT3 (118%). These results provide important evidence that FTO-variant linked obesity may be associated with altered metabolic functions through activation of downstream metabolic mediators including AMPk.

  10. Maternal mRNA knockdown studies: antisense experiments using the host-transfer technique in X. laevis and X. tropicalis

    PubMed Central

    Olson, David J.; Hulstrand, Alissa M.; Houston, Douglas W.

    2014-01-01

    SUMMARY The ability to inhibit the activity of maternally stored gene products in Xenopus has led to numerous insights into early developmental mechanisms. Oocytes can be cultured and manipulated in vitro and then implanted into the body cavity of a host female to make them competent for fertilization. Here, we summarize the methods for obtaining, culturing and fertilizing Xenopus oocytes, with the goal of inhibiting maternal gene function through antisense oligonucleotide-mediated mRNA knockdown. We describe a simplified technique for implanting donor oocytes into host females using intraperitoneal injection. Also, we present optimized methods for performing the host-transfer procedure with X. tropicalis oocytes. PMID:22956088

  11. Expression of IARS2 gene in colon cancer and effect of its knockdown on biological behavior of RKO cells

    PubMed Central

    Zhong, Ling; Zhang, Yi; Yang, Jing-Yu; Xiong, Liang-Fa; Shen, Tao; Sa, Ya-Lian; O’Yang, Yi-Ming; Zhao, Si-Hui; Chen, Jia-Yong

    2015-01-01

    Objective: To investigate the expression level of IARS2 gene in colon cancer tissues and various cell strains of the cancer; to explore cytologically the effect of IARS2 gene knockdown on proliferation, apoptosis and cell cycle of RKO cells in the cancer. Methods: Real-time, fluorescence-based quantitative PCR (qPCR) was used to detect the expression of IARS2 gene in human colon cancer and surrounding tissues and in various cell strains of the cancer; the RNA interference target of IARS2 gene was designed and the target was detected by Western blot; the IARS2-siRNA lentiviral vector was established and used to infect the RKO cells of colon cancer; qPCR was employed to determine the effect of gene knockdown; changes of the RKO cells in growth, apoptosis, cell cycle and clone formation were observed after IARS2 gene knockdown. Results: The expression of IARS2 gene was higher in human colon cancer tissues than in surrounding tissues; there was expression of IARS2 gene in colon cancer cells, and the expression level of IARS2 gene mRNA was higher in the RKO cells than in the SW480, HCT116, DLD1, HT-29 and SW620 cells. After infection of the RKO cells with IARS2-siRNA lentivirus, the expression of IARS2 gene was inhibited in the level of mRNA; proliferation rate of the RKO cells was significantly inhibited; the G1 phase arrest of the RKO cells was increased with less RKO cells in S phase; the apoptotic RKO cells increased significantly; and the number of colonies of the RKO cells reduced. Conclusion: The expression of IARS2 gene is different in human colon cancer and surrounding tissues; after knockdown of IARS2 gene, proliferation of the RKO cells is inhibited; there are more cells in G phase and fewer cells in S phase; apoptosis of cells is increased; and formation of colonies is reduced. IARS2 gene is probably a cancer-promoting gene. PMID:26722399

  12. RNA interference knockdown of aminopeptidase N genes decrease the susceptibility of Chilo suppressalis larvae to Cry1Ab/Cry1Ac and Cry1Ca-expressing transgenic rice.

    PubMed

    Qiu, Lin; Fan, Jinxing; Zhang, Boyao; Liu, Lang; Wang, Xiaoping; Lei, Chaoliang; Lin, Yongjun; Ma, Weihua

    2017-03-06

    Transgenic rice expressing Bacillus thuringiensis (Bt) Cry toxins are resistant to lepidopteran pests, such as Chilo suppressalis, a major insect pest of rice in Asia. Understanding how these toxins interact with their hosts is crucial to understanding their insecticidal action. In this study, knockdown of two aminopeptidase N genes (APN1 and APN2) by RNA interference resulted in decreased susceptibility of C. suppressalis larvae to the Bt rice varieties TT51 (Cry1Ab and Cry1Ac fusion genes) and T1C-19 (Cry1Ca), but not T2A-1 (Cry2Aa). This suggests that APN1 and APN2 are receptors for Cry1A and Cry1C toxins in C. suppressalis.

  13. Malaria vectors resistance to insecticides in Benin: current trends and mechanisms involved.

    PubMed

    Gnanguenon, Virgile; Agossa, Fiacre R; Badirou, Kefilath; Govoetchan, Renaud; Anagonou, Rodrigue; Oke-Agbo, Fredéric; Azondekon, Roseric; AgbanrinYoussouf, Ramziath; Attolou, Roseline; Tokponnon, Filemon T; Aïkpon, Rock; Ossè, Razaki; Akogbeto, Martin C

    2015-04-12

    Insecticides are widely used to control malaria vectors and have significantly contributed to the reduction of malaria-caused mortality. In addition, the same classes of insecticides were widely introduced and used in agriculture in Benin since 1980s. These factors probably contributed to the selection of insecticide resistance in malaria vector populations reported in several localities in Benin. This insecticide resistance represents a threat to vector control tool and should be monitored. The present study reveals observed insecticide resistance trends in Benin to help for a better management of insecticide resistance. Mosquito larvae were collected in eight sites and reared in laboratory. Bioassays were conducted on the adult mosquitoes upon the four types of insecticide currently used in public health in Benin. Knock-down resistance, insensitive acetylcholinesterase-1 resistance, and metabolic resistance analysis were performed in the mosquito populations based on molecular and biochemical analysis. The data were mapped using Geographical Information Systems (GIS) with Arcgis software. Mortalities observed with Deltamethrin (pyrethroid class) were less than 90% in 5 locations, between 90-97% in 2 locations, and over 98% in one location. Bendiocarb (carbamate class) showed mortalities ranged 90-97% in 2 locations and were over 98% in the others locations. A complete susceptibility to Pirimiphos methyl and Fenitrothion (organophosphate class) was observed in all locations with 98-100% mortalities. Knock-down resistance frequencies were high (0.78-0.96) and similar between Anopheles coluzzii, Anopheles gambiae, Anopheles arabiensis, and Anopheles melas. Insensitive acetylcholinesterase-1 was rare (0.002-0.1) and only detected in Anopheles gambiae in concomitance with Knock-down resistance mutation. The maps showed a large distribution of Deltamethrin resistance, Knock-down mutation and metabolic resistance throughout the country, a suspected resistance to

  14. CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.

    PubMed

    Miura, Hiromi; Gurumurthy, Channabasavaiah B; Sato, Takehito; Sato, Masahiro; Ohtsuka, Masato

    2015-08-05

    Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.

  15. Induction of leg-shaking, knock-down and killing responses by gamma-ray irradiation in Shaker mutants of Drosophila melanogaster.

    PubMed

    Megumi, T; Gamo, S; Ohonishi, T; Tanaka, Y

    1995-06-01

    Induction of membrane-associated responses, a leg-shaking, a knock-down and a killing, by gamma-ray irradiation was investigated in Shaker (Sh) mutants of Drosophila melanogaster in which the gene cords for the A-current K+ channel. Sh mutants were more sensitive in the knock-down response after gamma-ray irradiation than wild types. There were a great amount of sex difference in the knock-down response, males being more sensitive than females, but not in the killing response. The sex difference was larger than gene dosage effect on X chromosome in females. Genetical analysis revealed that the sensitivity of the knock-down response is an incompletely dominant character without maternal effects. The leg-shaking response, which had previously been reported to be induced by ether treatment, was demonstrated in the head-removed flies of Sh mutants. It was found to be the most sensitive among the responses tested, and may involve changes in K+ channel. The knock-down response may be related to expansion of the leg-shaking response. The killing response should have c