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Sample records for interfering rna increases

  1. 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.

  2. Polymers in Small-Interfering RNA Delivery

    PubMed Central

    Singha, Kaushik; Namgung, Ran

    2011-01-01

    This review will cover the current strategies that are being adopted to efficiently deliver small interfering RNA using nonviral vectors, including the use of polymers such as polyethylenimine, poly(lactic-co-glycolic acid), polypeptides, chitosan, cyclodextrin, dendrimers, and polymers-containing different nanoparticles. The article will provide a brief and concise account of underlying principle of these polymeric vectors and their structural and functional modifications which were intended to serve different purposes to affect efficient therapeutic outcome of small-interfering RNA delivery. The modifications of these polymeric vectors will be discussed with reference to stimuli-responsiveness, target specific delivery, and incorporation of nanoconstructs such as carbon nanotubes, gold nanoparticles, and silica nanoparticles. The emergence of small-interfering RNA as the potential therapeutic agent and its mode of action will also be mentioned in a nutshell. PMID:21749290

  3. Unzipping and binding of small interfering RNA with single walled carbon nanotube: A platform for small interfering RNA delivery

    NASA Astrophysics Data System (ADS)

    Santosh, Mogurampelly; Panigrahi, Swati; Bhattacharyya, Dhananjay; Sood, A. K.; Maiti, Prabal K.

    2012-02-01

    In an effort to design efficient platform for siRNA delivery, we combine all atom classical and quantum simulations to study the binding of small interfering RNA (siRNA) by pristine single wall carbon nanotube (SWCNT). Our results show that siRNA strongly binds to SWCNT surface via unzipping its base-pairs and the propensity of unzipping increases with the increase in the diameter of the SWCNTs. The unzipping and subsequent wrapping events are initiated and driven by van der Waals interactions between the aromatic rings of siRNA nucleobases and the SWCNT surface. However, molecular dynamics (MD) simulations of double strand DNA (dsDNA) of the same sequence show that the dsDNA undergoes much less unzipping and wrapping on the SWCNT in the simulation time scale of 70 ns. This interesting difference is due to smaller interaction energy of thymidine of dsDNA with the SWCNT compared to that of uridine of siRNA, as calculated by dispersion corrected density functional theory (DFT) methods. After the optimal binding of siRNA to SWCNT, the complex is very stable which serves as one of the major mechanisms of siRNA delivery for biomedical applications. Since siRNA has to undergo unwinding process with the effect of RNA-induced silencing complex, our proposed delivery mechanism by SWCNT possesses potential advantages in achieving RNA interference.

  4. Small interfering RNA delivery through positively charged polymer nanoparticles

    NASA Astrophysics Data System (ADS)

    Dragoni, Luca; Ferrari, Raffaele; Lupi, Monica; Cesana, Alberto; Falcetta, Francesca; Ubezio, Paolo; D'Incalci, Maurizio; Morbidelli, Massimo; Moscatelli, Davide

    2016-03-01

    Small interfering RNA (siRNA) is receiving increasing attention with regard to the treatment of many genetic diseases, both acquired and hereditary, such as cancer and diabetes. Being a high molecular weight (MW) polyanion, siRNA is not able to cross a cell membrane, and in addition it is unstable in physiological conditions. Accordingly, a biocompatible nanocarrier able to deliver siRNA into cells is needed. In this work, we synthesized biocompatible positively charged nanoparticles (NPs) following a two-step process that involves ring opening polymerization (ROP) and emulsion free radical polymerization (EFRP). Firstly, we proved the possibility of fine tuning the NPs’ characteristics (e.g. size and surface charge) by changing the synthetic process parameters. Then the capability in loading and delivering undamaged siRNA into a cancer cell cytoplasm has been shown. This latter process occurs through the biodegradation of the polymer constituting the NPs, whose kinetics can be tuned by adjusting the polymer’s MW. Finally, the ability of NPs to carry siRNA inside the cells in order to inhibit their target gene has been demonstrated using green flourescent protein positive cells.

  5. Therapeutic potential of small interfering RNAs/micro interfering RNA in hepatocellular carcinoma

    PubMed Central

    Farra, Rossella; Grassi, Mario; Grassi, Gabriele; Dapas, Barbara

    2015-01-01

    Hepatocellular carcinoma (HCC) is the predominant form of primary liver cancer and represents the third leading cause of cancer-related death worldwide. Current available therapeutic approaches are poorly effective, especially for the advanced forms of the disease. In the last year, short double stranded RNA molecules termed small interfering RNAs (siRNAs) and micro interfering RNAs (miRNA), emerged as interesting molecules with potential therapeutic value for HCC. The practical use of these molecules is however limited by the identification of optimal molecular targets and especially by the lack of effective and targeted HCC delivery systems. Here we focus our discussion on the most recent advances in the identification of siRNAs/miRNAs molecular targets and on the development of suitable siRNA/miRNAs delivery systems. PMID:26290628

  6. Delivery of small interfering RNA (siRNA) using the sleeping beauty transposon.

    PubMed

    Fletcher, Bradley S

    2010-11-01

    RNA interference (RNAi) is an evolutionarily conserved process that silences gene expression through double-stranded RNA species in a sequence-specific manner. Small interfering RNAs (siRNAs) can promote sequence-specific degradation and/or translational repression of target RNA by activation of the RNA-induced silencing complex (RISC). Traditionally, silencing in mammalian cells had been achieved by transfection of synthetically derived siRNA duplexes, resulting in transient gene suppression of the target sequence. As the technology was advanced, inhibitory short-hairpin-shaped RNAs (shRNAs) could be produced by transcription from RNA polymerase-III (pol-III)-driven promoters, such as H1, U6, or cytomegalovirus (CMV)-enhanced pol III promoters. Following transcription, the shRNAs are processed by the enzyme Dicer into active siRNA. This approach allows for the continuous production of siRNA within cells using a DNA template and offers increased options for delivery of the pol-III-driven transcriptional units. A number of different viral vectors, as well as plasmid DNAs, have been utilized to deliver shRNA to mammalian cells. Here, the Tc1/mariner DNA transposon Sleeping Beauty (SB) is used as a tool to deliver shRNA-encoding transcriptional units. The SB transposon system uses a "cut-and-paste" mechanism to insert the transposon into random TA dinucleotides within the target genome. The shRNAs are then processed and used for gene knockdown. PMID:21041394

  7. Functional Nanostructures for Effective Delivery of Small Interfering RNA Therapeutics

    PubMed Central

    Hong, Cheol Am; Nam, Yoon Sung

    2014-01-01

    Small interfering RNA (siRNA) has proved to be a powerful tool for target-specific gene silencing via RNA interference (RNAi). Its ability to control targeted gene expression gives new hope to gene therapy as a treatment for cancers and genetic diseases. However, siRNA shows poor pharmacological properties, such as low serum stability, off-targeting, and innate immune responses, which present a significant challenge for clinical applications. In addition, siRNA cannot cross the cell membrane for RNAi activity because of its anionic property and stiff structure. Therefore, the development of a safe, stable, and efficient system for the delivery of siRNA therapeutics into the cytoplasm of targeted cells is crucial. Several nanoparticle platforms for siRNA delivery have been developed to overcome the major hurdles facing the therapeutic uses of siRNA. This review covers a broad spectrum of non-viral siRNA delivery systems developed for enhanced cellular uptake and targeted gene silencing in vitro and in vivo and discusses their characteristics and opportunities for clinical applications of therapeutic siRNA. PMID:25285170

  8. Pulmonary administration of small interfering RNA: The route to go?

    PubMed

    Ruigrok, M J R; Frijlink, H W; Hinrichs, W L J

    2016-08-10

    Ever since the discovery of RNA interference (RNAi), which is a post-transcriptional gene silencing mechanism, researchers have been studying the therapeutic potential of using small interfering RNA (siRNA) to treat diseases that are characterized by excessive gene expression. Excessive gene expression can be particularly harmful if it occurs in a vulnerable organ such as the lungs as they are essential for physiological respiration. Consequently, RNAi could offer an approach to treat such lung diseases. Parenteral administration of siRNA has been shown to be difficult due to degradation by nucleases in the systemic circulation and excretion by the kidneys. To avoid these issues and to achieve local delivery and local effects, pulmonary administration has been proposed as an alternative administration route. Regarding this application, various animal studies have been conducted over the past few years. Therefore, this review presents a critical analysis of publications where pulmonary administration of siRNA in animals has been reported. Such an analysis is necessary to determine the feasibility of this administration route and to define directions for future research. First, we provide background information on lungs, pulmonary administration, and delivery vectors. Thereafter, we present and discuss relevant animal studies. Though nearly all publications reported positive outcomes, several reoccurring challenges were identified. They relate to 1) the necessity, efficacy, and safety of delivery vectors, 2) the biodistribution of siRNA in tissues other than the lungs, 3) the poor correlation between in vitro and in vivo models, and 4) the long-term effects upon (repeated) administration of siRNA. Finally, we present recommendations for future research to define the route to go: towards safer and more effective pulmonary administration of siRNA. PMID:27235976

  9. Short interfering RNA guide strand modifiers from computational screening.

    PubMed

    Onizuka, Kazumitsu; Harrison, Jason G; Ball-Jones, Alexi A; Ibarra-Soza, José M; Zheng, Yuxuan; Ly, Diana; Lam, Walter; Mac, Stephanie; Tantillo, Dean J; Beal, Peter A

    2013-11-13

    Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered "undruggable". However, properties associated with the native RNA structure limit drug development, and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5'-end using computational screening with the high-resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5'-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5'-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5'-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5'-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogues for siRNAs. PMID:24152142

  10. Short Interfering RNA Guide Strand Modifiers from Computational Screening

    PubMed Central

    Onizuka, Kazumitsu; Harrison, Jason G.; Ball-Jones, Alexi A.; Ibarra-Soza, José M.; Zheng, Yuxuan; Ly, Diana; Lam, Walter; Mac, Stephanie; Tantillo, Dean J.; Beal, Peter A.

    2013-01-01

    Short interfering RNAs (siRNAs) are promising drug candidates for a wide range of targets including those previously considered “undruggable”. However, properties associated with the native RNA structure limit drug development and chemical modifications are necessary. Here we describe the structure-guided discovery of functional modifications for the guide strand 5’ end using computational screening with the high resolution structure of human Ago2, the key nuclease on the RNA interference pathway. Our results indicate the guide strand 5’-end nucleotide need not engage in Watson-Crick (W/C) H-bonding but must fit the general shape of the 5’-end binding site in MID/PIWI domains of hAgo2 for efficient knockdown. 1,2,3-Triazol-4-yl bases formed from the CuAAC reaction of azides and 1-ethynylribose, which is readily incorporated into RNA via the phosphoramidite, perform well at the guide strand 5’-end. In contrast, purine derivatives with modified Hoogsteen faces or N2 substituents are poor choices for 5’-end modifications. Finally, we identified a 1,2,3-triazol-4-yl base incapable of W/C H-bonding that performs well at guide strand position 12, where base pairing to target was expected to be important. This work expands the repertoire of functional nucleotide analogs for siRNAs. PMID:24152142

  11. Characterization of defective interfering RNAs associated with RNA plant viruses

    SciTech Connect

    Morris, T.J. . School of Biological Sciences); Jackson, A.O. . Dept. of Plant Pathology)

    1993-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  12. Interfering cancer with polymeric siRNA nanomedicines.

    PubMed

    Tiram, Galia; Scomparin, Anna; Ofek, Paula; Satchi-Fainaro, Ronit

    2014-01-01

    The ability to specifically silence genes using RNA interference (RNAi) has wide therapeutic applications for the treatment of disease. Numerous studies have demonstrated global gene and protein signatures distinguishing malignant and nonmalignant tissues. This worldwide pursuit of optimal cancer targets has so far provided a wide list of potential targets for each cancer type and for each patient, for which RNAi-based therapies can be applied. Nevertheless, due to poor stability of RNAi molecules in physiological conditions and their inability to cross cellular membranes, the delivery of siRNA and microRNA (miRNA) in vivo holds a great challenge and remains a crucial issue for their therapeutic success. Supramolecular carriers are often used in order to improve the physicochemical and biopharmaceutical properties of RNAi. Nano-sized delivery systems enable the accumulation of drugs and oligonucleotides (ONTs) in angiogenesis-dependent areas due to the enhanced permeability and retention (EPR) effect, and are able to cross cellular membranes and release the siRNA/miRNA only inside the target cell. In addition, a targeting moiety can increase the selectivity and specific uptake in the target tissue. Several vehicles (dendrimers, nanoparticles, liposomes, polyplex, lipoplex, polymeric nanoconjugates) are being developed for siRNA/miRNA delivery. These vehicles provide an important tool for exploiting the full potential of ONTs as therapeutic agents. In this review we will focus on the polymer-based approaches to deliver siRNA to cancer in vivo. PMID:24724498

  13. Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.

    PubMed

    Singhania, Richa; Pavey, Sandra; Payne, Elizabeth; Gu, Wenyi; Clancy, Jennifer; Jubair, Luqman; Preiss, Thomas; Saunders, Nicholas; McMillan, Nigel A J

    2016-08-01

    Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing. PMID:27321990

  14. Colored petri net modeling of small interfering RNA-mediated messenger RNA degradation

    PubMed Central

    Nickaeen, Niloofar; Moein, Shiva; Heidary, Zarifeh; Ghaisari, Jafar

    2016-01-01

    Background: Mathematical modeling of biological systems is an attractive way for studying complex biological systems and their behaviors. Petri Nets, due to their ability to model systems with various levels of qualitative information, have been wildly used in modeling biological systems in which enough qualitative data may not be at disposal. These nets have been used to answer questions regarding the dynamics of different cell behaviors including the translation process. In one stage of the translation process, the RNA sequence may be degraded. In the process of degradation of RNA sequence, small-noncoding RNA molecules known as small interfering RNA (siRNA) match the target RNA sequence. As a result of this matching, the target RNA sequence is destroyed. Materials and Methods: In this context, the process of matching and destruction is modeled using Colored Petri Nets (CPNs). The model is constructed using CPNs which allow tokens to have a value or type on them. Thus, CPN is a suitable tool to model string structures in which each element of the string has a different type. Using CPNs, long RNA, and siRNA strings are modeled with a finite set of colors. The model is simulated via CPN Tools. Results: A CPN model of the matching between RNA and siRNA strings is constructed in CPN Tools environment. Conclusion: In previous studies, a network of stoichiometric equations was modeled. However, in this particular study, we modeled the mechanism behind the silencing process. Modeling this kind of mechanisms provides us with a tool to examine the effects of different factors such as mutation or drugs on the process. PMID:27376039

  15. Targeted delivery of small interfering RNA to colon cancer cells using chitosan and PEGylated chitosan nanoparticles.

    PubMed

    Rudzinski, Walter E; Palacios, Adriana; Ahmed, Abuzar; Lane, Michelle A; Aminabhavi, Tejraj M

    2016-08-20

    Small interfering RNA (siRNA) molecules specifically target messenger RNA species, decreasing intracellular protein levels. β-Catenin protein concentrations are increased in 70-80% of colon tumors, promoting tumor progression. Chitosan exhibits low levels of toxicity and can be transported across mucosal membranes; therefore, our objective was to develop chitosan and poly(ethylene glycol)-grafted (PEGylated) chitosan nanoparticles, 100-150nm in diameter, encapsulating anti-β-catenin siRNA for transfection into colon cancer cells. Encapsulation efficiencies up to 97% were observed. Confocal microscopy visualized the entry of fluorescently-tagged siRNA into cells. Western blot analysis showed that both chitosan and PEGylated chitosan nanoparticles containing anti-β-catenin siRNA decreased β-catenin protein levels in cultured colon cancer cells. These results indicate that nanoparticles made with chitosan and PEGylated chitosan can successfully enter colon cancer cells and decrease the level of a protein that promotes tumor progression. These or similar nanoparticles may prove beneficial for the treatment of colon cancer in humans. PMID:27178938

  16. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    PubMed

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. PMID:23913913

  17. Current preclinical small interfering RNA (siRNA)-based conjugate systems for RNA therapeutics.

    PubMed

    Lee, Soo Hyeon; Kang, Yoon Young; Jang, Hyo-Eun; Mok, Hyejung

    2016-09-01

    Recent promising clinical results of RNA therapeutics have drawn big attention of academia and industries to RNA therapeutics and their carrier systems. To improve their feasibility in clinics, systemic evaluations of currently available carrier systems under clinical trials and preclinical studies are needed. In this review, we focus on recent noticeable preclinical studies and clinical results regarding siRNA-based conjugates for clinical translations. Advantages and drawbacks of siRNA-based conjugates are discussed, compared to particle-based delivery systems. Then, representative siRNA-based conjugates with aptamers, peptides, carbohydrates, lipids, polymers, and nanostructured materials are introduced. To improve feasibility of siRNA conjugates in preclinical studies, several considerations for the rational design of siRNA conjugates in terms of cleavability, immune responses, multivalent conjugations, and mechanism of action are also presented. Lastly, we discuss lessons from previous preclinical and clinical studies related to siRNA conjugates and perspectives of their clinical applications. PMID:26514375

  18. Delivery of small interfering RNA for inhibition of endothelial cell apoptosis by hypoxia and serum deprivation

    SciTech Connect

    Cho, Seung-Woo; Hartle, Lauren; Son, Sun Mi; Yang, Fan; Goldberg, Michael; Xu, Qiaobing; Langer, Robert; Anderson, Daniel G.

    2008-11-07

    RNA interference (RNAi) for anti-angiogenic or pro-apoptotic factors in endothelial cells (ECs) has great potential for the treatment of ischemic diseases by promoting angiogenesis or inhibiting apoptosis. Here, we report the utility of small interfering RNA (siRNA) in inhibiting EC apoptosis induced by tumor necrosis factor-{alpha} (TNF-{alpha}). siRNA was designed and synthesized targeting tumor necrosis factor-{alpha} receptor-1 (TNFR-1) and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1). Human umbilical vein endothelial cells (HUVECs) were cultured under in vitro hypoxic and serum-deprived conditions to simulate in vivo ischemic conditions. Two days after liposomal delivery of siRNA targeting TNFR-1 and SHP-1, significant silencing of each target (TNFR-1; 76.5% and SHP-1; 97.2%) was detected. Under serum-deprived hypoxic (1% oxygen) conditions, TNF-{alpha} expression in HUVECs increased relative to normoxic (20% oxygen) and serum-containing conditions. Despite enhanced TNF-{alpha} expression, suppression of TNFR-1 or SHP-1 by siRNA delivery not only enhanced expression of angiogenic factors (KDR/Flk-1 and eNOS) and anti-apoptotic factor (Bcl-xL) but also reduced expression of a pro-apoptotic factor (Bax). Transfection of TNFR-1 or SHP-1 siRNA significantly decreased the HUVEC apoptosis while significantly enhancing HUVEC proliferation and capillary formation. The present study demonstrates that TNFR-1 and SHP-1 may be useful targets for the treatment of myocardial or hindlimb ischemia.

  19. Mutant p53 inhibits miRNA biogenesis by interfering with the microprocessor complex.

    PubMed

    Garibaldi, F; Falcone, E; Trisciuoglio, D; Colombo, T; Lisek, K; Walerych, D; Del Sal, G; Paci, P; Bossi, G; Piaggio, G; Gurtner, A

    2016-07-21

    Downregulation of microRNAs (miRNAs) is commonly observed in cancers and promotes tumorigenesis suggesting that miRNAs may function as tumor suppressors. However, the mechanism through which miRNAs are regulated in cancer, and the connection between oncogenes and miRNA biogenesis remain poorly understood. The TP53 tumor-suppressor gene is mutated in half of human cancers resulting in an oncogene with gain-of-function activities. Here we demonstrate that mutant p53 (mutp53) oncoproteins modulate the biogenesis of a subset of miRNAs in cancer cells inhibiting their post-transcriptional maturation. Interestingly, among these miRNAs several are also downregulated in human tumors. By confocal, co-immunoprecipitation and RNA-chromatin immunoprecipitation experiments, we show that endogenous mutp53 binds and sequesters RNA helicases p72/82 from the microprocessor complex, interfering with Drosha-pri-miRNAs association. In agreement with this, the overexpression of p72 leads to an increase of mature miRNAs levels. Moreover, functional experiments demonstrate the oncosuppressive role of mutp53-dependent miRNAs (miR-517a, -519a, -218, -105). Our study highlights a previously undescribed mechanism by which mutp53 interferes with Drosha-p72/82 association leading, at least in part, to miRNA deregulation observed in cancer. PMID:26996669

  20. Short interfering RNA-mediated gene silencing in Globodera pallida and Meloidogyne incognita infective stage juveniles.

    PubMed

    Dalzell, Johnathan J; McMaster, Steven; Fleming, Colin C; Maule, Aaron G

    2010-01-01

    The analysis of gene function through RNA interference (RNAi)-based reverse genetics in plant parasitic nematodes (PPNs) remains inexplicably reliant on the use of long double-stranded RNA (dsRNA) silencing triggers; a practice inherently disadvantageous due to the introduction of superfluous dsRNA sequence, increasing chances of aberrant or off-target gene silencing through interactions between nascent short interfering RNAs (siRNAs) and non-cognate mRNA targets. Recently, we have shown that non-nematode, long dsRNAs have a propensity to elicit profound impacts on the phenotype and migrational abilities of both root knot and cyst nematodes. This study presents, to our knowledge for the first time, gene-specific knockdown of FMRFamide-like peptide (flp) transcripts, using discrete 21bp siRNAs in potato cyst nematode Globodera pallida, and root knot nematode Meloidogyne incognita infective (J2) stage juveniles. Both knockdown at the transcript level through quantitative (q)PCR analysis and functional data derived from migration assay, indicate that siRNAs targeting certain areas of the FMRFamide-like peptide (FLP) transcripts are potent and specific in the silencing of gene function. In addition, we present a method of manipulating siRNA activity through the management of strand thermodynamics. Initial evaluation of strand thermodynamics as a determinant of RNA-Induced Silencing Complex (RISC) strand selection (inferred from knockdown efficacy) in the siRNAs presented here suggested that the purported influence of 5' stand stability on guide incorporation may be somewhat promiscuous. However, we have found that on strategically incorporating base mismatches in the sense strand of a G. pallida-specific siRNA, we could specifically increase or decrease the knockdown of its target (specific to the antisense strand), presumably through creating more favourable thermodynamic profiles for incorporation of either the sense (non-target-specific) or antisense (target

  1. Synthetic protection short interfering RNA screen reveals glyburide as a novel radioprotector.

    PubMed

    Jiang, Jianfei; McDonald, Peter R; Dixon, Tracy M; Franicola, Darcy; Zhang, Xichen; Nie, Suhua; Epperly, Laura D; Huang, Zhentai; Kagan, Valerian E; Lazo, John S; Epperly, Michael W; Greenberger, Joel S

    2009-10-01

    To assist in screening existing drugs for use as potential radioprotectors, we used a human unbiased 16,560 short interfering RNA (siRNA) library targeting the druggable genome. We performed a synthetic protection screen that was designed to identify genes that, when silenced, protected human glioblastoma T98G cells from gamma-radiation-induced cell death. We identified 116 candidate protective genes, then identified 10 small molecule inhibitors of 13 of these candidate gene products and tested their radioprotective effects. Glyburide, a clinically used second-generation hypoglycemic drug, effectively decreased radiation-induced cell death in several cell lines including T98G, glioblastoma U-87 MG, and normal lung epithelial BEAS-2B and in primary cultures of astrocytes. Glyburide significantly increased the survival of 32D cl3 murine hematopoietic progenitor cells when administrated before irradiation. Glyburide was radioprotective in vivo (90% of C57BL/6NHsd female mice pretreated with 10 mg/kg glyburide survived 9.5 Gy total-body irradiation compared to 42% of irradiated controls, P = 0.0249). These results demonstrate the power of unbiased siRNA synthetic protection screening with a druggable genome library to identify new radioprotectors. PMID:19772462

  2. Small interfering RNA delivery by polyethylenimine-functionalised porous silicon nanoparticles.

    PubMed

    Hasanzadeh Kafshgari, M; Alnakhli, M; Delalat, B; Apostolou, S; Harding, F J; Mäkilä, E; Salonen, J J; Kuss, B J; Voelcker, N H

    2015-12-01

    In this study, thermally hydrocarbonised porous silicon nanoparticles (THCpSiNPs) capped with polyethylenimine (PEI) were fabricated, and their potential for small interfering RNA (siRNA) delivery was investigated in an in vitro glioblastoma model. PEI coating following siRNA loading enhanced the sustained release of siRNA, and suppressed burst release effects. The positively-charged surface improved the internalisation of the nanoparticles across the cell membrane. THCpSiNP-mediated siRNA delivery reduced mRNA expression of the MRP1 gene, linked to the resistence of glioblastoma to chemotherapy, by 63% and reduced MRP1-protein levels by 70%. MRP1 siRNA loaded nanoparticles did not induce cytotoxicity in glioblastoma cells, but markedly reduced cell proliferation. In summary, the results demonstrated that non-cytotoxic cationic THCpSiNPs are promising vehicles for therapeutic siRNA delivery. PMID:26343506

  3. A nanoparticle system specifically designed to deliver short interfering RNA inhibits tumor growth in vivo.

    PubMed

    Yagi, Nobuhiro; Manabe, Ichiro; Tottori, Tsuneaki; Ishihara, Atsushi; Ogata, Fusa; Kim, Jong Heon; Nishimura, Satoshi; Fujiu, Katsuhito; Oishi, Yumiko; Itaka, Keiji; Kato, Yasuki; Yamauchi, Masahiro; Nagai, Ryozo

    2009-08-15

    Use of short interfering RNA (siRNA) is a promising new approach thought to have a strong potential to lead to rapid development of gene-oriented therapies. Here, we describe a newly developed, systemically injectable siRNA vehicle, the "wrapsome" (WS), which contains siRNA and a cationic lipofection complex in a core that is fully enveloped by a neutral lipid bilayer and hydrophilic polymers. WS protected siRNA from enzymatic digestion, providing a long half-life in the systemic circulation. Moreover, siRNA/WS leaked from blood vessels within tumors into the tumor tissue, where it accumulated and was subsequently transfected into the tumor cells. Because the transcription factor KLF5 is known to play a role in tumor angiogenesis, we designed KLF5-siRNA to test the antitumor activity of siRNA/WS. KLF5-siRNA/WS exhibited significant antitumor activity, although neither WS containing control scrambled-siRNA nor saline containing KLF5-siRNA affected tumor growth. KLF5-siRNA/WS inhibited Klf5 expression within tumors at both mRNA and protein levels, significantly reducing angiogenesis, and we detected no significant acute or long-term toxicity. Our findings support the idea that siRNA/WS can be used to knock down specific genes within tumors and thereby exert therapeutic effects against cancers. PMID:19654315

  4. Chitosan/interfering RNA nanoparticle mediated gene silencing in disease vector mosquito larvae

    PubMed Central

    Zhang, Xin; Mysore, Keshava; Flannery, Ellen; Michel, Kristin; Severson, David W.; Zhu, Kun Yan

    2015-01-01

    SHORT ABSTRACT Here we describe a procedure for inhibiting gene function in disease vector mosquitoes through the use of chitosan/interfering RNA nanoparticles that are ingested by larvae. LONG ABSTRACT Vector mosquitoes inflict more human suffering than any other organism—and kill more than one million people each year. The mosquito genome projects facilitated research in new facets of mosquito biology, including functional genetic studies in the primary African malaria vector Anopheles gambiae and the dengue and yellow fever vector Aedes aegypti. RNA interference- (RNAi-) mediated gene silencing has been used to target genes of interest in both of these disease vector mosquito species. Here, we describe a procedure for preparation of chitosan/interfering RNA nanoparticles that are combined with food and ingested by larvae. This technically straightforward, high-throughput, and relatively inexpensive methodology, which is compatible with long double stranded RNA (dsRNA) or small interfering RNA (siRNA) molecules, has been used for the successful knockdown of a number of different genes in A. gambiae and A. aegypti larvae. Following larval feedings, knockdown, which is verified through qRT-PCR or in situ hybridization, can persist at least through the late pupal stage. This methodology may be applicable to a wide variety of mosquito and other insect species, including agricultural pests, as well as other non-model organisms. In addition to its utility in the research laboratory, in the future, chitosan, an inexpensive, non-toxic and biodegradable polymer, could potentially be utilized in the field. PMID:25867635

  5. Interfering passages of Sindbis virus: concomitant appearance of interference, morphological variants, and trucated viral RNA.

    PubMed Central

    Johnston, R E; Tovell, D R; Brown, D T; Faulkner, P

    1975-01-01

    Serial passage of Sindbis at high multiplicities of infection resulted in cyclical variations in virus titer. Decreases in virus titer were correlated with the appearance of smaller-sized virions, interference and truncated viral RNA. The smaller particles were 37 nm in diameter, exclusive of the hemagglutinin spikes as compared with a diameter of 50 nm for standard virions. Passages which contained 37-nm partilces also interfered with infectious center formation by standard, plaque-purified virus. Polyacrylamide gel analysis of RNA isolated from virions present in interfering passages demonstrated the sequential appearance of three RNA species smaller than standard RNA with approximate molecular weights of 3.3 X 106, 2.7 X 106, and 2.2 X 106. The 3.3 X 106 RNA was evident in passage 5, by passage 8 both the 3.3 X 106 and 2.7 X 106 RNAs were present, and by passage 13 all three were present with the 2.2 X 106 RNA predominating. Images PMID:1165599

  6. Characterization of homologous defective interfering RNA during persistent infection of Vero cells with Japanese encephalitis virus.

    PubMed

    Yoon, Sung Wook; Lee, Sang-Yong; Won, Sung-Yong; Park, Sun-Hee; Park, Soo-Young; Jeong, Yong Seok

    2006-02-28

    It has been suggested that defective interfering (DI) RNA contributes to the persistence of Japanese en-cephalitis virus (JEV). In this study, we characterized molecular and biological aspects of the DI RNA and its relation to viral persistence. We identified a homolo-gous DI virus intimately associated with JEV persis-tence in Vero cells. The production of DI RNA during undiluted serial passages of JEV coincided with the appearance of cells refractory to acute infection with JEV. We also established a Vero cell clone with a per-sistent JEV infection in which the DI RNA co-replicated efficiently at the expense of helper virus. The infectious virus yield of the clone fluctuated dur-ing its growth depending upon the amount of DI RNA accumulated in the previous replication cycle. Identifi-cation of the corresponding negative-sense RNA of the DI RNA indicated that the DI RNA functioned as a replication unit. Most of the DI RNA molecules re-tained their open reading frames despite a large dele-tion, encompassing most of the prM, the entire E, and the 5' half of the NS1 gene. Taken together, these ob-servations suggest that the generation of homologous DI RNA during successive JEV acute infections in Vero cells probably participates actively in persistent JEV infection. PMID:16511353

  7. Development of a software tool and criteria evaluation for efficient design of small interfering RNA.

    PubMed

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-01

    RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA. PMID:21145307

  8. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    NASA Astrophysics Data System (ADS)

    Wei, Zonghui; Luijten, Erik

    2015-12-01

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  9. Systematic coarse-grained modeling of complexation between small interfering RNA and polycations

    SciTech Connect

    Wei, Zonghui; Luijten, Erik

    2015-12-28

    All-atom molecular dynamics simulations can provide insight into the properties of polymeric gene-delivery carriers by elucidating their interactions and detailed binding patterns with nucleic acids. However, to explore nanoparticle formation through complexation of these polymers and nucleic acids and study their behavior at experimentally relevant time and length scales, a reliable coarse-grained model is needed. Here, we systematically develop such a model for the complexation of small interfering RNA (siRNA) and grafted polyethyleneimine copolymers, a promising candidate for siRNA delivery. We compare the predictions of this model with all-atom simulations and demonstrate that it is capable of reproducing detailed binding patterns, charge characteristics, and water release kinetics. Since the coarse-grained model accelerates the simulations by one to two orders of magnitude, it will make it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  10. Recent In Vivo Evidences of Particle-Based Delivery of Small-Interfering RNA (siRNA) into Solid Tumors

    PubMed Central

    2014-01-01

    Small-interfering RNA (siRNA) is both a powerful tool in research and a promising therapeutic platform to modulate expression of disease-related genes. Malignant tumors are attractive disease targets for nucleic acid-based therapies. siRNA directed against oncogenes, and genes driving metastases or angiogenesis have been evaluated in animal models and in some cases, in humans. The outcomes of these studies indicate that drug delivery is a significant limiting factor. This review provides perspectives on in vivo validated nanoparticle-based siRNA delivery systems. Results of recent advances in liposomes and polymeric and inorganic formulations illustrate the need for mutually optimized attributes for performance in systemic circulation, tumor interstitial space, plasma membrane, and endosomes. Physiochemical properties conducive to efficient siRNA delivery are summarized and directions for future research are discussed. PMID:25221632

  11. Plasma exosomes can deliver exogenous short interfering RNA to monocytes and lymphocytes

    PubMed Central

    Wahlgren, Jessica; Karlson, Tanya De L.; Brisslert, Mikael; Vaziri Sani, Forugh; Telemo, Esbjörn; Sunnerhagen, Per; Valadi, Hadi

    2012-01-01

    Despite the promise of RNA interference (RNAi) and its potential, e.g. for use in cancer therapy, several technical obstacles must first be overcome. The major hurdle of RNAi-based therapeutics is to deliver nucleic acids across the cell’s plasma membrane. This study demonstrates that exosome vesicles derived from humans can deliver short interfering RNA (siRNA) to human mononuclear blood cells. Exosomes are nano-sized vesicles of endocytic origin that are involved in cell-to-cell communication, i.e. antigen presentation, tolerance development and shuttle RNA (mainly mRNA and microRNA). Having tested different strategies, an optimized method (electroporation) was used to introduce siRNA into human exosomes of various origins. Plasma exosomes (exosomes from peripheral blood) were used as gene delivery vector (GDV) to transport exogenous siRNA to human blood cells. The vesicles effectively delivered the administered siRNA into monocytes and lymphocytes, causing selective gene silencing of mitogen-activated protein kinase 1. These data suggest that human exosomes can be used as a GDV to provide cells with heterologous nucleic acids such as therapeutic siRNAs. PMID:22618874

  12. Optimized in vivo transfer of small interfering RNA targeting dermal tissue using in vivo surface electroporation.

    PubMed

    Broderick, Kate E; Chan, Amy; Lin, Feng; Shen, Xuefei; Kichaev, Gleb; Khan, Amir S; Aubin, Justin; Zimmermann, Tracy S; Sardesai, Niranjan Y

    2012-01-01

    Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner. PMID:23344722

  13. Optimized In Vivo Transfer of Small Interfering RNA Targeting Dermal Tissue Using In Vivo Surface Electroporation

    PubMed Central

    Broderick, Kate E; Chan, Amy; Lin, Feng; Shen, Xuefei; Kichaev, Gleb; Khan, Amir S; Aubin, Justin; Zimmermann, Tracy S; Sardesai, Niranjan Y.

    2012-01-01

    Electroporation (EP) of mammalian tissue is a technique that has been used successfully in the clinic for the delivery of genetic-based vaccines in the form of DNA plasmids. There is great interest in platforms which efficiently deliver RNA molecules such as messenger RNA and small interfering RNA (siRNA) to mammalian tissue. However, the in vivo delivery of RNA enhanced by EP has not been extensively characterized. This paper details the optimization of electrical parameters for a novel low-voltage EP method to deliver oligonucleotides (both DNA and RNA) to dermal tissue in vivo. Initially, the electrical parameters were optimized for dermal delivery of plasmid DNA encoding green fluorescent protein (GFP) using this novel surface dermal EP device. While all investigated parameters resulted in visible transfection, voltage parameters in the 10 V range elicited the most robust signal. The parameters optimized for DNA, were then assessed for translation of successful electrotransfer of siRNA into dermal tissue. Robust tagged-siRNA transfection in skin was detected. We then assessed whether these parameters translated to successful transfer of siRNA resulting in gene knockdown in vivo. Using a reporter gene construct encoding GFP and tagged siRNA targeting the GFP message, we show simultaneous transfection of the siRNA to the skin via EP and the concomitant knockdown of the reporter gene signal. The siRNA delivery was accomplished with no evidence of injection site inflammation or local tissue damage. The minimally invasive low-voltage EP method is thus capable of efficiently delivering both DNA and RNA molecules to dermal tissue in a tolerable manner. PMID:23344722

  14. An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons

    PubMed Central

    Luo, Miaw-Chyi; Zhang, Dong-Qin; Ma, Shou-Wu; Huang, Yuan-Yuan; Shuster, Sam J; Porreca, Frank; Lai, Josephine

    2005-01-01

    We have developed a highly effective method for in vivo gene silencing in the spinal cord and dorsal root ganglia (DRG) by a cationic lipid facilitated delivery of synthetic, small interfering RNA (siRNA). A siRNA to the delta opioid receptor (DOR), or a mismatch RNA, was mixed with the transfection reagent, i-Fect™ (vehicle), and delivered as repeated daily bolus doses (0.5 μg to 4 μg) via implanted intrathecal catheter to the lumbar spinal cord of rats. Twenty-four hours after the last injection, rats were tested for antinociception by the DOR selective agonist, [D-Ala2, Glu4]deltorphin II (DELT), or the mu opioid receptor (MOR) selective agonist, [D-Ala2, N-Me-Phe4, Gly-ol5]enkephalin (DAMGO). Pretreatment with the siRNA, but not the mismatch RNA or vehicle alone, blocked DELT antinociception dose-dependently. The latter was concomitant with a reduction in the spinal immunoreactivity and receptor density of DOR, and in DOR transcripts in the lumbar DRG and spinal dorsal horn. Neither siRNA nor mismatch RNA pretreatment altered spinal immunoreactivity of MOR or antinociception by spinal DAMGO, and had no effect on the baseline thermal nociceptive threshold. The inhibition of function and expression of DOR by siRNA was reversed by 72 hr after the last RNA injection. The uptake of fluorescence-tagged siRNA was detected in both DRG and spinal cord. The low effective dose of siRNA/i-Fect™ complex reflects an efficient delivery of the siRNA to peripheral and spinal neurons, produced no behavioral signs of toxicity. This delivery method may be optimized for other gene targets. PMID:16191203

  15. Comparison of small interfering RNA (siRNA) delivery into bovine monocyte-derived macrophages by transfection and electroporation

    PubMed Central

    Jensen, Kirsty; Anderson, Jennifer A.; Glass, Elizabeth J.

    2014-01-01

    The manipulation of the RNA interference pathway using small interfering RNA (siRNA) has become the most frequently used gene silencing method. However, siRNA delivery into primary cells, especially primary macrophages, is often considered challenging. Here we report the investigation of the suitability of two methodologies: transient transfection and electroporation, to deliver siRNA targeted against the putative immunomodulatory gene Mediterranean fever (MEFV) into primary bovine monocyte-derived macrophages (bMDM). Eleven commercial transfection reagents were investigated with variable results with respect to siRNA uptake, target gene knock-down, cell toxicity and type I interferon (IFN) response induction. Three transfection reagents: Lipofectamine 2000, Lipofectamine RNAiMAX and DharmaFECT 3, were found to consistently give the best results. However, all the transfection reagents tested induced an IFN response in the absence of siRNA, which could be minimized by reducing the transfection reagent incubation period. In addition, optimized siRNA delivery into bMDM by electroporation achieved comparable levels of target gene knock-down as transient transfection, without a detectable IFN response, but with higher levels of cell toxicity. The optimized transient transfection and electroporation methodologies may provide a starting point for optimizing siRNA delivery into macrophages derived from other species or other cells considered difficult to investigate with siRNA. PMID:24598124

  16. Adenovirus Virus-Associated RNA Is Processed to Functional Interfering RNAs Involved in Virus Production

    PubMed Central

    Aparicio, Oscar; Razquin, Nerea; Zaratiegui, Mikel; Narvaiza, Iñigo; Fortes, Puri

    2006-01-01

    Posttranscriptional gene silencing allows sequence-specific control of gene expression. Specificity is guaranteed by small antisense RNAs such as microRNAs (miRNAs) or small interfering RNAs (siRNAs). Functional miRNAs derive from longer double-stranded RNA (dsRNA) molecules that are cleaved to pre-miRNAs in the nucleus and are transported by exportin 5 (Exp 5) to the cytoplasm. Adenovirus-infected cells express virus-associated (VA) RNAs, which are dsRNA molecules similar in structure to pre-miRNAs. VA RNAs are also transported by Exp 5 to the cytoplasm, where they accumulate. Here we show that small RNAs derived from VA RNAs (svaRNAs), similar to miRNAs, can be found in adenovirus-infected cells. VA RNA processing to svaRNAs requires neither viral replication nor viral protein expression, as evidenced by the fact that svaRNA accumulation can be detected in cells transfected with VA sequences. svaRNAs are efficiently bound by Argonaute 2, the endonuclease of the RNA-induced silencing complex, and behave as functional siRNAs, in that they inhibit the expression of reporter genes with complementary sequences. Blocking svaRNA-mediated inhibition affects efficient adenovirus production, indicating that svaRNAs are required for virus viability. Thus, svaRNA-mediated silencing could represent a novel mechanism used by adenoviruses to control cellular or viral gene expression. PMID:16415015

  17. Establishing an Infrastructure for High-Throughput Short-Interfering RNA Screening.

    PubMed

    Yin, Hongwei; Sereduk, Chris; Tang, Nanyun

    2016-01-01

    RNA interference (RNAi) is a readily available research tool that can be used to accelerate the identification and functional validation of a multitude of new candidate drug targets by experimentally perturbing gene expression and function. High-throughput RNAi technology using libraries of short-interfering RNA (siRNA) makes it possible to rapidly identify genes and biomarkers associated with biological processes such as diseases or a cellular response to therapy. Thus, RNAi-based screening is an extremely powerful technology that can provide tremendous insights into the mechanisms of action and contexts of vulnerability of a particular drug treatment. This chapter describes the infrastructure requirements needed to successfully perform HT-RNAi screening. Information on the methodology, instrumentation, experimental design, and workflow aspects is provided, as well as insights on how to successfully implement a high-throughput RNAi screen. PMID:27581280

  18. Targeted Delivery of Small Interfering RNA Using Reconstituted High-Density Lipoprotein Nanoparticles12

    PubMed Central

    Shahzad, Mian MK; Mangala, Lingegowda S; Han, Hee Dong; Lu, Chunhua; Bottsford-Miller, Justin; Nishimura, Masato; Mora, Edna M; Lee, Jeong-Won; Stone, Rebecca L; Pecot, Chad V; Thanapprapasr, Duangmani; Roh, Ju-Won; Gaur, Puja; Nair, Maya P; Park, Yun-Yong; Sabnis, Nirupama; Deavers, Michael T; Lee, Ju-Seog; Ellis, Lee M; Lopez-Berestein, Gabriel; McConathy, Walter J; Prokai, Laszlo; Lacko, Andras G; Sood, Anil K

    2011-01-01

    RNA interference holds tremendous potential as a therapeutic approach, especially in the treatment of malignant tumors. However, efficient and biocompatible delivery methods are needed for systemic delivery of small interfering RNA (siRNA). To maintain a high level of growth, tumor cells scavenge high-density lipoprotein (HDL) particles by overexpressing its receptor: scavenger receptor type B1 (SR-B1). In this study, we exploited this cellular characteristic to achieve efficient siRNA delivery and established a novel formulation of siRNA by incorporating it into reconstituted HDL (rHDL) nanoparticles. Here, we demonstrate that rHDL nanoparticles facilitate highly efficient systemic delivery of siRNA in vivo, mediated by the SR-B1. Moreover, in therapeutic proof-of-concept studies, these nanoparticles were effective in silencing the expression of two proteins that are key to cancer growth and metastasis (signal transducer and activator of transcription 3 and focal adhesion kinase) in orthotopic mouse models of ovarian and colorectal cancer. These data indicate that an rHDL nanoparticle is a novel and highly efficient siRNA carrier, and therefore, this novel technology could serve as the foundation for new cancer therapeutic approaches. PMID:21472135

  19. High Density Lipoproteins for the Systemic Delivery of short interfering RNA

    PubMed Central

    McMahon, Kaylin M.; Thaxton, C. Shad

    2014-01-01

    Introduction RNA interference (RNAi) is a powerful mechanism for gene silencing with the potential to greatly impact the development of new therapies for many human diseases. Short interfering RNAs (siRNAs) may be the ideal molecules for therapeutic RNAi. However, therapeutic siRNAs face significant challenges that must be overcome prior to widespread clinical use. Many efforts have been made to overcome the hurdles associated with systemic administration of siRNA; however, current approaches are still limited. As such, there is an urgent need to develop new strategies for siRNA delivery that have the potential to impact a broad spectrum of systemic diseases. Areas covered This review focuses on the promise of siRNA therapies and highlights current siRNA delivery methods. With an eye toward new strategies, this review first introduces high density lipoproteins (HDL) and their natural functions, and then transitions into how HDLs may provide significant opportunities as next generation siRNA delivery vehicles. Importantly, this review describes how synthetic HDLs leverage the natural ability of HDL to stabilize and deliver siRNAs. Expert Opinion HDLs are natural nanoparticles that are critical to understanding the systemic delivery of therapeutic nucleic acids, like siRNA. Methods to synthesize biomimetic HDLs are being explored and data demonstrate that this type of delivery vehicle may be highly beneficial for targeted and efficacious systemic delivery of siRNAs. PMID:24313310

  20. A cis-acting function for the coronavirus leader in defective interfering RNA replication.

    PubMed Central

    Chang, R Y; Hofmann, M A; Sethna, P B; Brian, D A

    1994-01-01

    To test the hypothesis that the 65-nucleotide (nt) leader on subgenomic mRNAs suffices as a 5'-terminal cis-acting signal for RNA replication, a corollary to the notion that coronavirus mRNAs behave as replicons, synthetic RNA transcripts of a cloned, reporter-containing N mRNA (mRNA 7) of the bovine coronavirus with a precise 5' terminus and a 3' poly(A) of 68 nt were tested for replication after being transfected into helper virus-infected cells. No replication was observed, but synthetic transcripts of a cloned reporter-containing defective interfering (DI) RNA differing from the N mRNA construct by 433 nt of continuous 5'-proximal genomic sequence between the leader and the N open reading frame did replicate and become packaged, indicating the insufficiency of the leader alone as a 5' signal for replication of transfected RNA molecules. The leader was shown to be a necessary part of the cis-acting signal for DI RNA replication, however, since removal of terminal bases that destroyed a predicted intraleader stem-loop also destroyed replicating ability. Surprisingly, when the same stem-loop was disrupted by base substitutions, replication appeared only minimally impaired and the leader was found to have rapidly reverted to wild type during DI RNA replication, a phenomenon reminiscent of high-frequency leader switching in the mouse hepatitis coronavirus. These results suggest that once a minimal structural requirement for leader is fulfilled for initiation of DI RNA replication, the wild-type leader is strongly preferred for subsequent replication. They also demonstrate that, in contrast to reported natural mouse hepatitis coronavirus DI RNAs, the DI RNA of the bovine coronavirus does not require sequence elements originating from discontinuous downstream regions within the polymerase gene for replication or for packaging. Images PMID:7966615

  1. Subcellular distribution of small interfering RNA: directed delivery through RNA polymerase III expression cassettes and localization by in situ hybridization.

    PubMed

    Paul, Cynthia P

    2005-01-01

    Reduction in the expression of specific genes through small interfering RNAs (siRNAs) is dependent on the colocalization of siRNAs with other components of the RNA interference (RNAi) pathways within the cell. The expression of siRNAs within cells from cassettes that are derived from genes transcribed by RNA polymerase III (pol III) and provide for selective subcellular distribution of their products can be used to direct siRNAs to the cellular pathways. Expression from the human U6 promoter, resulting in siRNA accumulation in the nucleus, is effective in reducing gene expression, whereas cytoplasmic and nucleolar localization of the siRNA when expressed from the 5S or 7 SL promoters is not effective. The distribution of siRNA within the cell is determined by fluorescence in situ hybridization. Although the long uninterrupted duplex of siRNA makes it difficult to detect with DNA oligonucleotide probes, labeled oligonucleotide probes with 2'-O-methyl RNA backbones provide the stability needed for a strong signal. These methods contribute to studies of the interconnected cellular RNAi pathways and are useful in adapting RNAi as a tool to determine gene function and develop RNA-based therapeutics. PMID:15644179

  2. A novel albumin nanocomplex containing both small interfering RNA and gold nanorods for synergetic anticancer therapy

    NASA Astrophysics Data System (ADS)

    Choi, Jin-Ha; Hwang, Hai-Jin; Shin, Seung Won; Choi, Jeong-Woo; Um, Soong Ho; Oh, Byung-Keun

    2015-05-01

    Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au nanorods per BSA complex and were successively functionalized with polyethylene glycol (PEG) and anti-ErbB-2 antibodies to facilitate active targeting. The synergetic therapeutic activity originating from the two components effectively induced cell death (~80% reduction in viability compared with control cells) in target breast cancer cells after a single dose of laser irradiation. Intracellular SREB nanocomplex decomposition by proteolytic enzymes resulted in simultaneous RNA interference and thermal ablation, thus leading to apoptosis in the targeted cancer cells. Moreover, these therapeutic effects were sustained for approximately 72 hours. The intrinsic biocompatibility, multifunctionality, and potent in vitro anticancer properties of these SREB nanocomplexes indicate that they have great therapeutic potential for in vivo targeted cancer therapy, in addition to other areas of nanomedicine.Therapeutic nanocomplexes have been extensively developed for the effective treatment of aggressive cancers because of their outstanding versatility, easy manipulation, and low cytotoxicity. In this study, we describe the synthesis of a novel bovine serum albumin (BSA)-based nanocomplex harboring both Bcl-2-specific small interfering RNA (siRNA) and gold (Au) nanorods (siRNA and rods encapsulated in BSA; SREB) with the aim of developing a targeted breast cancer therapeutic. The SREB complexes contained 2 × 105 siRNA molecules and eight Au

  3. Development of a software tool and criteria evaluation for efficient design of small interfering RNA

    SciTech Connect

    Chaudhary, Aparna; Srivastava, Sonam; Garg, Sanjeev

    2011-01-07

    Research highlights: {yields} The developed tool predicted siRNA constructs with better thermodynamic stability and total score based on positional and other criteria. {yields} Off-target silencing below score 30 were observed for the best siRNA constructs for different genes. {yields} Immunostimulation and cytotoxicity motifs considered and penalized in the developed tool. {yields} Both positional and compositional criteria were observed to be important. -- Abstract: RNA interference can be used as a tool for gene silencing mediated by small interfering RNAs (siRNA). The critical step in effective and specific RNAi processing is the selection of suitable constructs. Major design criteria, i.e., Reynolds's design rules, thermodynamic stability, internal repeats, immunostimulatory motifs were emphasized and implemented in the siRNA design tool. The tool provides thermodynamic stability score, GC content and a total score based on other design criteria in the output. The viability of the tool was established with different datasets. In general, the siRNA constructs produced by the tool had better thermodynamic score and positional properties. Comparable thermodynamic scores and better total scores were observed with the existing tools. Moreover, the results generated had comparable off-target silencing effect. Criteria evaluations with additional criteria were achieved in WEKA.

  4. Intravaginal gene silencing using biodegradable polymer nanoparticles densely loaded with small-interfering RNA

    NASA Astrophysics Data System (ADS)

    Woodrow, Kim A.; Cu, Yen; Booth, Carmen J.; Saucier-Sawyer, Jennifer K.; Wood, Monica J.; Mark Saltzman, W.

    2009-06-01

    Vaginal instillation of small-interfering RNA (siRNA) using liposomes has led to silencing of endogenous genes in the genital tract and protection against challenge from infectious disease. Although siRNA lipoplexes are easily formulated, several of the most effective transfection agents available commercially may be toxic to the mucosal epithelia and none are able to provide controlled or sustained release. Here, we demonstrate an alternative approach using nanoparticles composed entirely of FDA-approved materials. To render these materials effective for gene silencing, we developed novel approaches to load them with high amounts of siRNA. A single dose of siRNA-loaded nanoparticles to the mouse female reproductive tract caused efficient and sustained gene silencing. Knockdown of gene expression was observed proximal (in the vaginal lumen) and distal (in the uterine horns) to the site of topical delivery. In addition, nanoparticles penetrated deep into the epithelial tissue. This is the first report demonstrating that biodegradable polymer nanoparticles are effective delivery vehicles for siRNA to the vaginal mucosa.

  5. The hub protein loquacious connects the microRNA and short interfering RNA pathways in mosquitoes

    PubMed Central

    Haac, Mary Etna; Anderson, Michelle A.E.; Eggleston, Heather; Myles, Kevin M.; Adelman, Zach N.

    2015-01-01

    Aedes aegypti mosquitoes vector several arboviruses of global health significance, including dengue viruses and chikungunya virus. RNA interference (RNAi) plays an important role in antiviral immunity, gene regulation and protection from transposable elements. Double-stranded RNA binding proteins (dsRBPs) are important for efficient RNAi; in Drosophila functional specialization of the miRNA, endo-siRNA and exo-siRNA pathway is aided by the dsRBPs Loquacious (Loqs-PB, Loqs-PD) and R2D2, respectively. However, this functional specialization has not been investigated in other dipterans. We were unable to detect Loqs-PD in Ae. aegypti; analysis of other dipteran genomes demonstrated that this isoform is not conserved outside of Drosophila. Overexpression experiments and small RNA sequencing following depletion of each dsRBP revealed that R2D2 and Loqs-PA cooperate non-redundantly in siRNA production, and that these proteins exhibit an inhibitory effect on miRNA levels. Conversely, Loqs-PB alone interacted with mosquito dicer-1 and was essential for full miRNA production. Mosquito Loqs interacted with both argonaute 1 and 2 in a manner independent of its interactions with dicer. We conclude that the functional specialization of Loqs-PD in Drosophila is a recently derived trait, and that in other dipterans, including the medically important mosquitoes, Loqs-PA participates in both the miRNA and endo-siRNA based pathways. PMID:25765650

  6. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes.

    PubMed

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic liposomes is able to reduce immune responses, cytotoxicity, and other side effects caused by viral vectors in clinical applications. PMID:22001930

  7. Octamer 4 small interfering RNA results in cancer stem cell-like cell apoptosis.

    PubMed

    Hu, Tingsong; Liu, Shanrong; Breiter, Deborah R; Wang, Fang; Tang, Ying; Sun, Shuhan

    2008-08-15

    Octamer 4 (Oct4), a member of the POU family of transcription factors, plays a key role in the maintenance of pluripotency and proliferation potential of embryonic stem cells. Cancer stem cell-like cells (CSCLC) are reported to be a minor population in tumors or even in tumor cell lines which also express Oct4. The role of Oct4 in CSCLCs still remains to be defined. In our study, we show that, in vitro, almost all murine Lewis lung carcinoma 3LL cells and human breast cancer MCF7 cells express Oct4 at high levels. This expression of Oct4 is successfully reduced by small interfering RNA, which eventually results in cell apoptosis. The signal pathway Oct4/Tcl1/Akt1 has been observed to be involved in this event. The repression of Oct4 reduces Tcl1 expression and further down-regulates the level of p-Ser.473-Akt1. In vivo, only approximately 5% of tumor cells were detected to express Oct4 in established 3LL and MCF7 tumor models, respectively. Small interfering RNA against Oct4 successfully decreases the CSCLCs and markedly inhibits tumor growth. In summary, we show that Oct4 might maintain the survival of CSCLCs partly through Oct4/Tcl1/Akt1 by inhibiting apoptosis, which strongly indicates that targeting Oct4 may have important clinical applications in cancer therapy. PMID:18701476

  8. Functional Specialization of the Small Interfering RNA Pathway in Response to Virus Infection

    PubMed Central

    Marques, Joao Trindade; Wang, Ji-Ping; Wang, Xiaohong; de Oliveira, Karla Pollyanna Vieira; Gao, Catherine; Aguiar, Eric Roberto Guimaraes Rocha; Jafari, Nadereh; Carthew, Richard W.

    2013-01-01

    In Drosophila, post-transcriptional gene silencing occurs when exogenous or endogenous double stranded RNA (dsRNA) is processed into small interfering RNAs (siRNAs) by Dicer-2 (Dcr-2) in association with a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs-PD). siRNAs are then loaded onto Argonaute-2 (Ago2) by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to siRNAs. Although Dcr-2, R2D2, and Ago2 are essential for innate antiviral defense, the mechanism of virus-derived siRNA (vsiRNA) biogenesis and viral target inhibition remains unclear. Here, we characterize the response mechanism mediated by siRNAs against two different RNA viruses that infect Drosophila. In both cases, we show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and, to a lesser extent, viral transcription. These vsiRNAs seem to preferentially target viral polyadenylated RNA to inhibit viral replication. Loqs-PD is completely dispensable for silencing of the viruses, in contrast to its role in silencing endogenous targets. Biogenesis of vsiRNAs is independent of both Loqs-PD and R2D2. R2D2, however, is required for sorting and loading of vsiRNAs onto Ago2 and inhibition of viral RNA expression. Direct injection of viral RNA into Drosophila results in replication that is also independent of Loqs-PD. This suggests that triggering of the antiviral pathway is not related to viral mode of entry but recognition of intrinsic features of virus RNA. Our results indicate the existence of a vsiRNA pathway that is separate from the endogenous siRNA pathway and is specifically triggered by virus RNA. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response. PMID:24009507

  9. Safe and effective delivery of small interfering RNA with polymer- and liposomes-based complexes.

    PubMed

    Kodama, Yukinobu; Harauchi, Satoe; Kawanabe, Saki; Ichikawa, Nobuhiro; Nakagawa, Hiroo; Muro, Takahiro; Higuchi, Norihide; Nakamura, Tadahiro; Kitahara, Takashi; Sasaki, Hitoshi

    2013-01-01

    We developed binary and ternary complexes based on polymers and liposomes for safe and effective delivery of small interfering RNA (siRNA). Anti-luciferase siRNA was used as a model of nucleic acid medicine. The binary complexes of siRNA were prepared with cationic polymers and cationic liposomes such as polyethylenimine (PEI), polyamidoamine (PAMAM) dendrimer, poly-L-arginine (PLA), trimethyl[2,3-(dioleoxy)-propyl]ammonium chloride (DOTMA), and cholesteryl 3β-N-(dimetylaminnoethyl)carbamate hydrochloride (DC-Chol). The ternary complexes were constructed by the addition of γ-polyglutamic acid (γ-PGA) to the binary complexes. The complexes were approximately 54-153 nm in particle size. The binary complexes showed a cationic surface charge although an anionic surface charge was observed in the ternary complexes. The polymer-based complexes did not show a silencing effect in the mouse colon carcinoma cell line expressing luciferase regularly (Colon26/Luc cells). The binary complexes based on liposomes and their ternary complexes coated by γ-PGA showed a significant silencing effect. The binary complexes showed significant cytotoxicity although the ternary complexes coated by γ-PGA did not show significant cytotoxicity. The ternary complexes coated by γ-PGA suppressed luciferase activity in the tumor after their direct injection into the tumors of mice bearing Colon26/Luc cells. Thus, we have newly identified safe and efficient ternary complexes of siRNA for clinical use. PMID:23727920

  10. Inhibition of pathologic immunoglobulin free light chain production by small interfering RNA molecules

    PubMed Central

    Phipps, Jonathan E.; Kestler, Daniel P.; Foster, James S.; Kennel, Stephen J.; Donnell, Robert; Weiss, Deborah T.; Solomon, Alan; Wall, Jonathan S.

    2010-01-01

    Objectives Morbidity and mortality occurring in patients with multiple myeloma, AL amyloidosis, and light chain deposition disease can result from the pathologic deposition of monoclonal Ig light chains (LCs) in kidneys and other organs. To reduce synthesis of such components, therapy for these disorders typically has involved anti-plasma cell agents; however, this approach is not always effective and can have adverse consequences. We have investigated another means to achieve this objective; namely, RNA interference (RNAi). Materials and Methods SP2/O mouse myeloma cells were stably transfected with a construct encoding a λ6 LC (Wil) under control of the CMV promoter, while λ2-producing myeloma cell line RPMI 8226 was purchased from the ATCC. Both were treated with small interfering RNA (siRNA) directed specifically to the V, J, or C portions of the molecules and then analyzed by ELISA, flow cytometry and real time PCR. Results Transfected cells were found to constitutively express detectable quantities of mRNA and protein Wil and, after exposure to siRNAs, an ~40% reduction in mRNA and LC production was evidenced at 48 hours. An even greater effect was seen with the 8226 cells. Conclusion Our results have shown that RNAi can markedly reduce LC synthesis and provide the basis for testing the therapeutic potential of this strategy using in vivo experimental models of multiple myeloma. PMID:20637260

  11. Failure of the Tomato Trans-Acting Short Interfering RNA Program to Regulate AUXIN RESPONSE FACTOR3 and ARF4 Underlies the Wiry Leaf Syndrome[C][W

    PubMed Central

    Yifhar, Tamar; Pekker, Irena; Peled, Dror; Friedlander, Gilgi; Pistunov, Anna; Sabban, Moti; Wachsman, Guy; Alvarez, John Paul; Amsellem, Ziva; Eshed, Yuval

    2012-01-01

    Interfering with small RNA production is a common strategy of plant viruses. A unique class of small RNAs that require microRNA and short interfering (siRNA) biogenesis for their production is termed trans-acting short interfering RNAs (ta-siRNAs). Tomato (Solanum lycopersicum) wiry mutants represent a class of phenotype that mimics viral infection symptoms, including shoestring leaves that lack leaf blade expansion. Here, we show that four WIRY genes are involved in siRNA biogenesis, and in their corresponding mutants, levels of ta-siRNAs that regulate AUXIN RESPONSE FACTOR3 (ARF3) and ARF4 are reduced, while levels of their target ARFs are elevated. Reducing activity of both ARF3 and ARF4 can rescue the wiry leaf lamina, and increased activity of either can phenocopy wiry leaves. Thus, a failure to negatively regulate these ARFs underlies tomato shoestring leaves. Overexpression of these ARFs in Arabidopsis thaliana, tobacco (Nicotiana tabacum), and potato (Solanum tuberosum) failed to produce wiry leaves, suggesting that the dramatic response in tomato is exceptional. As negative regulation of orthologs of these ARFs by ta-siRNA is common to land plants, we propose that ta-siRNA levels serve as universal sensors for interference with small RNA biogenesis, and changes in their levels direct species-specific responses. PMID:23001036

  12. Rapid delivery of small interfering RNA by biosurfactant MEL-A-containing liposomes

    SciTech Connect

    Inoh, Yoshikazu; Furuno, Tadahide; Hirashima, Naohide; Kitamoto, Dai; Nakanishi, Mamoru

    2011-10-28

    Highlights: Black-Right-Pointing-Pointer We use MEL-A-containing cationic liposomes for siRNA delivery. Black-Right-Pointing-Pointer MEL-A-containing cationic liposomes can efficiently and rapidly deliver siRNA into the cytoplasm. Black-Right-Pointing-Pointer Rapid delivery of siRNA is due to the membrane fusion between liposomes and plasma membrane. -- Abstract: The downregulation of gene expression by RNA interference holds great potential for genetic analysis and gene therapy. However, a more efficient delivery system for small interfering RNA (siRNA) into the target cells is required for wide fields such as cell biology, physiology, and clinical application. Non-viral vectors are stronger candidates than viral vectors because they are safer and easier to prepare. We have previously used a new method for gene transfection by combining cationic liposomes with the biosurfactant mannosylerythritol lipid-A (MEL-A). The novel MEL-A-containing cationic liposomes rapidly delivered DNA (plasmids and oligonucleotides) into the cytosol and nucleus through membrane fusion between liposomes and the plasma membrane, and consequently, enhanced the gene transfection efficiency. In this study, we determined the efficiency of MEL-A-containing cationic liposomes for siRNA delivery. We observed that exogenous and endogenous protein expression was suppressed by approximately 60% at 24 h after brief (30 min) incubation of target cells with MEL-A-containing cationic liposome/siRNA complexes. Confocal microscopic analysis showed that suppression of protein expression was caused by rapid siRNA delivery into the cytosol. We found that the MEL-A-containing cationic liposomes directly delivered siRNA into the cytoplasm by the membrane fusion in addition to endocytotic pathway whereas Lipofectamine Trade-Mark-Sign RNAiMax delivered siRNA only by the endocytotic pathway. It seems that the ability to rapidly and directly deliver siRNA into the cytosol using MEL-A-containing cationic

  13. Nanoparticle-based delivery of small interfering RNA: challenges for cancer therapy

    PubMed Central

    Miele, Evelina; Spinelli, Gian Paolo; Miele, Ermanno; Di Fabrizio, Enzo; Ferretti, Elisabetta; Tomao, Silverio; Gulino, Alberto

    2012-01-01

    During recent decades there have been remarkable advances and profound changes in cancer therapy. Many therapeutic strategies learned at the bench, including monoclonal antibodies and small molecule inhibitors, have been used at the bedside, leading to important successes. One of the most important advances in biology has been the discovery that small interfering RNA (siRNA) is able to regulate the expression of genes, by a phenomenon known as RNA interference (RNAi). RNAi is one of the most rapidly growing fields of research in biology and therapeutics. Much research effort has gone into the application of this new discovery in the treatment of various diseases, including cancer. However, even though these molecules may have potential and strong utility, some limitations make their clinical application difficult, including delivery problems, side effects due to off-target actions, disturbance of physiological functions of the cellular machinery involved in gene silencing, and induction of the innate immune response. Many researchers have attempted to overcome these limitations and to improve the safety of potential RNAi-based therapeutics. Nanoparticles, which are nanostructured entities with tunable size, shape, and surface, as well as biological behavior, provide an ideal opportunity to modify current treatment regimens in a substantial way. These nanoparticles could be designed to surmount one or more of the barriers encountered by siRNA. Nanoparticle drug formulations afford the chance to improve drug bioavailability, exploiting superior tissue permeability, payload protection, and the “stealth” features of these entities. The main aims of this review are: to explain the siRNA mechanism with regard to potential applications in siRNA-based cancer therapy; to discuss the possible usefulness of nanoparticle-based delivery of certain molecules for overcoming present therapeutic limitations; to review the ongoing relevant clinical research with its pitfalls and

  14. Small interfering RNA suppression of polyamine analog-induced spermidine/spermine n1-acetyltransferase.

    PubMed

    Chen, Ying; Kramer, Debora L; Jell, Jason; Vujcic, Slavoljub; Porter, Carl W

    2003-11-01

    N1,N11-diethylnorspermine (DENSPM) is a polyamine analog that down-regulates polyamine biosynthesis and potently upregulates the polyamine catabolic enzyme spermidine/spermine N1-acetyltransferase (SSAT). In certain cells, such as SKMEL-28 human melanoma cells, induction of SSAT is associated with rapid apoptosis. In this study, we used small interfering RNA (siRNA) to examine the role of SSAT induction in mediating polyamine pool depletion and apoptosis. siRNA duplexes were designed to target three independent sites in the SSAT mRNA coding region (siSSAT). When transfected under nontoxic conditions, two of the duplexes selectively reduced basal SSAT mRNA in HEK-293 cells by >80% and prevented DENSPM-induced SSAT mRNA by 95% in SK-MEL-28 cells. Treatment of SK-MEL-28 cells with 10 muM DENSPM in the presence of 83 nM siSSAT selectively prevented the 1400-fold induction of SSAT activity by approximately 90% and, in turn, prevented analog depletion of spermine (Spm) pools by approximately 35%. siSSAT also prevented DENSPM-induced cytochrome c release and caspase-3 cleavage at 36 h and apoptosis at 48 h as measured by annexin V staining. Overall, the data directly link analog induction of SSAT to Spm pool depletion and to caspase-dependent apoptosis in DENSPM-treated SK-MEL-28 cells. This represents the first use of siRNA technology directed toward a polyamine gene and the first unequivocal demonstration that SSAT induction initiates events leading to polyamine analog-induced apoptosis. PMID:14573765

  15. Phytophthora Have Distinct Endogenous Small RNA Populations That Include Short Interfering and microRNAs

    PubMed Central

    Fahlgren, Noah; Bollmann, Stephanie R.; Kasschau, Kristin D.; Cuperus, Josh T.; Press, Caroline M.; Sullivan, Christopher M.; Chapman, Elisabeth J.; Hoyer, J. Steen; Gilbert, Kerrigan B.; Grünwald, Niklaus J.; Carrington, James C.

    2013-01-01

    In eukaryotes, RNA silencing pathways utilize 20-30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focused on plant and animal RNA silencing systems. Phytophthora species belong to a phylogenetically distinct group of economically important plant pathogens that cause billions of dollars in yield losses annually as well as ecologically devastating outbreaks. We analyzed the small RNA-generating components of the genomes of P. infestans, P. sojae and P. ramorum using bioinformatics, genetic, phylogenetic and high-throughput sequencing-based methods. Each species produces two distinct populations of small RNAs that are predominantly 21- or 25-nucleotides long. The 25-nucleotide small RNAs were primarily derived from loci encoding transposable elements and we propose that these small RNAs define a pathway of short-interfering RNAs that silence repetitive genetic elements. The 21-nucleotide small RNAs were primarily derived from inverted repeats, including a novel microRNA family that is conserved among the three species, and several gene families, including Crinkler effectors and type III fibronectins. The Phytophthora microRNA is predicted to target a family of amino acid/auxin permeases, and we propose that 21-nucleotide small RNAs function at the post-transcriptional level. The functional significance of microRNA-guided regulation of amino acid/auxin permeases and the association of 21-nucleotide small RNAs with Crinkler effectors remains unclear, but this work provides a framework for testing the role of small RNAs in Phytophthora biology and pathogenesis in future work. PMID:24204767

  16. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-01-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  17. Delivery of antiviral small interfering RNA with gold nanoparticles inhibits dengue virus infection in vitro

    PubMed Central

    Paul, Amber M.; Shi, Yongliang; Acharya, Dhiraj; Douglas, Jessica R.; Cooley, Amanda; Anderson, John F.; Huang, Faqing

    2014-01-01

    Dengue virus (DENV) infection in humans can cause flu-like illness, life-threatening haemorrhagic fever or even death. There is no specific anti-DENV therapeutic or approved vaccine currently available, partially due to the possibility of antibody-dependent enhancement reaction. Small interfering RNAs (siRNAs) that target specific viral genes are considered a promising therapeutic alternative against DENV infection. However, in vivo, siRNAs are vulnerable to degradation by serum nucleases and rapid renal excretion due to their small size and anionic character. To enhance siRNA delivery and stability, we complexed anti-DENV siRNAs with biocompatible gold nanoparticles (AuNPs) and tested them in vitro. We found that cationic AuNP–siRNA complexes could enter Vero cells and significantly reduce DENV serotype 2 (DENV-2) replication and infectious virion release under both pre- and post-infection conditions. In addition, RNase-treated AuNP–siRNA complexes could still inhibit DENV-2 replication, suggesting that AuNPs maintained siRNA stability. Collectively, these results demonstrated that AuNPs were able to efficiently deliver siRNAs and control infection in vitro, indicating a novel anti-DENV strategy. PMID:24828333

  18. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide

    NASA Astrophysics Data System (ADS)

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-05-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis.

  19. Codelivery of paclitaxel and small interfering RNA by octadecyl quaternized carboxymethyl chitosan-modified cationic liposome for combined cancer therapy.

    PubMed

    Zhang, Ran; Wang, Shi-Bin; Chen, Ai-Zheng; Chen, Wei-Guang; Liu, Yuan-Gang; Wu, Wen-Guo; Kang, Yong-Qiang; Ye, Shi-Fu

    2015-09-01

    Conventional therapeutic approaches for cancer are limited by cancer cell resistance, which has impeded their clinical applications. The main goal of this work was to investigate the combined antitumor effect of paclitaxel with small interfering RNA modified by cationic liposome formed from modified octadecyl quaternized carboxymethyl chitosan. The cationic liposome was composed of 3β-[N-(N', N'-dimethylaminoethane)-carbamoyl]-cholesterol, dioleoylphosphatidylethanolamine, and octadecyl quaternized carboxymethyl chitosan. The cationic liposome properties were characterized by Fourier transform infrared spectroscopy, dynamic light scattering and zeta potential measurements, transmission electron microscopy, atomic force microscopy, and gel retardation assay. The cationic liposome exhibited good properties, such as a small particle size, a narrow particle size distribution, a good spherical shape, a smooth surface, and a good binding ability with small interfering RNA. Most importantly, when combined with paclitaxel and small interfering RNA, the composite cationic liposome induced a great enhancement in the antitumor activity, which showed a significantly higher in vitro cytotoxicity in Bcap-37 cells than liposomal paclitaxel or small interfering RNA alone. In conclusion, the results indicate that cationic liposome could be further developed as a codelivery system for chemotherapy drugs and therapeutic small interfering RNAs. PMID:25838353

  20. Nanotechnologies and controlled release systems for the delivery of antisense oligonucleotides and small interfering RNA

    PubMed Central

    Fattal, Elias; Barratt, Gillian

    2009-01-01

    Antisense oligonucleotides and small interfering RNA have enormous potential for the treatment of a number of diseases, including cancer. However, several impediments to their widespread use as drugs still have to be overcome: in particular their lack of stability in physiological fluids and their poor penetration into cells. Association with or encapsulation within nano-and microsized drug delivery systems could help to solve these problems. In this review, we describe the progress that has been made using delivery systems composed of natural or synthetic polymers in the form of complexes, nanoparticles or microparticles. This article is part of a themed section on Vector Design and Drug Delivery. For a list of all articles in this section see the end of this paper, or visit: http://www3.interscience.wiley.com/journal/121548564/issueyear?year=200 PMID:19366348

  1. Trans-acting small interfering RNA4: key to nutraceutical synthesis in 1 grape development?

    PubMed Central

    Rock, Christopher D.

    2013-01-01

    The facility and versatility of microRNAs (miRNAs) to evolve and change likely underlies how they have become dominant constituents of eukaryotic genomes. In this opinion article I propose that trans-acting small interfering RNA gene 4 (TAS4) evolution may be important for biosynthesis of polyphenolics, arbuscular symbiosis, and bacterial pathogen etiologies. Expression-based and phylogenetic evidence shows that TAS4 targets two novel grape (Vitis vinifera L.) MYB transcription factors (VvMYBA6, VvMYBA7) that spawn phased siRNAs and likely function in nutraceutical bioflavonoid biosynthesis and fruit development. Characterization of the molecular mechanisms of TAS4 control of plant development and integration into biotic and abiotic stress- and nutrient signaling regulatory networks has applicability to molecular breeding and development of strategies for engineering healthier foods. PMID:23993483

  2. Characterization of defective interfering RNAs associated with RNA plant viruses. Progress report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1993-04-01

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since the original observation with TBSV, we discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), and many other reports have now appeared characterizing DI and DI-like RNAs in other plant viral infections. We are seeking to improve our understanding of the mechanisms of DI generation and the precise nature of the RNA sequences necessary for DI replication and encapsidation. We also want to address the nature of the DI mediated symptom attenuation and interference effects in plants, and to determine the feasibility of using transgenic plants constitutively expressing DI RNAs for disease control. The progress made on each of these objectives is summarized along with the proposed experiments for the continuation period.

  3. Coarse-grained Simulation of Complexation between Small Interfering RNA and Polycations

    NASA Astrophysics Data System (ADS)

    Wei, Zonghui; Ren, Yong; Williford, John-Michael; Mao, Hai-Quan; Luijten, Erik; Northwestern University Collaboration; Johns Hopkins University Collaboration

    Nanoparticles formed through self-assembly of polycations and nucleic acids are promising systems for gene delivery. A full understanding of the behavior of these particles in physiological context requires detailed knowledge of their physical properties. All-atom molecular dynamics simulations can provide insight into the interaction of polymeric carriers with genomic material, but only at limited time and length scales. To overcome these limitations and explore the full complexation process, a reliable coarse-grained model is needed. Here, we systematically develop such a model for a system comprised of small interfering RNA (siRNA) and polyethyleneimine-based carriers, and evaluate the quality of the coarse-grained model through comparison with all-atom simulations. We show that our coarse-grained model provides a reliable description of detailed binding pictures, charge characteristics, and water dynamics, while accelerating the simulations by two orders of magnitude. This makes it possible to quantitatively investigate nanoparticle formation involving multiple siRNA molecules and cationic copolymers.

  4. Silencing of uncoupling protein 2 by small interfering RNA aggravates mitochondrial dysfunction in cardiomyocytes under septic conditions

    PubMed Central

    ZHENG, GUILANG; LYU, JUANJUAN; LIU, SHU; HUANG, JINDA; LIU, CUI; XIANG, DAN; XIE, MEIYAN; ZENG, QIYI

    2015-01-01

    Uncoupling protein 2 (UCP2) regulates the production of mitochondrial reactive oxygen species (ROS) and cellular energy transduction under physiological or pathological conditions. In this study, we aimed to determine whether mitochondrial UCP2 plays a protective role in cardiomyocytes under septic conditions. In order to mimic the septic condition, rat embryonic cardiomyoblast-derived H9C2 cells were cultured in the presence of lipopolysaccharide (LPS) plus peptidoglycan G (PepG) and small interfering RNA (siRNA) against UCP2 (siUCP2) was used to suppress UCP2 expression. Reverse transcription quantitative-polymerase chain reaction (RT-qPCR), western blot analysis, transmission electron microscopy (TEM), confocal microscopy and flow cytometry (FCM) were used to detect the mRNA levels, protein levels, mitochondrial morphology and mitochondrial membrane potential (MMP or ΔΨm) in qualitative and quantitative analyses, respectively. Indicators of cell damage [lactate dehydrogenase (LDH), creatine kinase (CK), interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the culture supernatant] and mitochondrial function [ROS, adenosine triphosphate (ATP) and mitochondrial DNA (mtDNA)] were detected. Sepsis enhanced the mRNA and protein expression of UCP2 in the H9C2 cells, damaged the mitochondrial ultrastructure, increased the forward scatter (FSC)/side scatter (SSC) ratio, increased the CK, LDH, TNF-α and IL-6 levels, and lead to the dissipation of MMP, as well as the overproduction of ROS; in addition, the induction of sepsis led to a decrease in ATP levels and the deletion of mtDNA. The silencing of UCP2 aggravated H9C2 cell damage and mitochondrial dysfunction. In conclusion, our data demonstrate that mitochondrial morphology and funtion are damaged in cardiomyocytes under septic conditions, while the silencing of UCP2 using siRNA aggravated this process, indicating that UCP2 may play a protective role in cardiomyocytes under septic conditions. PMID:25873251

  5. MicroRNA superfamilies descended from miR390 and their roles in secondary small interfering RNA Biogenesis in Eudicots.

    PubMed

    Xia, Rui; Meyers, Blake C; Liu, Zhongchi; Beers, Eric P; Ye, Songqing; Liu, Zongrang

    2013-05-01

    Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA-TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. PMID:23695981

  6. Caenorhabditis elegans RSD-2 and RSD-6 promote germ cell immortality by maintaining small interfering RNA populations.

    PubMed

    Sakaguchi, Aisa; Sarkies, Peter; Simon, Matt; Doebley, Anna-Lisa; Goldstein, Leonard D; Hedges, Ashley; Ikegami, Kohta; Alvares, Stacy M; Yang, Liwei; LaRocque, Jeannine R; Hall, Julie; Miska, Eric A; Ahmed, Shawn

    2014-10-14

    Germ cells are maintained in a pristine non-aging state as they proliferate over generations. Here, we show that a novel function of the Caenorhabditis elegans RNA interference proteins RNAi spreading defective (RSD)-2 and RSD-6 is to promote germ cell immortality at high temperature. rsd mutants cultured at high temperatures became progressively sterile and displayed loss of small interfering RNAs (siRNAs) that target spermatogenesis genes, simple repeats, and transposons. Desilencing of spermatogenesis genes occurred in late-generation rsd mutants, although defective spermatogenesis was insufficient to explain the majority of sterility. Increased expression of repetitive loci occurred in both germ and somatic cells of late-generation rsd mutant adults, suggesting that desilencing of many heterochromatic segments of the genome contributes to sterility. Nuclear RNAi defective (NRDE)-2 promotes nuclear silencing in response to exogenous double-stranded RNA, and our data imply that RSD-2, RSD-6, and NRDE-2 function in a common transgenerational nuclear silencing pathway that responds to endogenous siRNAs. We propose that RSD-2 and RSD-6 promote germ cell immortality at stressful temperatures by maintaining transgenerational epigenetic inheritance of endogenous siRNA populations that promote genome silencing. PMID:25258416

  7. Identification of Druggable Targets for Radiation Mitigation Using a Small Interfering RNA Screening Assay

    PubMed Central

    Zellefrow, Crystal D.; Sharlow, Elizabeth R.; Epperly, Michael W.; Reese, Celeste E.; Shun, Tongying; Lira, Ana; Greenberger, Joel S.; Lazo, John S.

    2013-01-01

    Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D0 = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D0 = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D0 = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation

  8. Identification of druggable targets for radiation mitigation using a small interfering RNA screening assay.

    PubMed

    Zellefrow, Crystal D; Sharlow, Elizabeth R; Epperly, Michael W; Reese, Celeste E; Shun, Tongying; Lira, Ana; Greenberger, Joel S; Lazo, John S

    2012-09-01

    Currently, there is a serious absence of pharmaceutically attractive small molecules that mitigate the lethal effects of an accidental or intentional public exposure to toxic doses of ionizing radiation. Moreover, cellular systems that emulate the radiobiologically relevant cell populations and that are suitable for high-throughput screening have not been established. Therefore, we examined two human pluripotent embryonal carcinoma cell lines for use in an unbiased phenotypic small interfering RNA (siRNA) assay to identify proteins with the potential of being drug targets for the protection of human cell populations against clinically relevant ionizing radiation doses that cause acute radiation syndrome. Of the two human cell lines tested, NCCIT cells had optimal growth characteristics in a 384 well format, exhibited radiation sensitivity (D(0) = 1.3 ± 0.1 Gy and ñ = 2.0 ± 0.6) comparable to the radiosensitivity of stem cell populations associated with human death within 30 days after total-body irradiation. Moreover, they internalized siRNA after 4 Gy irradiation enabling siRNA library screening. Therefore, we used the human NCCIT cell line for the radiation mitigation study with a siRNA library that silenced 5,520 genes known or hypothesized to be potential therapeutic targets. Exploiting computational methodologies, we identified 113 siRNAs with potential radiomitigative properties, which were further refined to 29 siRNAs with phosphoinositide-3-kinase regulatory subunit 1 (p85α) being among the highest confidence candidate gene products. Colony formation assays revealed radiation mitigation when the phosphoinositide-3-kinase inhibitor LY294002 was given after irradiation of 32D cl 3 cells (D(0) = 1.3 ± 0.1 Gy and ñ = 2.3 ± 0.3 for the vehicle control treated cells compared to D(0) = 1.2 ± 0.1 Gy and ñ = 6.0 ± 0.8 for the LY294002 treated cells, P = 0.0004). LY294002 and two other PI3K inhibitors, PI 828 and GSK 1059615, also mitigated radiation

  9. MicroRNA Superfamilies Descended from miR390 and Their Roles in Secondary Small Interfering RNA Biogenesis in Eudicots[W

    PubMed Central

    Xia, Rui; Meyers, Blake C.; Liu, Zhongchi; Beers, Eric P.; Ye, Songqing; Liu, Zongrang

    2013-01-01

    Trans-acting small interfering RNAs (tasiRNAs) are a major class of small RNAs performing essential biological functions in plants. The first reported tasiRNA pathway, that of miR173-TAS1/2, produces tasiRNAs regulating a set of pentatricopeptide repeat (PPR) genes and has been characterized only in Arabidopsis thaliana to date. Here, we demonstrate that the microRNA (miRNA)-trans-acting small interfering RNA gene (TAS)-pentatricopeptide repeat-containing gene (PPR)-small interfering RNA pathway is a highly dynamic and widespread feature of eudicots. Nine eudicot plants, representing six different plant families, have evolved similar tasiRNA pathways to initiate phased small interfering RNA (phasiRNA) production from PPR genes. The PPR phasiRNA production is triggered by different 22-nucleotide miRNAs, including miR7122, miR1509, and fve-PPRtri1/2, and through distinct mechanistic strategies exploiting miRNA direct targeting or indirect targeting through TAS-like genes (TASL), one-hit or two-hit, or even two layers of tasiRNA–TASL interactions. Intriguingly, although those miRNA triggers display high sequence divergence caused by the occurrence of frequent point mutations and splicing shifts, their corresponding MIRNA genes show pronounced identity to the Arabidopsis MIR173, implying a common origin of this group of miRNAs (super-miR7122). Further analyses reveal that super-miR7122 may have evolved from a newly defined miR4376 superfamily, which probably originated from the widely conserved miR390. The elucidation of this evolutionary path expands our understanding of the course of miRNA evolution, especially for relatively conserved miRNA families. PMID:23695981

  10. Non-Viral Nanoparticle Delivers Small Interfering RNA to Macrophages In Vitro and In Vivo

    PubMed Central

    Zhang, Mei; Gao, Yunxiang; Caja, Kevin; Zhao, Bocheng; Kim, Julian A.

    2015-01-01

    Macrophages are increasingly being viewed as therapeutic target for various cancers and many inflammatory diseases. Sequence specific gene reduction by siRNA represents an attractive approach to modulate macrophage function. However, delivery of the therapeutic siRNA into macrophages by non-viral nanoparticles has been a major technical challenge. In this study, we developed a glucan-based siRNA carrier system (BG34-10-Re-I) and demonstrated that the BG34-10-Re-I can effectively assemble siRNA into uniformly distributed nanoparticles of the novel core-shell structure. The BG34-10-Re-I/siRNA nanoparticles effectively reduced gene expression of macrophage migration inhibitory factor (MIF) in primary macrophages at both protein and mRNA level. The nanoparticles also mediated a sustained reduction of MIF within primary macrophages. Moreover, systemic injection of the nanoparticles into the Balb/c mice bearing 4T1 mammary tumors resulted in the MIF reduction in tumor-associated macrophages. Mechanistic studies demonstrated that the glucan-shell and the siRNA-core structure contribute to the effective delivery of MIF siRNA to macrophages both in vitro and in vivo. This study represents the first development of the primary macrophage MIF gene targeted non-viral nanoparticle system for both in vitro and in vivo applications. PMID:25799489

  11. Arabidopsis RNA-dependent RNA polymerases and dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection.

    PubMed

    Garcia-Ruiz, Hernan; Takeda, Atsushi; Chapman, Elisabeth J; Sullivan, Christopher M; Fahlgren, Noah; Brempelis, Katherine J; Carrington, James C

    2010-02-01

    Plants respond to virus infections by activation of RNA-based silencing, which limits infection at both the single-cell and system levels. Viruses encode RNA silencing suppressor proteins that interfere with this response. Wild-type Arabidopsis thaliana is immune to silencing suppressor (HC-Pro)-deficient Turnip mosaic virus, but immunity was lost in the absence of DICER-LIKE proteins DCL4 and DCL2. Systematic analysis of susceptibility and small RNA formation in Arabidopsis mutants lacking combinations of RNA-dependent RNA polymerase (RDR) and DCL proteins revealed that the vast majority of virus-derived small interfering RNAs (siRNAs) were dependent on DCL4 and RDR1, although full antiviral defense also required DCL2 and RDR6. Among the DCLs, DCL4 was sufficient for antiviral silencing in inoculated leaves, but DCL2 and DCL4 were both involved in silencing in systemic tissues (inflorescences). Basal levels of antiviral RNA silencing and siRNA biogenesis were detected in mutants lacking RDR1, RDR2, and RDR6, indicating an alternate route to form double-stranded RNA that does not depend on the three previously characterized RDR proteins. PMID:20190077

  12. Suppression of Breast Cancer Cell Migration by Small Interfering RNA Delivered by Polyethylenimine-Functionalized Graphene Oxide.

    PubMed

    Huang, Yuan-Pin; Hung, Chao-Ming; Hsu, Yi-Chiang; Zhong, Cai-Yan; Wang, Wan-Rou; Chang, Chi-Chang; Lee, Mon-Juan

    2016-12-01

    The carbon-based nanomaterial graphene can be chemically modified to associate with various molecules such as chemicals and biomolecules and developed as novel carriers for drug and gene delivery. In this study, a nonviral gene transfection reagent was produced by functionalizing graphene oxide (GO) with a polycationic polymer, polyethylenimine (PEI), to increase the biocompatibility of GO and to transfect small interfering RNA (siRNA) against C-X-C chemokine receptor type 4 (CXCR4), a biomarker associated with cancer metastasis, into invasive breast cancer cells. PEI-functionalized GO (PEI-GO) was a homogeneous aqueous solution that remained in suspension during storage at 4 °C for at least 6 months. The particle size of PEI-GO was 172 ± 4.58 and 188 ± 5.00 nm at 4 and 25 °C, respectively, and increased slightly to 262 ± 17.6 nm at 37 °C, but remained unaltered with time. Binding affinity of PEI-GO toward siRNA was assessed by electrophoretic mobility shift assay (EMSA), in which PEI-GO and siRNA were completely associated at a PEI-GO:siRNA weight ratio of 2:1 and above. The invasive breast cancer cell line, MDA-MB-231, was transfected with PEI-GO in complex with siRNAs against CXCR4 (siCXCR4). Suppression of the mRNA and protein expression of CXCR4 by the PEI-GO/siCXCR4 complex was confirmed by real-time PCR and western blot analysis. In addition, the metastatic potential of MDA-MB-231 cells was attenuated by the PEI-GO/siCXCR4 complex as demonstrated in wound healing assay. Our results suggest that PEI-GO is effective in the delivery of siRNA and may contribute to targeted gene therapy to suppress cancer metastasis. PMID:27173676

  13. One-pot synthesis of pH-responsive hybrid nanogel particles for the intracellular delivery of small interfering RNA.

    PubMed

    Khaled, Sm Z; Cevenini, Armando; Yazdi, Iman K; Parodi, Alessandro; Evangelopoulos, Michael; Corbo, Claudia; Scaria, Shilpa; Hu, Ye; Haddix, Seth G; Corradetti, Bruna; Salvatore, Francesco; Tasciotti, Ennio

    2016-05-01

    This report describes a novel, one-pot synthesis of hybrid nanoparticles formed by a nanostructured inorganic silica core and an organic pH-responsive hydrogel shell. This easy-to-perform, oil-in-water emulsion process synthesizes fluorescently-doped silica nanoparticles wrapped within a tunable coating of cationic poly(2-diethylaminoethyl methacrylate) hydrogel in one step. Transmission electron microscopy and dynamic light scattering analysis demonstrated that the hydrogel-coated nanoparticles are uniformly dispersed in the aqueous phase. The formation of covalent chemical bonds between the silica and the polymer increases the stability of the organic phase around the inorganic core as demonstrated by thermogravimetric analysis. The cationic nature of the hydrogel is responsible for the pH buffering properties of the nanostructured system and was evaluated by titration experiments. Zeta-potential analysis demonstrated that the charge of the system was reversed when transitioned from acidic to basic pH and vice versa. Consequently, small interfering RNA (siRNA) can be loaded and released in an acidic pH environment thereby enabling the hybrid particles and their payload to avoid endosomal sequestration and enzymatic degradation. These nanoparticles, loaded with specific siRNA molecules directed towards the transcript of the membrane receptor CXCR4, significantly decreased the expression of this protein in a human breast cancer cell line (i.e., MDA-MB-231). Moreover, intravenous administration of siRNA-loaded nanoparticles demonstrated a preferential accumulation at the tumor site that resulted in a reduction of CXCR4 expression. PMID:26901429

  14. Effective Small Interfering RNA Therapy to Treat CLCN7-dependent Autosomal Dominant Osteopetrosis Type 2

    PubMed Central

    Capulli, Mattia; Maurizi, Antonio; Ventura, Luca; Rucci, Nadia; Teti, Anna

    2015-01-01

    In about 70% of patients affected by autosomal dominant osteopetrosis type 2 (ADO2), osteoclast activity is reduced by heterozygous mutations of the CLCN7 gene, encoding the ClC-7 chloride/hydrogen antiporter. CLCN7G215R-, CLCN7R767W-, and CLCN7R286W-specific siRNAs silenced transfected mutant mRNA/EGFP in HEK293 cells, in RAW264.7 cells and in human osteoclasts, with no change of CLCN7WT mRNA and no effect of scrambled siRNA on the mutant transcripts. Osteoclasts from Clcn7G213R ADO2 mice showed reduced bone resorption, a condition rescued by Clcn7G213R-specific siRNA. Treatment of ADO2 mice with Clcn7G213R-specific siRNA induced increase of bone resorption variables and decrease of trabecular bone mass, leading to an overall improvement of the osteopetrotic bone phenotype. Treatment did not induce overt adverse effects and was effective also with siRNAs specific for other mutants. These results demonstrate that a siRNA-based experimental treatment of ADO2 is feasible, and underscore a translational impact for future strategy to cure this therapeutically neglected form of osteopetrosis. PMID:26325626

  15. Small interfering RNA pathway modulates persistent infection of a plant virus in its insect vector

    PubMed Central

    Lan, Hanhong; Wang, Haitao; Chen, Qian; Chen, Hongyan; Jia, Dongsheng; Mao, Qianzhuo; Wei, Taiyun

    2016-01-01

    Plant reoviruses, rhabdoviruses, tospoviruses, and tenuiviruses are transmitted by insect vectors in a persistent-propagative manner. How such persistent infection of plant viruses in insect vectors is established and maintained remains poorly understood. In this study, we used rice gall dwarf virus (RGDV), a plant reovirus, and its main vector leafhopper Recilia dorsalis as a virus–insect system to determine how the small interference (siRNA) pathway modulates persistent infection of a plant virus in its insect vector. We showed that a conserved siRNA antiviral response was triggered by the persistent replication of RGDV in cultured leafhopper cells and in intact insects, by appearance of virus-specific siRNAs, primarily 21-nt long, and the increased expression of siRNA pathway core components Dicer-2 and Argonaute-2. Silencing of Dicer-2 using RNA interference strongly suppressed production of virus-specific siRNAs, promoted viral accumulation, and caused cytopathological changes in vitro and in vivo. When the viral accumulation level rose above a certain threshold of viral genome copy (1.32 × 1014 copies/μg insect RNA), the infection of the leafhopper by RGDV was lethal rather than persistent. Taken together, our results revealed a new finding that the siRNA pathway in insect vector can modulate persistent infection of plant viruses. PMID:26864546

  16. DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana.

    PubMed

    Xie, Zhixin; Allen, Edwards; Wilken, April; Carrington, James C

    2005-09-01

    Arabidopsis thaliana contains four DICER-LIKE (DCL) genes with specialized functions in small RNA biogenesis for RNA interference-related processes. A mutant with defects in DCL4 was identified and analyzed for microRNA- and endogenous, small interfering RNA (siRNA)-related functions. The dcl4-2 mutant contained normal or near-normal levels of microRNAs (21 nt) and heterochromatin-associated siRNAs (24 nt). In contrast, this mutant lacked each of three families of 21-nt trans-acting siRNAs (ta-siRNAs) and possessed elevated levels of ta-siRNA target transcripts. The dcl4-2 mutant resembled an rna-dependent RNA polymerase 6 mutant in that both mutants lacked ta-siRNAs and displayed heterochronic defects in which vegetative phase change was accelerated. Double mutant analyses with dcl2-1, dcl3-1, and dcl4-2 alleles revealed hierarchical redundancy among DCL activities, leading to alternative processing of ta-siRNA precursors in the absence of DCL4. These data support the concept that plants have specialized and compartmentalized DCL functions for biogenesis of distinct small RNA classes. PMID:16129836

  17. Effect of Interleukin-8 Gene Silencing With Liposome-Encapsulated Small Interfering RNA on Ovarian Cancer Cell Growth

    PubMed Central

    Merritt, William M.; Lin, Yvonne G.; Spannuth, Whitney A.; Fletcher, Mavis S.; Kamat, Aparna A.; Han, Liz Y.; Landen, Charles N.; Jennings, Nicholas; De Geest, Koen; Langley, Robert R.; Villares, Gabriel; Sanguino, Angela; Lutgendorf, Susan K.; Lopez-Berestein, Gabriel; Bar-Eli, Menashe M.; Sood, Anil K.

    2009-01-01

    Background Interleukin-8 (IL-8) is a proangiogenic cytokine that is overexpressed in many human cancers. We investigated the clinical and biologic significance of IL-8 in ovarian carcinoma using human samples and orthotopic mouse models. Methods Tumor expression of IL-8 was assessed by immunohistochemistry among ovarian cancer patients (n = 102) with available clinical and survival data. We examined the effect of IL-8 gene silencing with small interfering RNAs incorporated into neutral liposomes (siRNA-DOPCs), alone and in combination with docetaxel, on in vivo tumor growth, angiogenesis (microvessel density), and tumor cell proliferation in mice (n = 10 per treatment group) bearing orthotopic taxane-sensitive (HeyA8 and SKOV3ip1) and taxane-resistant (SKOV3ip2.TR) ovarian tumors. All statistical tests were two-sided. Results Of the 102 cancer specimens, 43 (42%) had high IL-8 expression and 59 (58%) had low or no IL-8 expression; high IL-8 expression was associated with advanced tumor stage (P = .019), high tumor grade (P = .031), and worse survival (median survival for patients with high vs low IL-8 expression: 1.62 vs 3.79 years; P < .001). Compared with empty liposomes, IL-8 siRNA-DOPC reduced the mean tumor weight by 32% (95% confidence interval [CI] = 14% to 50%; P = .03) and 52% (95% CI = 27% to 78%; P = .03) in the HeyA8 and SKOV3ip1 mouse models, respectively. In all three mouse models, treatment with IL-8 siRNA-DOPC plus the taxane docetaxel reduced tumor growth the most compared with empty liposomes (77% to 98% reduction in tumor growth; P < .01 for all). In the HeyA8 and SKOV3ip1 models, tumors from mice treated with IL-8 siRNA-DOPC alone had lower microvessel density than tumors from mice treated with empty liposomes (HeyA8: 34% lower, 95% CI = 32% to 36% lower [P = .002]; SKOV3ip1: 39% lower, 95% CI = 34% to 44% lower [P = .007]). Compared with empty liposomes, IL-8 siRNA-DOPC plus docetaxel reduced tumor cell proliferation by 35% (95% CI = 25% to 44

  18. In silico reconstruction of viral genomes from small RNAs improves virus-derived small interfering RNA profiling.

    PubMed

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-11-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  19. In Silico Reconstruction of Viral Genomes from Small RNAs Improves Virus-Derived Small Interfering RNA Profiling ▿ † ‡

    PubMed Central

    Vodovar, Nicolas; Goic, Bertsy; Blanc, Hervé; Saleh, Maria-Carla

    2011-01-01

    RNA interference (RNAi) is the essential component of antiviral immunity in invertebrates and plants. One of the landmarks of the antiviral RNAi response is the production of virus-derived small interfering RNA (vsiRNA) from viral double-stranded RNA (dsRNA). vsiRNAs constitute a fragmented image of the viral genome sequence that results from Dicer cleavage. vsiRNA sequence profiling is used extensively as a surrogate to study the antiviral RNAi response by determining the nature of the viral dsRNA molecules exposed to and processed by the RNAi machinery. The accuracy of these profiles depends on the actual viral genome sequence used as a reference to align vsiRNA reads, and the interpretation of inaccurate profiles can be misleading. Using Flock house virus and Drosophila melanogaster as a model RNAi-competent organism, we show accurate reconstruction of full-length virus reference sequence from vsiRNAs and prediction of the structure of defective interfering particles (DIs). We developed a Perl script, named Paparazzi, that reconstitutes viral genomes through an iterative alignment/consensus call procedure using a related reference sequence as scaffold. As prevalent DI-derived reads introduce artifacts during reconstruction, Paparazzi eliminates DI-specific reads to improve the quality of the reconstructed genome. Paparazzi constitutes a promising alternative to Sanger sequencing in this context and an effective tool to study antiviral RNAi mechanisms by accurately quantifying vsiRNA along the replicating viral genome. We further discuss Paparazzi as a companion tool for virus discovery as it provides full-length genome sequences and corrects for potential artifacts of assembly. PMID:21880776

  20. Effect of small interfering RNA against Paracin 1.7 bacteriocin produced by Lactobacillus paracasei HD1-7.

    PubMed

    Ge, Jingping; Wang, Yang; Gao, Dongni; Ping, Wenxiang

    2015-09-01

    Lactobacillus paracasei HD1-7 (CCTCCM 205015), isolated from Chinese sauerkraut fermentation broth, contains the bacteriocin Paracin 1.7 which possesses broad-spectrum antibacterial activity. The gene-silencing effect of small interfering RNA (siRNA) is a potential strategy for further understanding the mechanism of production of Paracin 1.7 by L. paracasei HD1-7. In this study, the effect of siRNA on the expression of the most important proteins in the production of Paracin 1.7, sensor kinase (prcK) and response regulator (prcR), was investigated. SiRNA were designed against prcK and prcR, and qRT-PCR was performed to examine the expression of prcK and prcR mRNA. The efficacy of siRNA was determined by comparing the level of antimicrobial activity of the strains. qRT-PCR showed that siRNA-K4 and siRNA-K5 significantly inhibited the expression of prcK mRNA, and siRNA-R4 and siRNA-R6 significantly inhibited the expression of prcR mRNA. The proteins levels and antibacterial activities of mutant strains were lower than the original and control groups, respectively. The results demonstrate that siRNA inhibited both mRNA expression and the production of Paracin 1.7 in L. paracasei HD1-7. Targeting of prcK and prcR with siRNA appears to be a novel strategy for researching the mechanism of Paracin 1.7 production by L. paracasei HD1-7. PMID:26011063

  1. Small Interfering RNA Targeted to ASPP2 Promotes Progression of Experimental Proliferative Vitreoretinopathy

    PubMed Central

    Bai, Yu-Jing; Huang, Lv-Zhen; Li, Xiao-Xin

    2016-01-01

    Background. Epithelial-mesenchymal transition (EMT) of retinal pigment epithelium (RPE) is vital in proliferative vitreoretinopathy (PVR) development. Apoptosis-stimulating proteins of p53 (ASPP2) have recently been reported to participate in EMT. However, the role of ASPP2 in PVR pathogenesis has not been identified. Methods. Immunohistochemistry was used to investigate the expression of ASPP2 in epiretinal membranes of PVR patients. ARPE-19 cells were transfected with ASPP2-siRNA, followed with measurement of cell cytotoxicity, proliferation, and migration ability. EMT markers and related inflammatory and fibrosis cytokines were measured by western blot or flow cytometry. Additionally, PVR rat models were induced by intravitreal injection of ARPE-19 cells transfected with ASPP2-siRNA and evaluated accordingly. Results. Immunofluorescence analysis revealed less intense expression of ASPP2 in PVR membranes. ASPP2 knockdown facilitated the proliferation and migration of RPE cells and enhanced the expression of mesenchymal markers such as alpha smooth muscle actin, fibronectin, and ZEB1. Meanwhile, ASPP2-siRNA increased EMT-related and inflammatory cytokines, including TGF-β, CTGF, VEGF, TNF-α, and interleukins. PVR severities were more pronounced in the rat models with ASPP2-siRNA treatment. Conclusions. ASPP2 knockdown promoted EMT of ARPE-19 cells in vitro and exacerbated the progression of experimental PVR in vivo, possibly via inflammatory and fibrosis cytokines. PMID:27378826

  2. Small interfering RNA targeting m2 gene induces effective and long term inhibition of influenza A virus replication.

    PubMed

    Sui, Hong-Yan; Zhao, Guang-Yu; Huang, Jian-Dong; Jin, Dong-Yan; Yuen, Kwok-Yung; Zheng, Bo-Jian

    2009-01-01

    RNA interference (RNAi) provides a powerful new means to inhibit viral infection specifically. However, the selection of siRNA-resistant viruses is a major concern in the use of RNAi as antiviral therapeutics. In this study, we conducted a lentiviral vector with a H1-short hairpin RNA (shRNA) expression cassette to deliver small interfering RNAs (siRNAs) into mammalian cells. Using this vector that also expresses enhanced green fluorescence protein (EGFP) as surrogate marker, stable shRNA-expressing cell lines were successfully established and the inhibition efficiencies of rationally designed siRNAs targeting to conserved regions of influenza A virus genome were assessed. The results showed that a siRNA targeting influenza M2 gene (siM2) potently inhibited viral replication. The siM2 was not only effective for H1N1 virus but also for highly pathogenic avian influenza virus H5N1. In addition to its M2 inhibition, the siM2 also inhibited NP mRNA accumulation and protein expression. A long term inhibition effect of the siM2 was demonstrated and the emergence of siRNA-resistant mutants in influenza quasispecies was not observed. Taken together, our study suggested that M2 gene might be an optimal RNAi target for antiviral therapy. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for human influenza virus infection. PMID:19479060

  3. Biodistribution of Small Interfering RNA at the Organ and Cellular Levels after Lipid Nanoparticle-mediated Delivery

    PubMed Central

    Shi, Bin; Keough, Ed; Matter, Andrea; Leander, Karen; Young, Stephanie; Carlini, Ed; Sachs, Alan B.; Tao, Weikang; Abrams, Marc; Howell, Bonnie; Sepp-Lorenzino, Laura

    2011-01-01

    Chemically stabilized small interfering RNA (siRNA) can be delivered systemically by intravenous injection of lipid nanoparticles (LNPs) in rodents and primates. The biodistribution and kinetics of LNP–siRNA delivery in mice at organ and cellular resolution have been studied using immunofluorescence (IF) staining and quantitative polymerase chain reaction (qPCR). At 0.5 and 2 hr post tail vein injection of Cy5-labeled siRNA encapsulated in LNP, the organ rank-order of siRNA levels is liver > spleen > kidney, with only negligible accumulation in duodenum, lung, heart, and brain. Similar conclusions were drawn by using qPCR to measure tissue siRNA levels as a secondary end point. siRNA levels in these tissues decreased by more than 10-fold after 24 hr. Within the liver, LNPs delivered siRNA to hepatocytes, Kupffer cells, and sinusoids in a time-dependent manner, as revealed by IF staining and signal quantitation methods established using OPERA/Columbus software. siRNA first accumulated in liver sinusoids and trafficked to hepatocytes by 2 hr post dose, corresponding to the onset of target mRNA silencing. Fluorescence in situ hybridization methods were used to detect both strands of siRNA in fixed tissues. Collectively, the authors have implemented a platform to evaluate biodistribution of siRNA across cell types and across tissues in vivo, with the objective of elucidating the pharmacokinetic and pharmacodynamic relationship to guide optimization of delivery vehicles. PMID:21804077

  4. Structural features of microRNA (miRNA) precursors and their relevance to miRNA biogenesis and small interfering RNA/short hairpin RNA design.

    PubMed

    Krol, Jacek; Sobczak, Krzysztof; Wilczynska, Urszula; Drath, Maria; Jasinska, Anna; Kaczynska, Danuta; Krzyzosiak, Wlodzimierz J

    2004-10-01

    We have established the structures of 10 human microRNA (miRNA) precursors using biochemical methods. Eight of these structures turned out to be different from those that were computer-predicted. The differences localized in the terminal loop region and at the opposite side of the precursor hairpin stem. We have analyzed the features of these structures from the perspectives of miRNA biogenesis and active strand selection. We demonstrated the different thermodynamic stability profiles for pre-miRNA hairpins harboring miRNAs at their 5'- and 3'-sides and discussed their functional implications. Our results showed that miRNA prediction based on predicted precursor structures may give ambiguous results, and the success rate is significantly higher for the experimentally determined structures. On the other hand, the differences between the predicted and experimentally determined structures did not affect the stability of termini produced through "conceptual dicing." This result confirms the value of thermodynamic analysis based on mfold as a predictor of strand section by RNAi-induced silencing complex (RISC). PMID:15292246

  5. Inhibition of PARP1 by small interfering RNA enhances docetaxel activity against human prostate cancer PC3 cells

    SciTech Connect

    Wu, Wenqi; Kong, Zhenzhen; Duan, Xiaolu; Zhu, Hanliang; Li, Shujue; Zeng, Shaohua; Liang, Yeping; Iliakis, George; Gui, Zhiming; Yang, Dong

    2013-12-06

    Highlights: •PARP1 siRNA enhances docetaxel’s activity against PC3 cells. •PARP1 siRNA enhances docetaxel’s activity against EGFR/Akt/FOXO1 pathway. •PARP1 siRNA and PARP1 inhibitor differently affect the phosphorylation and expression of FOXO1. -- Abstract: Though poly(ADP-ribose) polymerase 1 (PARP1) inhibitors have benefits in combination with radiotherapy in prostate cancers, few is known about the exactly role and underlying mechanism of PARP1 in combination with chemotherapy agents. Here our data revealed that inhibition of PARP1 by small interfering RNA (siRNA) could enhance docetaxel’s activity against PC3 cells, which is associated with an accelerate repression of EGF/Akt/FOXO1 signaling pathway. Our results provide a novel role of PARP1 in transcription regulation of EGFR/Akt/FOXO1 signaling pathway and indicate that PARP1 siRNA combined with docetaxel can be an innovative treatment strategy to potentially improve outcomes in CRPC patients.

  6. Host-virus interaction: the antiviral defense function of small interfering RNAs can be enhanced by host microRNA-7 in vitro

    PubMed Central

    Zhang, Xiaoying; Liu, Dongyun; Zhang, Sheng; Wei, Xiujuan; Song, Jie; Zhang, Yupei; Jin, Min; Shen, Zhiqiang; Wang, Xinwei; Feng, Zhichun; Li, Junwen

    2015-01-01

    Small interfering RNAs (siRNAs) directed against poliovirus (PV) and other viruses effectively inhibit viral replication and have been developed as antiviral agents. Here, we demonstrate that a specific siRNA targeting the region between nucleotides 100–125 (siRNA-100) from the 5′-untranslated region (5′-UTR) of PV plays a critical role in inhibiting PV replication. Our data demonstrate that siRNA-100 treatment can greatly reduce PV titers, resulting in up-regulation of host microRNA-7 (miR-7), which in turn, leads to enhance inhibition of PV infection further. Moreover, our results suggest that siRNA-100 can also impair the spread of PV to uninfected cells by increasing host resistance to PV, resulting in decreasing necrosis and cytopathic effects (CPE) levels, as well as prolonging the survival of infected cells. Indeed, the active antiviral effect of siRNA-100 was potentially supplemented by the activity of miR-7, and both of them can serve as stabilizing factors for maintenance of cellular homeostasis. Results of this study identify a molecular mechanism of RNAi for antiviral defense, and extend our knowledge of the complex interplay between host and PV, which will provide a basis for the development of effective RNAi-based therapies designed to inhibit PV replication and protect host cells. PMID:26067353

  7. P-SAMS: a web site for plant artificial microRNA and synthetic trans-acting small interfering RNA design

    PubMed Central

    Fahlgren, Noah; Hill, Steven T.; Carrington, James C.; Carbonell, Alberto

    2016-01-01

    Summary: The Plant Small RNA Maker Site (P-SAMS) is a web tool for the simple and automated design of artificial miRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) for efficient and specific targeted gene silencing in plants. P-SAMS includes two applications, P-SAMS amiRNA Designer and P-SAMS syn-tasiRNA Designer. The navigation through both applications is wizard-assisted, and the job runtime is relatively short. Both applications output the sequence of designed small RNA(s), and the sequence of the two oligonucleotides required for cloning into ‘B/c’ compatible vectors. Availability and implementation: The P-SAMS website is available at http://p-sams.carringtonlab.org. Contact: acarbonell@ibmcp.upv.es or nfahlgren@danforthcenter.org PMID:26382195

  8. The p122 Subunit of Tobacco Mosaic Virus Replicase Is a Potent Silencing Suppressor and Compromises both Small Interfering RNA- and MicroRNA-Mediated Pathways▿

    PubMed Central

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-01-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3′ overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised. PMID:17715232

  9. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways.

    PubMed

    Csorba, Tibor; Bovi, Aurelie; Dalmay, Tamás; Burgyán, József

    2007-11-01

    One of the functions of RNA silencing in plants is to defend against molecular parasites, such as viruses, retrotransposons, and transgenes. Plant viruses are inducers, as well as targets, of RNA silencing-based antiviral defense. Replication intermediates or folded viral RNAs activate RNA silencing, generating small interfering RNAs (siRNAs), which are the key players in the antiviral response. Viruses are able to counteract RNA silencing by expressing silencing-suppressor proteins. It has been shown that many of the identified silencing-suppressor proteins bind long double-stranded RNA or siRNAs and thereby prevent assembly of the silencing effector complexes. In this study, we show that the 122-kDa replicase subunit (p122) of crucifer-infecting Tobacco mosaic virus (cr-TMV) is a potent silencing-suppressor protein. We found that the p122 protein preferentially binds to double-stranded 21-nucleotide (nt) siRNA and microRNA (miRNA) intermediates with 2-nt 3' overhangs inhibiting the incorporation of siRNA and miRNA into silencing-related complexes (e.g., RNA-induced silencing complex [RISC]) both in vitro and in planta but cannot interfere with previously programmed RISCs. In addition, our results also suggest that the virus infection and/or sequestration of the siRNA and miRNA molecules by p122 enhances miRNA accumulation despite preventing its methylation. However, the p122 silencing suppressor does not prevent the methylation of certain miRNAs in hst-15 mutants, in which the nuclear export of miRNAs is compromised. PMID:17715232

  10. Small interfering RNA targeting S100A4 sensitizes non-small-cell lung cancer cells (A549) to radiation treatment

    PubMed Central

    Qi, Ruixue; Qiao, Tiankui; Zhuang, Xibing

    2016-01-01

    Objective This study aimed to investigate the impact of S100A4-small interfering RNA (S100A4-siRNA) on apoptosis and enhanced radiosensitivity in non-small-cell lung cancer (A549) cells. We also explored the mechanisms of radiosensitization and identified a new target to enhance radiosensitivity and gene therapy for non-small-cell lung cancer. Methods RNA interference is a powerful tool for gene silencing. In this study, we constructed an effective siRNA to knock down S100A4. A549 cells were randomly divided into three groups: blank, negative control, and S100A4-siRNA. To investigate the effect of S100A4-siRNA, the expression of S100A4, E-cadherin, and p53 proteins and their messenger RNA (mRNA) was detected by Western blot and quantitative real-time polymerase chain reaction. Transwell chambers were used to assess cell invasion. Cell cycle and apoptosis were analyzed by flow cytometry. Radiosensitivity was determined by colony formation ability. Results Our results demonstrate that S100A4-siRNA effectively silenced the S100A4 gene. When siRNA against S100A4 was used, S100A4 protein expression was downregulated, whereas the expressions of E-cadherin and p53 were upregulated. In addition, a clear reduction in S100A4 mRNA levels was noted compared with the blank and negative control groups, whereas E-cadherin and p53 mRNA levels increased. Transfection with S100A4-siRNA significantly reduced the invasiveness of A549 cells. S100A4 silencing induced immediate G2/M arrest in cell cycle studies and increased apoptosis rates in A549 cells. In clonogenic assays, we used a multitarget, single-hit model to detect radiosensitivity after S100A4 knockdown. All parameters (D0, Dq, α, β) indicated that the downregulation of S100A4 enhanced radiosensitivity in A549 cells. Furthermore, S100A4-siRNA upregulated p53 expression, suggesting that S100A4 may promote A549 cell proliferation, invasion, and metastasis by regulating the expression of other proteins. Therefore, siRNA

  11. Zwitterionic Poly(carboxybetaine)-based Cationic Liposomes for Effective Delivery of Small Interfering RNA Therapeutics without Accelerated Blood Clearance Phenomenon

    PubMed Central

    Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis. PMID:25825598

  12. Zwitterionic poly(carboxybetaine)-based cationic liposomes for effective delivery of small interfering RNA therapeutics without accelerated blood clearance phenomenon.

    PubMed

    Li, Yan; Liu, Ruiyuan; Shi, Yuanjie; Zhang, Zhenzhong; Zhang, Xin

    2015-01-01

    For efficient delivery of small interfering RNA (siRNA) to the target diseased site in vivo, it is important to design suitable vehicles to control the blood circulation of siRNA. It has been shown that surface modification of cationic liposome/siRNA complexes (lipoplexes) with polyethylene glycol (PEG) could enhance the circulation time of lipoplexes. However, the first injection of PEGylated lipoplexes in vivo induces accelerated blood clearance and enhances hepatic accumulation of the following injected PEGylated lipoplexes, which is known as the accelerated blood clearance (ABC) phenomenon. Herein, we developed zwitterionic poly(carboxybetaine) (PCB) modified lipoplexes for the delivery of siRNA therapeutics, which could avoid protein adsorption and enhance the stability of lipoplexes as that for PEG. Quite different from the PEGylation, the PCBylated lipoplexes could avoid ABC phenomenon, which extended the blood circulation time and enhanced the tumor accumulation of lipoplexes in vivo. After accumulation in tumor site, the PCBylation could promote the cellular uptake and endosomal/lysosomal escape of lipoplexes due to its unique chemical structure and pH-sensitive ability. With excellent tumor accumulation, cellular uptake and endosomal/lysosomal escape abilities, the PCBylated lipoplexes significantly inhibited tumor growth and induced tumor cell apoptosis. PMID:25825598

  13. Phytophthora have distinct endogenous small RNA populations that include short interfering and microRNAs

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In eukaryotes, RNA silencing pathways utilize 20–30-nucleotide small RNAs to regulate gene expression, specify and maintain chromatin structure, and repress viruses and mobile genetic elements. RNA silencing was likely present in the common ancestor of modern eukaryotes, but most research has focuse...

  14. Cloned Defective Interfering Influenza RNA and a Possible Pan-Specific Treatment of Respiratory Virus Diseases

    PubMed Central

    Dimmock, Nigel J.; Easton, Andrew J.

    2015-01-01

    Defective interfering (DI) genomes are characterised by their ability to interfere with the replication of the virus from which they were derived, and other genetically compatible viruses. DI genomes are synthesized by nearly all known viruses and represent a vast natural reservoir of antivirals that can potentially be exploited for use in the clinic. This review describes the application of DI virus to protect from virus-associated diseases in vivo using as an example a highly active cloned influenza A DI genome and virus that protects broadly in preclinical trials against different subtypes of influenza A and against non-influenza A respiratory viruses. This influenza A-derived DI genome protects by two totally different mechanisms: molecular interference with influenza A replication and by stimulating innate immunity that acts against non-influenza A viruses. The review considers what is needed to develop DI genomes to the point of entry into clinical trials. PMID:26184282

  15. Characterization of a defective interfering RNA that contains a mosaic of a plant viral genome. Final report

    SciTech Connect

    Morris, T.J.; Jackson, A.O.

    1991-12-31

    Our lab was the first to describe and characterize a defective interfering RNA (DI RNAs or DIs) in association with a small RNA plant virus. The features of the DIs that we discovered in infections of tomato bushy stunt virus were compatible with the properties of DIs identified in many animal virus infections. Animal virologists have generally recognized the importance of studying DIs because they are invaluable tools for identifying cis-acting sequences important in virus multiplication and because they offer the opportunity to elucidate mechanisms involved in viral persistence and disease attenuation. Hence our discovery offered a comparably valuable tool for use in plant virus studies for the first time. Since then, we have also discovered the second example of plant viral DI RNAs associated with turnip crinkle virus (TCV), a virus structurally related to TBSV. We proposed a thorough characterization of this unique class of symptom modulating RNAs with the overall objective of identifying viral RNA nucleotide, sequences involved in such fundamental processes as virus replication and encapsidation as well as the degree of symptom expression resulting from the viral-DI-host interaction. The proposed research focused on the molecular characterization of the DI RNAs and the helper virus. We had demonstrated that the DIs were collinear deletion mutants of the genome of a cherry strain of tomato bushy stunt virus (TBSV). We had also shown that these low molecular weight RNAs interfered with the helper plant virus and modulated disease expression by preventing the development of a lethal necrotic disease in susceptible host plants. We also suggested that by exploring the mechanisms associated with the symptom attenuation effect, we might be able to devise novel strategies useful for engineering viral disease resistance.

  16. Comprehensive Annotation of Physcomitrella patens Small RNA Loci Reveals That the Heterochromatic Short Interfering RNA Pathway Is Largely Conserved in Land Plants[OPEN

    PubMed Central

    Coruh, Ceyda; Cho, Sung Hyun; Shahid, Saima; Liu, Qikun; Wierzbicki, Andrzej; Axtell, Michael J.

    2015-01-01

    Many plant small RNAs are sequence-specific negative regulators of target mRNAs and/or chromatin. In angiosperms, the two most abundant endogenous small RNA populations are usually 21-nucleotide microRNAs (miRNAs) and 24-nucleotide heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs are derived from repetitive regions and reinforce DNA methylation at targeted loci. The existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Using small RNA-sequencing (RNA-seq) of the moss Physcomitrella patens, we identified 1090 loci that produce mostly 23- to 24-nucleotide siRNAs. These loci are mostly in intergenic regions with dense DNA methylation. Accumulation of siRNAs from these loci depends upon P. patens homologs of DICER-LIKE3 (DCL3), RNA-DEPENDENT RNA POLYMERASE2, and the largest subunit of DNA-DEPENDENT RNA POLYMERASE IV, with the largest subunit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23-nucleotide siRNA accumulation. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and P. patens, with the notable exception of the P. patens-specific use of mDCL to produce 23-nucleotide siRNAs. PMID:26209555

  17. Small interfering RNA targeting of Recepteur d'Origine Nantais induces apoptosis via modulation of nuclear factor-kappaB and Bcl-2 family in gastric cancer cells.

    PubMed

    Park, Jung Sun; Park, Ji Hye; Lee, Soong; Joo, Young Eun; Jung, Young Do

    2010-09-01

    The abnormal accumulation and activation of the receptor tyrosine kinase, Recepteur d'Origine Nantais (RON), has been implicated in tumorigenesis and metastasis in epithelial tumors including gastric cancer. This study examined whether the sequence-specific small interfering RNA (siRNA) suppression of the RON expression could induce apoptotic cell death, and investigated the involved molecular mechanisms. Sequence-specific siRNA effectively suppressed the RON expression at both the mRNA and protein levels. Silencing of the RON expression significantly inhibited gastric cancer cell proliferation and induced apoptosis in a time-dependent manner. The induction of apoptosis was confirmed by the ladder-patterned DNA fragmentation, the presence of cleaved and condensed nuclear chromatin and the increased number of annexin V-positive cells. RON-targeted siRNA effectively inhibited the constitutive nuclear factor-kappaB (NF-kappaB) activation as revealed by an altered electrophoretic mobility shift. In agreement with this, silencing of the RON expression resulted in a decrease in the nuclear level of the p65 subunit of NF-kappaB. The transfection of siRNA, which blocked the RON expression, also caused a change in the ratio of Bax/Bcl-2 in a manner that favored apoptosis. The siRNA silencing of RON induced cytochrome c release and the activation of caspase-8 and caspase-9. These results indicate that RON-targeted siRNA could be therapeutically efficacious by inducing cell apoptosis through the modulation of the NF-kappaB and Bcl-2 family in gastric cancer cells. PMID:20664977

  18. In vitro evaluation of inhibitory effect of nuclear factor-kappaB activity by small interfering RNA on pro-tumor characteristics of M2-like macrophages.

    PubMed

    Kono, Yusuke; Kawakami, Shigeru; Higuchi, Yuriko; Yamashita, Fumiyoshi; Hashida, Mitsuru

    2014-01-01

    Tumor-associated macrophages (TAMs) have an alternatively activated macrophage phenotype (M2) and promote cancer cell proliferation, angiogenesis and metastasis. Nuclear factor-kappaB (NF-κB) is one of the master regulators of macrophage polarization. Here, we investigated the effect of inhibition of NF-κB activity by small interfering RNA (siRNA) on the pro-tumor response of macrophages located in the tumor microenvironment in vitro. We used mouse peritoneal macrophages cultured in conditioned medium from colon-26 cancer cells as an in vitro TAM model (M2-like macrophages). Transfection of NF-κB (p50) siRNA into M2-like macrophages resulted in a significant decrease in the secretion of interleukin (IL)-10 (a T helper 2 (Th2) cytokine) and a significant increase of T helper 1 (Th1) cytokine production (IL-12, tumor necrosis factor-α, and IL-6). Furthermore, vascular endothelial growth factor production and matrix metalloproteinase-9 mRNA expression in M2-like macrophages were suppressed by inhibition of NF-κB expression with NF-κB (p50) siRNA. In addition, there was a reduction of arginase mRNA expression and increase in nitric oxide production. The cytokine secretion profiles of macrophages cultured in conditioned medium from either B16BL6 or PAN-02 cancer cells were also converted from M2 to classically activated (M1) macrophages by transfection of NF-κB (p50) siRNA. These results suggest that inhibition of NF-κB activity in M2-like macrophages alters their phenotype toward M1. PMID:24141263

  19. Targeting melanoma growth and metastasis with systemic delivery of liposome-incorporated protease-activated receptor-1 small interfering RNA.

    PubMed

    Villares, Gabriel J; Zigler, Maya; Wang, Hua; Melnikova, Vladislava O; Wu, Hong; Friedman, Ran; Leslie, Michael C; Vivas-Mejia, Pablo E; Lopez-Berestein, Gabriel; Sood, Anil K; Bar-Eli, Menashe

    2008-11-01

    The thrombin receptor [protease-activated receptor-1 (PAR-1)] is overexpressed in highly metastatic melanoma cell lines and in patients with metastatic lesions. Activation of PAR-1 leads to cell signaling and up-regulation of genes involved in adhesion, invasion, and angiogenesis. Herein, we stably silence PAR-1 through the use of lentiviral short hairpin RNA and found significant decreases in both tumor growth (P < 0.01) and metastasis (P < 0.001) of highly metastatic melanoma cell lines in vivo. The use of viruses for therapy is not ideal as it can induce toxic immune responses and possible gene alterations following viral integration. Therefore, we also used systemic delivery of PAR-1 small interfering RNA (siRNA) incorporated into neutral liposomes [1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)] to decrease melanoma growth and metastasis in vivo. Significant decreases in tumor growth, weight, and metastatic lung colonies (P < 0.001 for all) were found in mice treated with PAR-1 siRNA-DOPC. The in vivo effects of PAR-1 on invasion and angiogenesis were analyzed via immunohistochemistry. Concomitant decreases in vascular endothelial growth factor, interleukin-8, and matrix metalloproteinase-2 expression levels, as well as decreased blood vessel density (CD31), were found in tumor samples from PAR-1 siRNA-treated mice, suggesting that PAR-1 is a regulator of melanoma cell growth and metastasis by affecting angiogenic and invasive factors. We propose that siRNA incorporated into DOPC nanoparticles could be delivered systemically and used as a new modality for melanoma treatment. PMID:18974154

  20. Interference of hepatitis C virus RNA replication by short interfering RNAs

    NASA Astrophysics Data System (ADS)

    Kapadia, Sharookh B.; Brideau-Andersen, Amy; Chisari, Francis V.

    2003-02-01

    Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Current therapy of patients with chronic HCV infection includes treatment with IFN in combination with ribavirin. Because most treated patients do not resolve the infection, alternative treatment is essential. RNA interference (RNAi) is a recently discovered antiviral mechanism present in plants and animals that induces double-stranded RNA degradation. Using a selectable subgenomic HCV replicon cell culture system, we have shown that RNAi can specifically inhibit HCV RNA replication and protein expression in Huh-7 cells that stably replicate the HCV genome, and that this antiviral effect is independent of IFN. These results suggest that RNAi may represent a new approach for the treatment of persistent HCV infection.

  1. Identification of genes required for Cf-dependent hypersensitive cell death by combined proteomic and RNA interfering analyses

    PubMed Central

    Xu, Qiu-Fang; Cheng, Wei-Shun; Zhang, Zhi-Xin; Xu, You-Ping; Zhou, Xue-Ping; Cai, Xin-Zhong

    2012-01-01

    Identification of hypersensitive cell death (HCD) regulators is essential to dissect the molecular mechanisms underlying plant disease resistance. In this study, combined proteomic and RNA interfering (RNAi) analyses were employed to identify genes required for the HCD conferred by the tomato resistance gene Cf-4 and the Cladosporium fulvum avirulence gene Avr4. Forty-nine proteins differentially expressed in the tomato seedlings mounting and those not mounting Cf-4/Avr4-dependent HCD were identified through proteomic analysis. Among them were a variety of defence-related proteins including a cysteine protease, Pip1, an operative target of another C. fulvum effector, Avr2. Additionally, glutathione-mediated antioxidation is a major response to Cf-4/Avr4-dependent HCD. Functional analysis through tobacco rattle virus-induced gene silencing and transient RNAi assays of the chosen 16 differentially expressed proteins revealed that seven genes, which encode Pip1 homologue NbPip1, a SIPK type MAP kinase Nbf4, an asparagine synthetase NbAsn, a trypsin inhibitor LeMir-like protein NbMir, a small GTP-binding protein, a late embryogenesis-like protein, and an ASR4-like protein, were required for Cf-4/Avr4-dependent HCD. Furthermore, the former four genes were essential for Cf-9/Avr9-dependent HCD; NbPip1, NbAsn, and NbMir, but not Nbf4, affected a nonadaptive bacterial pathogen Xanthomonas oryzae pv. oryzae-induced HCD in Nicotiana benthamiana. These data demonstrate that Pip1 and LeMir may play a general role in HCD and plant immunity and that the application of combined proteomic and RNA interfering analyses is an efficient strategy to identify genes required for HCD, disease resistance, and probably other biological processes in plants. PMID:22275387

  2. Automated parallel synthesis of 5'-triphosphate oligonucleotides and preparation of chemically modified 5'-triphosphate small interfering RNA.

    PubMed

    Zlatev, Ivan; Lackey, Jeremy G; Zhang, Ligang; Dell, Amy; McRae, Kathy; Shaikh, Sarfraz; Duncan, Richard G; Rajeev, Kallanthottathil G; Manoharan, Muthiah

    2013-02-01

    A fully automated chemical method for the parallel and high-throughput solid-phase synthesis of 5'-triphosphate and 5'-diphosphate oligonucleotides is described. The desired full-length oligonucleotides were first constructed using standard automated DNA/RNA solid-phase synthesis procedures. Then, on the same column and instrument, efficient implementation of an uninterrupted sequential cycle afforded the corresponding unmodified or chemically modified 5'-triphosphates and 5'-diphosphates. The method was readily translated into a scalable and high-throughput synthesis protocol compatible with the current DNA/RNA synthesizers yielding a large variety of unique 5'-polyphosphorylated oligonucleotides. Using this approach, we accomplished the synthesis of chemically modified 5'-triphosphate oligonucleotides that were annealed to form small-interfering RNAs (ppp-siRNAs), a potentially interesting class of novel RNAi therapeutic tools. The attachment of the 5'-triphosphate group to the passenger strand of a siRNA construct did not induce a significant improvement in the in vitro RNAi-mediated gene silencing activity nor a strong specific in vitro RIG-I activation. The reported method will enable the screening of many chemically modified ppp-siRNAs, resulting in a novel bi-functional RNAi therapeutic platform. PMID:23260577

  3. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral.

    PubMed

    Smith, Claire M; Scott, Paul D; O'Callaghan, Christopher; Easton, Andrew J; Dimmock, Nigel J

    2016-01-01

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. PMID:27556481

  4. A Defective Interfering Influenza RNA Inhibits Infectious Influenza Virus Replication in Human Respiratory Tract Cells: A Potential New Human Antiviral

    PubMed Central

    Smith, Claire M.; Scott, Paul D.; O’Callaghan, Christopher; Easton, Andrew J.; Dimmock, Nigel J.

    2016-01-01

    Defective interfering (DI) viruses arise during the replication of influenza A virus and contain a non-infective version of the genome that is able to interfere with the production of infectious virus. In this study we hypothesise that a cloned DI influenza A virus RNA may prevent infection of human respiratory epithelial cells with infection by influenza A. The DI RNA (244/PR8) was derived by a natural deletion process from segment 1 of influenza A/PR/8/34 (H1N1); it comprises 395 nucleotides and is packaged in the DI virion in place of a full-length genome segment 1. Given intranasally, 244/PR8 DI virus protects mice and ferrets from clinical influenza caused by a number of different influenza A subtypes and interferes with production of infectious influenza A virus in cells in culture. However, evidence that DI influenza viruses are active in cells of the human respiratory tract is lacking. Here we show that 244/PR8 DI RNA is replicated by an influenza A challenge virus in human lung diploid fibroblasts, bronchial epithelial cells, and primary nasal basal cells, and that the yield of challenge virus is significantly reduced in a dose-dependent manner indicating that DI influenza virus has potential as a human antiviral. PMID:27556481

  5. A small interfering RNA targeting Lnk accelerates bone fracture healing with early neovascularization.

    PubMed

    Kawakami, Yohei; Ii, Masaaki; Matsumoto, Tomoyuki; Kawamoto, Atsuhiko; Kuroda, Ryosuke; Akimaru, Hiroshi; Mifune, Yutaka; Shoji, Taro; Fukui, Tomoaki; Asahi, Michio; Kurosaka, Masahiro; Asahara, Takayuki

    2013-09-01

    Lnk, an intracellular adapter protein, is expressed in hematopoietic cell lineages, which has recently been proved as an essential inhibitory signaling molecule for stem cell self-renewal in the stem cell factor-c-Kit signaling pathway with enhanced hematopoietic and osteogenic reconstitution in Lnk-deficient mice. Moreover, the therapeutic potential of hematopoietic stem/endothelial progenitor cells (EPCs) for fracture healing has been demonstrated with mechanistic insight into vasculogenesis/angiogenesis and osteogenesis enhancement in the fracture sites. We report here, Lnk siRNA-transfected endothelial commitment of c-kit+/Sca-1+/lineage- subpopulations of bone marrow cells have high EPC colony-forming capacity exhibiting endothelial markers, VE-Cad, VEGF and Ang-1. Lnk siRNA-transfected osteoblasts also show highly osteoblastic capacity. In vivo, locally transfected Lnk siRNA could successfully downregulate the expression of Lnk at the fracture site up to 1 week, and radiological and histological examination showed extremely accelerated fracture healing in Lnk siRNA-transfected mice. Moreover, Lnk siRNA-transfected mice exhibited sufficient therapeutic outcomes with intrinstic enhancement of angiogenesis and osteogenesis, specifically, the mice demonstrated better blood flow recovery in the sites of fracture. In our series of experiments, we clarified that a negatively regulated Lnk system contributed to a favorable circumstance for fracture healing by enhancing vasculogenesis/angiogenesis and osteogenesis. These findings suggest that downregulation of Lnk system may have the clinical potential for faster fracture healing, which contributes to the reduction of delayed unions or non-unions. PMID:23897412

  6. Efficient delivery of small interfering RNA into injured spinal cords in rats by photomechanical waves

    NASA Astrophysics Data System (ADS)

    Ando, Takahiro; Sato, Shunichi; Toyooka, Terushige; Kobayashi, Hiroaki; Nawashiro, Hiroshi; Ashida, Hiroshi; Obara, Minoru

    2011-03-01

    In the central nervous system, lack of axonal regeneration leads to permanent functional disabilities. In spinal cord injury (SCI), the over-expressions of intermediate filament (IF) proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are mainly involved in glial scar formation; these proteins work as both physical and biochemical barriers to axonal regeneration. Thus, silencing of these IF proteins would be an attractive strategy to treat SCI. In this study, we first attempted to deliver fluorescent probe-labeled siRNAs into injured spinal cords in rats by applying photomechanical waves (PMWs) to examine the capability of PMWs as a tool for siRNA delivery. Intense fluorescence from siRNAs was observed in much broader regions in the spinal cords with PMW application when compared with those with siRNA injection alone. Based on this result, we delivered siRNAs for GFAP and vimentin into injured spinal tissues in rats by applying PMWs. The treatment resulted in efficient silencing of the proteins at five days after SCI and a decrease of the cavity area in the injured tissue at three weeks after SCI when compared with those with siRNA injection alone. These results demonstrate the capability of PMWs for efficient delivery of siRNAs into injured spinal cords and treatment of SCIs.

  7. Mu opioid receptor knockdown in the substantia nigra/ventral tegmental area by synthetic small interfering RNA blocks the rewarding and locomotor effects of heroin

    PubMed Central

    Zhang, Yong; Landthaler, Markus; Schlussman, Stefan D.; Yuferov, Vadim; Ho, Ann; Tuschl, Thomas; Kreek, Mary Jeanne

    2014-01-01

    Mu opioid receptors (MOP-r) play an important role in the rewarding and locomotor stimulatory effects of heroin. The aim of the current study was to determine whether infusion of small interfering RNAs (siRNA) targeting MOP-r into the midbrain could knock down MOP-r mRNA and affect heroin-induced locomotor activity or heroin-induced conditioned place preference. Ten week old male C57BL/6J mice were surgically implanted bilaterally with guide cannulae directed between the substantia nigra and ventral tegmental area. After 4 days recovery, mice were infused bilaterally with siRNAs that target the MOP-r (2mM × 0.75 μl/side/day for 3 days) or control siRNA. Seven days after the last infusion, a procedure for conditioned place preference was begun with four heroin (3mg/kg i.p.) administration sessions alternating with four saline sessions. While heroin induced an increase in locomotor activity in all groups, siRNAs targeting specific regions of MOP-r significantly attenuated this effect. Of particular interest, mice infused with specific siRNAs targeting the MOP-r failed to develop and express conditioned place preference to heroin, or showed a significantly attenuated preference. These alterations in reward related behaviors are likely due to the reduction in MOP-r mRNA and protein, shown in separate studies by in situ hybridization and autoradiography using the same MOP-r- siRNA infusions. Taken together, these studies demonstrate the utility of siRNA in the neurobiological study of specific components of the reward system and should contribute to the study of other complex behaviors. PMID:18938225

  8. Ribozyme-enhanced single-stranded Ago2-processed interfering RNA triggers efficient gene silencing with fewer off-target effects

    PubMed Central

    Shang, Renfu; Zhang, Fengjuan; Xu, Beiying; Xi, Hairui; Zhang, Xue; Wang, Weihua; Wu, Ligang

    2015-01-01

    Short-hairpin RNAs (shRNAs) are widely used to produce small-interfering RNAs (siRNAs) for gene silencing. Here we design an alternative siRNA precursor, named single-stranded, Argonaute 2 (Ago2)-processed interfering RNA (saiRNA), containing a 16–18 bp stem and a loop complementary to the target transcript. The introduction of a self-cleaving ribozyme derived from hepatitis delta virus to the 3′ end of the transcribed saiRNA dramatically improves its silencing activity by generating a short 3′ overhang that facilitates the efficient binding of saiRNA to Ago2. The same ribozyme also enhances the activity of Dicer-dependent shRNAs. Unlike a classical shRNA, the strand-specific cleavage of saiRNA by Ago2 during processing eliminates the passenger strand and prevents the association of siRNA with non-nucleolytic Ago proteins. As a result, off-target effects are reduced. In addition, saiRNA exhibits less competition with the biogenesis of endogenous miRNAs. Therefore, ribozyme-enhanced saiRNA provides a reliable tool for RNA interference applications. PMID:26455506

  9. Novel epithelial cell adhesion molecule antibody conjugated polyethyleneimine-capped gold nanoparticles for enhanced and targeted small interfering RNA delivery to retinoblastoma cells

    PubMed Central

    Mitra, Moutushy; Kandalam, Mallikarjuna; Rangasamy, Judith; Shankar, Balaji; Maheswari, Uma K.; Swaminathan, Sethuraman

    2013-01-01

    Background Several nanoconjugates have been designed to deliver nucleic acids such as small interfering RNA (siRNA) and DNA to cells to study silencing and expression efficacies. In the present study, we prepared novel epithelial cell adhesion molecule (EpCAM) monoclonal antibody conjugated polyethyleneimine (PEI) capped gold nanoparticles (AuNPs) loaded with EpCAM-specific siRNA molecules to knock-down the EpCAM gene in retinoblastoma (RB) cells. We chose EpCAM as a target moiety to deliver siRNA because this molecule is highly expressed in various epithelial cancers and is an ideal target as it is highly expressed in the apical surface of tumor cells while showing basolateral expression in normal cells. Methods The EpCAM antibody was conjugated to AuNP-PEI loaded with siRNA molecules to specifically deliver siRNA to EpCAM-expressing RB cells. Conjugation efficiencies were confirmed with ultraviolet-visible spectrophotometry, Fourier transform infrared spectroscopy, and agarose and SDS–polyacrylamide gel electrophoresis. The size and zeta potential were measured using a Zeta sizer analyzer. Nanoparticle internalization and uptake were studied using fluorescent microscopy and flow cytometry. Gene silencing efficacy was monitored with western blot analysis and real-time quantitative PCR. Results Optimal size and neutral zeta potential properties of the AuNP-PEI- EpCAM antibody (EpAb) antibody were achieved for the transfection studies. The AuNP-PEI nanoparticles did not show any cytotoxicity to the cells, which means these nanomaterials are suitable for intracellular delivery of siRNA for therapeutic interventions. With EpCAM antibody conjugation, PEI-capped AuNPs loaded with EpCAM siRNA were significantly internalized in the Y79 cells as observed with fluorescence microscopy and flow cytometry and induced a highly significant reduction in the cell viability of the Y79 cells. Through increased binding of EpCAM antibody–conjugated AuNP-PEI nanoparticles

  10. Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification.

    PubMed

    Lippman, Zachary; May, Bruce; Yordan, Cristy; Singer, Tatjana; Martienssen, Rob

    2003-12-01

    Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation. PMID:14691539

  11. A Short Open Reading Frame Encompassing the MicroRNA173 Target Site Plays a Role in trans-Acting Small Interfering RNA Biogenesis.

    PubMed

    Yoshikawa, Manabu; Iki, Taichiro; Numa, Hisataka; Miyashita, Kyoko; Meshi, Tetsuo; Ishikawa, Masayuki

    2016-05-01

    trans-Acting small interfering RNAs (tasiRNAs) participate in the regulation of organ morphogenesis and determination of developmental timing in plants by down-regulating target genes through mRNA cleavage. The production of tasiRNAs is triggered by microRNA173 (miR173) and other specific microRNA-mediated cleavage of 5'-capped and 3'-polyadenylated primary TAS transcripts (pri-TASs). Although pri-TASs are not thought to encode functional proteins, they contain multiple short open reading frames (ORFs). For example, the primary TAS2 transcript (pri-TAS2) contains 11 short ORFs, and the third ORF from the 5' terminus (ORF3) encompasses the miR173 target site. Here, we show that nonsense mutations in ORF3 of pri-TAS2 upstream of the miR173 recognition site suppress tasiRNA accumulation and that ORF3 is translated in vitro. Glycerol gradient centrifugation analysis of Arabidopsis (Arabidopsis thaliana) plant extracts revealed that pri-TAS2 and its miR173-cleaved 5' and 3' fragments are fractionated together in the polysome fractions. These and previous results suggest that the 3' fragment of pri-TAS2, which is a source of tasiRNAs, forms a huge complex containing SGS3, miR173-programmed AGO1 RNA-induced silencing complex, the 5' fragment, and ribosomes. This complex overaccumulated, moderately accumulated, and did not accumulate in rdr6, sde5, and sgs3 mutants, respectively. The sgs3 sde5 and rdr6 sde5 double mutants showed phenotypes similar to those of sgs3 and sde5 single mutants, respectively, with regard to the TAS2-related RNA accumulation, suggesting that the complex is formed in an SGS3-dependent manner, somehow modified and stabilized by SDE5, and becomes competent for RDR6 action. Ribosomes in this complex likely play an important role in this process. PMID:26966170

  12. SINGLE-WALLED CARBON NANOTUBE–MEDIATED SMALL INTERFERING RNA DELIVERY AND SILENCING GASTRIN-RELEASING PEPTIDE RECEPTOR IN HUMAN NEUROBLASTOMA CELLS

    PubMed Central

    Qiao, Jingbo; Hong, Tu; Guo, Honglian; Xu, Ya-Qiong; Chung, Dai H.

    2015-01-01

    Small interfering RNA (siRNA) has the potential to influence expression with a high degree of target gene specificity. However, the clinical application of siRNA therapeutics has proven to be less promising as evidenced by poor intracellular uptake, instability in vivo, and non-specific immune stimulations. Recently, we have demonstrated that single-walled carbon nanotube (SWNT)-mediated siRNA delivery can enhance the efficiency of siRNA-mediated gastrin-releasing peptide receptor (GRP-R) gene silencing by stabilizing siRNA while selectively targeting tumor tissues. Based on our recent findings, we introduce a novel technique to silence specific gene(s) in human neuroblastoma through SWNT-mediated siRNA delivery in vitro and in vivo. PMID:23749575

  13. A genome-wide survey of small interfering RNA and micro RNA pathway genes in a galling insect

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Mayetiola destructor (Say), Hessian fly, is a significant pest of wheat in most production regions worldwide. Deployment of resistance (R) genes is the most effective control for this pest; however, deployment of R genes results in an increased frequency of pest genotypes that display virulence to t...

  14. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry1[OPEN

    PubMed Central

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C.; Liu, Zhongchi

    2015-01-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. PMID:26143249

  15. Novel and Recently Evolved MicroRNA Clusters Regulate Expansive F-BOX Gene Networks through Phased Small Interfering RNAs in Wild Diploid Strawberry.

    PubMed

    Xia, Rui; Ye, Songqing; Liu, Zongrang; Meyers, Blake C; Liu, Zhongchi

    2015-09-01

    The wild strawberry (Fragaria vesca) has recently emerged as an excellent model for cultivated strawberry (Fragaria × ananassa) as well as other Rosaceae fruit crops due to its short seed-to-fruit cycle, diploidy, and sequenced genome. Deep sequencing and parallel analysis of RNA ends were used to identify F. vesca microRNAs (miRNAs) and their target genes, respectively. Thirty-eight novel and 31 known miRNAs were identified. Many known miRNAs targeted not only conserved mRNA targets but also developed new target genes in F. vesca. Significantly, two new clusters of miRNAs were found to collectively target 94 F-BOX (FBX) genes. One of the miRNAs in the new cluster is 22 nucleotides and triggers phased small interfering RNA production from six FBX genes, which amplifies the silencing to additional FBX genes. Comparative genomics revealed that the main novel miRNA cluster evolved from duplications of FBX genes. Finally, conserved trans-acting siRNA pathways were characterized and confirmed with distinct features. Our work identified novel miRNA-FBX networks in F. vesca and shed light on the evolution of miRNAs/phased small interfering RNA networks that regulate large gene families in higher plants. PMID:26143249

  16. A small interfering RNA targeting vascular endothelial growth factor efficiently inhibits growth of VX2 cells and VX2 tumor model of hepatocellular carcinoma in rabbit by transarterial embolization-mediated siRNA delivery

    PubMed Central

    Zou, Yu; Guo, Chuan-Gen; Yang, Zheng-Gang; Sun, Jun-Hui; Zhang, Min-Ming; Fu, Cai-Yun

    2016-01-01

    Introduction Hepatocellular carcinoma is currently the second leading cause of cancer-related deaths worldwide with an increasing incidence. Objective The objective of this study is to investigate the effect of vascular endothelial growth factor small interfering RNA (VEGF-siRNA) on rabbit VX2 carcinoma cell viability in vitro and the effect of transarterial embolization (TAE)-mediated VEGF-siRNA delivery on the growth of rabbit VX2 liver-transplanted model in vivo. Methods Quantitative reverse transcription polymerase chain reaction, enzyme-linked immunosorbent assay, and Western blot technologies were used to detect the expression level of VEGF. TAE and computed tomography scan were used to deliver the VEGF-siRNA and detect the tumor volume in vivo, respectively. Microvessel density was detected by immunohistochemistry with CD34 antibody. A biochemical autoanalyzer was used to evaluate the hepatic and renal toxicity. Results The designed VEGF-siRNAs could effectively decrease the expression levels of VEGF mRNA and protein in vitro and in vivo. In vitro, the viability of rabbit VX2 carcinoma cells was reduced by 38.5%±7.3% (VEGF-siRNA no 1) and 30.0%±5.8% (VEGF-siRNA no 3) at 48 hours after transfection. Moreover, in rabbit VX2 liver-transplanted model, the growth ratios of tumors at 28 days after TAE-mediated siRNA delivery were 155.18%±19.42% in the control group, 79.67%±19.63% in the low-dose group, and 36.09%±15.73% in the high-dose group, with significant differences among these three groups. Microvessel density dropped to 34.22±4.01 and 22.63±4.07 in the low-dose group and high-dose group, respectively, compared with the control group (57.88±5.67), with significant differences among these three groups. Furthermore, inoculation of VX2 tumor into the liver itself at later stage induced significant increase in alanine aminotransferase and aspartate aminotransferase, indicating an obvious damage of liver functions, while treatment of VX2 tumor via TAE

  17. Insights into the therapeutic potential of hypoxia-inducible factor-1α small interfering RNA in malignant melanoma delivered via folate-decorated cationic liposomes

    PubMed Central

    Chen, Zhongjian; Zhang, Tianpeng; Wu, Baojian; Zhang, Xingwang

    2016-01-01

    Malignant melanoma (MM) represents the most dangerous form of skin cancer, and its incidence is expected to rise in the coming time. However, therapy for MM is limited by low topical drug concentration and multidrug resistance. This article aimed to develop folate-decorated cationic liposomes (fc-LPs) for hypoxia-inducible factor-1α (HIF-1α) small interfering (siRNA) delivery, and to evaluate the potential of such siRNA/liposome complexes in MM therapy. HIF-1α siRNA-loaded fc-LPs (siRNA-fc-LPs) were prepared by a film hydration method followed by siRNA incubation. Folate decoration of liposomes was achieved by incorporation of folate/oleic acid-diacylated oligochitosans. The resulting siRNA-fc-LPs were 95.3 nm in size with a ζ potential of 2.41 mV. The liposomal vectors exhibited excellent loading capacity and protective effect toward siRNA. The in vitro cell transfection efficiency was almost parallel to the commercially available Lipofectamine™ 2000. Moreover, the anti-melanoma activity of HIF-1α siRNA was significantly enhanced through fc-LPs. Western blot analysis and apoptosis test demonstrated that siRNA-fc-LPs substantially reduced the production of HIF-1α-associated protein and induced the apoptosis of hypoxia-tolerant melanoma cells. Our designed liposomal vectors might be applicable as siRNA delivery vehicle to systemically or topically treat MM. PMID:27042054

  18. [Corrigendum] Transient transfection of macrophage migration inhibitory factor small interfering RNA disrupts the biological behavior of oral squamous carcinoma cells.

    PubMed

    Zeng, Jie; Quan, Jingjing; Xia, Xuefeng

    2016-07-01

    Due to an inability to contact various of the contributors to this study at the time of submission and a desire to publish this work, the published article did not include the full complement of authors who should have been credited on the paper. All of the existing authors have agreed that the following authors, whose names were omitted, should also have been included as co-authors: Professor Jin Gao (now at the School of Dentistry and Oral Health, Griffith University, Queensland, Australia), who was involved with project design and revisions of the manuscript; Dr Shuyu Luo (now at the School and Hospital of Stomatology, Tianjin Medical University, Tianjin, China), who was involved in project development, data collection (Figs 3 and 6) and manuscript writing; and Dr Jianming Zhang (now at the Department of Stomatology, General Hospital of Tianjin Medical University, Tianjin, China), who was involved in project development, data collection and analysis (Fig. 4) The full author list for this paper is presented below, showing the corrected order of the authors as they should have appeared on the paper. We regret the omission of the three aforementioned authors on the published article. Note that Professor Jin Gao should be considered as the co-corresponding author (with Xuefeng Xia), and Jie Zeng and Shuyu Luo contributed equally to this study. [the original article was published in the Molecular Medicine Reports 13: 174‑180, 2015; DOI: 10.3892/mmr.2015.4525] Transient transfection of macrophage migration inhibitory factor small interfering RNA disrupts the biological behavior of oral squamous carcinoma cells Jie Zeng1*, Shuyu Luo2*, Jianming Zhang3, Jingjing Quan4, Xuefeng Xia1 and Jin Gao5 1The Third Affiliated Hospital, Guangzhou Medical University, Guangzhou, Guangdong 510150; 2School and Hospital of Stomatology, Tianjin Medical University, Tianjin 300070, 3Department of Stomatology, General Hospital of Tianjin Medical University, Tianjin 300052; 4Guanghua

  19. Short interfering RNA against STAT1 attenuates cisplatin-induced ototoxicity in the rat by suppressing inflammation

    PubMed Central

    Kaur, T; Mukherjea, D; Sheehan, K; Jajoo, S; Rybak, L P; Ramkumar, V

    2011-01-01

    Cisplatin is widely used for treating various solid tumors. However, this drug produces dose-limiting ototoxicity and nephrotoxicity, which significantly reduce the quality of life of cancer patients. While nephrotoxicity could be alleviated by diuresis, there is currently no approved treatment for hearing loss. Previous studies show that the ROS and inflammation are major contributors to cisplatin-induced hearing loss. In this study, we show that ROS trigger the inflammatory process in the cochlea by activating signal transducer and activator of transcription-1 (STAT1). Activation of STAT1 activation was dependent on ROS generation through NOX3 NADPH oxidase, knockdown of which by siRNA reduced STAT1 activation. Moreover, STAT1 siRNA protected against activation of p53, reduced apoptosis, reduced damage to OHCs and preserved hearing in rats. STAT1 siRNA attenuated the increase in inflammatory mediators, such as TNF-α, inhibition of which protected cells from cisplatin-mediated apoptosis. Finally, we showed that trans-tympanic administration of etanercept, a TNF-α antagonist, protected against OHC damage and cisplatin-induced hearing loss. These studies suggest that controlling inflammation by inhibition of STAT1-dependent pathways in the cochlea could serve as an effective approach to treat cisplatin ototoxicity and improve the overall quality of life for cancer patients. PMID:21776018

  20. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis

    PubMed Central

    Kanazawa, Takanori; Shizawa, Yuki; Takeuchi, Mayu; Tamano, Kuniko; Ibaraki, Hisako; Seta, Yasuo; Takashima, Yuuki; Okada, Hiroaki

    2015-01-01

    The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. PMID:26371030

  1. Topical Anti-Nuclear Factor-Kappa B Small Interfering RNA with Functional Peptides Containing Sericin-Based Hydrogel for Atopic Dermatitis.

    PubMed

    Kanazawa, Takanori; Shizawa, Yuki; Takeuchi, Mayu; Tamano, Kuniko; Ibaraki, Hisako; Seta, Yasuo; Takashima, Yuki; Okada, Hiroaki

    2015-01-01

    The small interfering RNA (siRNA) is suggested to offer a novel means of treating atopic dermatitis (AD) because it allows the specific silencing of genes related to AD pathogenesis. In our previous study, we found that siRNA targeted against RelA, an important nuclear factor-kappa B (NF-κB) subdomain, with functional peptides, showed therapeutic effects in a mouse model of AD. In the present study, to develop a topical skin application against AD, we prepared a hydrogel containing anti-RelA siRNA and functional peptides and determined the intradermal permeation and the anti-AD effects in an AD mouse model. We selected the silk protein, sericin (SC), which is a versatile biocompatible biomaterial to prepare hydrogel as an aqueous gel base. We found that the siRNA was more widely delivered to the site of application in AD-induced ear skin of mice after topical application via the hydrogel containing functional peptides than via the preparation without functional peptides. In addition, the ear thickness and clinical skin severity of the AD-induced mice treated with hydrogel containing anti-RelA siRNA with functional peptides improved more than that of mice treated with the preparation formulated with negative siRNA. PMID:26371030

  2. PACT- and RIG-I-Dependent Activation of Type I Interferon Production by a Defective Interfering RNA Derived from Measles Virus Vaccine

    PubMed Central

    Ho, Ting-Hin; Kew, Chun; Lui, Pak-Yin; Chan, Chi-Ping; Satoh, Takashi; Akira, Shizuo

    2015-01-01

    ABSTRACT The live attenuated measles virus vaccine is highly immunostimulatory. Identification and characterization of its components that activate the innate immune response might provide new strategies and agents for the rational design and development of chemically defined adjuvants. In this study, we report on the activation of type I interferon (IFN) production by a defective interfering (DI) RNA isolated from the Hu-191 vaccine strain of measles virus. We found that the Hu-191 virus induced IFN-β much more potently than the Edmonston strain. In the search for IFN-inducing species in Hu-191, we identified a DI RNA specifically expressed by this strain. This DI RNA, which was of the copy-back type, was predicted to fold into a hairpin structure with a long double-stranded stem region of 206 bp, and it potently induced the expression of IFN-β. Its IFN-β-inducing activity was further enhanced when both cytoplasmic RNA sensor RIG-I and its partner, PACT, were overexpressed. On the contrary, this activity was abrogated in cells deficient in PACT or RIG-I. The DI RNA was found to be associated with PACT in infected cells. In addition, both the 5′-di/triphosphate end and the double-stranded stem region on the DI RNA were essential for its activation of PACT and RIG-I. Taken together, our findings support a model in which a viral DI RNA is sensed by PACT and RIG-I to initiate an innate antiviral response. Our work might also provide a foundation for identifying physiological PACT ligands and developing novel adjuvants or antivirals. IMPORTANCE The live attenuated measles virus vaccine is one of the most successful human vaccines and has largely contained the devastating impact of a highly contagious virus. Identifying the components in this vaccine that stimulate the host immune response and understanding their mechanism of action might help to design and develop better adjuvants, vaccines, antivirals, and immunotherapeutic agents. We identified and characterized

  3. Controlled Delivery of T-box21 Small Interfering RNA Ameliorates Autoimmune Alopecia (Alopecia Areata) in a C3H/HeJ Mouse Model

    PubMed Central

    Nakamura, Motonobu; Jo, Jun-ichiro; Tabata, Yasuhiko; Ishikawa, Osamu

    2008-01-01

    Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-γ antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-γ antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-γ mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin–small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease. PMID:18245811

  4. Controlled delivery of T-box21 small interfering RNA ameliorates autoimmune alopecia (Alopecia Areata) in a C3H/HeJ mouse model.

    PubMed

    Nakamura, Motonobu; Jo, Jun-ichiro; Tabata, Yasuhiko; Ishikawa, Osamu

    2008-03-01

    Autoimmune alopecia (alopecia areata) is considered to be triggered by a collapse of immune privilege in hair follicles. Here we confirmed that infiltrating CD4 T lymphocytes around hair follicles of patients with alopecia areata were primarily CCR5-positive with few CCR4-positive cells, suggesting a dominant role of Th1 cells in the alopecic lesion. Given this finding, we sought to elucidate the effect of cytokine therapy in C3H/HeJ mice, a mouse model of alopecia areata, by applying recombinant interleukin-4 and neutralizing anti-interferon-gamma antibody. We found that local injections of both interleukin-4 and neutralizing anti-interferon-gamma antibody effectively treated alopecia in C3H/HeJ mice. Results from immunohistochemistry and semiquantitative reverse transcription-polymerase chain reaction demonstrated that intralesional injection of interleukin-4 suppressed CD8 T cell infiltrates around the hair follicles and repressed enhanced interferon-gamma mRNA expression in the affected alopecic skin. Furthermore, Th1 transcription factor T-box21 small interfering RNAs conjugated to cationized gelatin showed mitigating effects on alopecia in C3H/HeJ mice, resulting in the restoration of hair shaft elongation. Taken together, the use of gelatin-small interfering RNA conjugates promises to be a novel, efficient, and safe tool as an alternative gene therapy for the treatment of various human diseases. To our knowledge, this is the first report of effective controlled delivery of small interfering RNA using biodegradable cationized gelatin microspheres in an animal model of disease. PMID:18245811

  5. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot.

    PubMed

    Moon, Jae-Su; Lee, Seung-Hoon; Kim, Eun-Jung; Cho, Hee; Lee, Wooseong; Kim, Geon-Woo; Park, Hyun-Ji; Cho, Seung-Woo; Lee, Choongho; Oh, Jong-Won

    2016-01-01

    The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277-343. Based on their antiviral activity, we mapped a druggable region (nts 313-343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5' or 3' direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. PMID:26751678

  6. Inhibition of Hepatitis C Virus in Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence in Viral IRES Pseudoknot

    PubMed Central

    Kim, Eun-Jung; Cho, Hee; Lee, Wooseong; Kim, Geon-Woo; Park, Hyun-Ji; Cho, Seung-Woo; Lee, Choongho; Oh, Jong-Won

    2016-01-01

    The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277–343. Based on their antiviral activity, we mapped a druggable region (nts 313–343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5′ or 3′ direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. PMID:26751678

  7. Cytoskeleton-Dependent Transport as a Potential Target for Interfering with Post-transcriptional HuR mRNA Regulons.

    PubMed

    Eberhardt, Wolfgang; Badawi, Amel; Biyanee, Abhiruchi; Pfeilschifter, Josef

    2016-01-01

    The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision protein family has a critical impact on the post-transcriptional control of AU-rich element bearing mRNA regulons implied in inflammation, senescence, and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression, and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets ("HuR mRNA regulons") encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskeleton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated post-transcriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy. PMID:27582706

  8. Cytoskeleton-Dependent Transport as a Potential Target for Interfering with Post-transcriptional HuR mRNA Regulons

    PubMed Central

    Eberhardt, Wolfgang; Badawi, Amel; Biyanee, Abhiruchi; Pfeilschifter, Josef

    2016-01-01

    The ubiquitous mRNA binding protein human antigen R (HuR), a member of the embryonal lethal abnormal vision protein family has a critical impact on the post-transcriptional control of AU-rich element bearing mRNA regulons implied in inflammation, senescence, and carcinogenesis. HuR in addition to mRNA stability can affect many other aspects of mRNA processing including splicing, polyadenylation, translation, modulation of miRNA repression, and intracellular mRNA trafficking. Since many of the identified HuR mRNA targets (“HuR mRNA regulons”) encode tumor-related proteins, HuR is not only discussed as an useful biomarker but also as promising therapeutic target for treatment of various human cancers. HuR which is most abundantly localized in the nucleus is translocated to the cytoplasm which is fundamental for most of the described HuR functions on target mRNAs. Accordingly, an elevation in cytoplasmic HuR was found in many tumors and correlated with a high grade of malignancy and a poor prognosis of patients. Therefore, direct interference with the intracellular trafficking of HuR offers an attractive approach to intervene with pathologically deregulated HuR functions. Data from several laboratories implicate that the integrity of the cytoskeleton is critical for HuR-mediated intracellular mRNA localization and translation. This review will particularly focus on drugs which have proven a direct inhibitory effect on HuR translocation. Based on the results from those studies, we will also discuss on the principle value of targeting cytoskeleton-dependent transport of HuR by natural or synthetic inhibitors as a potential therapeutic avenue for interfering with dysregulated post-transcriptional HuR mRNA regulons and related tumor cell functions. In spite of that, interfering with cytoplasmic HuR transport could highlight a so far underestimated action of microtubule inhibitors clinically used for cancer chemotherapy. PMID:27582706

  9. A novel betapartitivirus RnPV6 from Rosellinia necatrix tolerates host RNA silencing but is interfered by its defective RNAs.

    PubMed

    Chiba, Sotaro; Lin, Yu-Hsin; Kondo, Hideki; Kanematsu, Satoko; Suzuki, Nobuhiro

    2016-07-01

    The family Partitiviridae comprises of five genera with bi-segmented dsRNA genomes that accommodate members infecting plants, fungi or protists. All partitiviruses with only a few exceptions cause asymptomatic infections. We report the characterization of a novel betapartitivirus termed Rosellinia necatrix partitivirus 6 (RnPV6) from a field isolate of a plant pathogenic fungus, white root rot fungus. RnPV6 has typical partitivirus features: dsRNA1 and dsRNA2 are 2462 and 2499bps in length encoding RNA-dependent RNA polymerase and capsid protein. Purified particles are spherical with a diameter of 30nm. Taking advantage of infectivity as virions, RnPV6 was introduced into a model filamentous fungal host, chestnut blight fungus to investigate virus/host interactions. Unlike other partitiviruses tested previously, RnPV6 induced profound phenotypic alterations with symptoms characterized by a reduced growth rate and enhanced pigmentation and was tolerant to host RNA silencing. In addition, a variety of defective RNAs derived from dsRNA1 appear after virion transfection. These sub-viral RNAs were shown to interfere with RnPV6 replication, at least for that of cognate segment dsRNA1. Presence of these sub-viral elements resulted in reduced symptom expression by RnPV6, suggesting their nature as defective-interfering RNAs. The features of RnPV6 are similar to but distinct from those of a previously reported alphapartitivirus, Rosellinia necatrix partitivirus 2 that is susceptible to RNA silencing. PMID:26494168

  10. Arabidopsis RNASE THREE LIKE2 Modulates the Expression of Protein-Coding Genes via 24-Nucleotide Small Interfering RNA-Directed DNA Methylation[OPEN

    PubMed Central

    Hachet, Mélanie; Comella, Pascale; Zytnicki, Matthias; Vaucheret, Hervé

    2016-01-01

    RNaseIII enzymes catalyze the cleavage of double-stranded RNA (dsRNA) and have diverse functions in RNA maturation. Arabidopsis thaliana RNASE THREE LIKE2 (RTL2), which carries one RNaseIII and two dsRNA binding (DRB) domains, is a unique Arabidopsis RNaseIII enzyme resembling the budding yeast small interfering RNA (siRNA)-producing Dcr1 enzyme. Here, we show that RTL2 modulates the production of a subset of small RNAs and that this activity depends on both its RNaseIII and DRB domains. However, the mode of action of RTL2 differs from that of Dcr1. Whereas Dcr1 directly cleaves dsRNAs into 23-nucleotide siRNAs, RTL2 likely cleaves dsRNAs into longer molecules, which are subsequently processed into small RNAs by the DICER-LIKE enzymes. Depending on the dsRNA considered, RTL2-mediated maturation either improves (RTL2-dependent loci) or reduces (RTL2-sensitive loci) the production of small RNAs. Because the vast majority of RTL2-regulated loci correspond to transposons and intergenic regions producing 24-nucleotide siRNAs that guide DNA methylation, RTL2 depletion modifies DNA methylation in these regions. Nevertheless, 13% of RTL2-regulated loci correspond to protein-coding genes. We show that changes in 24-nucleotide siRNA levels also affect DNA methylation levels at such loci and inversely correlate with mRNA steady state levels, thus implicating RTL2 in the regulation of protein-coding gene expression. PMID:26764378

  11. Silencing tissue inhibitors of metalloproteinases (TIMPs) with short interfering RNA reveals a role for TIMP-1 in hepatic stellate cell proliferation

    SciTech Connect

    Fowell, Andrew J.; Collins, Jane E.; Duncombe, Dale R.; Pickering, Judith A.; Rosenberg, William M.C.; Benyon, R. Christopher

    2011-04-08

    Research highlights: {yields} Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis. {yields} We used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. {yields} Specific silencing of TIMP-1, but not TIMP-2, significantly reduces HSC proliferation and is associated with reduced Akt phosphorylation. {yields} TIMP-1 is localised in part to the HSC nucleus. {yields} TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. -- Abstract: Myofibroblastic, activated hepatic stellate cells (HSC) play a pivotal role in the development of liver fibrosis through the secretion of fibrillar collagens and the tissue inhibitors of metalloproteinase (TIMP)-1 and -2. TIMPs are believed to promote hepatic fibrosis by inhibiting both matrix degradation and apoptosis of HSC. In other cell types, there is evidence that TIMP-1 has effects on proliferation, however the role of TIMPs in the regulation of HSC proliferation remains unexplored. Therefore, we have used short interfering RNA (siRNA) to investigate the effects of autocrine TIMP-1 and -2 on HSC proliferation. TIMP-1 and -2 siRNA were highly effective, producing peak target protein knockdown compared to negative control siRNA of 92% and 63%, respectively. Specific silencing of TIMP-1, using siRNA, significantly reduced HSC proliferation. TIMP-1 was localised in part to the HSC nucleus and TIMP-1 siRNA resulted in loss of both cytoplasmic and nuclear TIMP-1. Attenuated proliferation was associated with reduced Akt phosphorylation and was partially rescued by addition of recombinant TIMP-1. We have revealed a novel autocrine mitogenic effect of TIMP-1 on HSC, which may involve Akt-dependent and specific nuclear mechanisms of action. We suggest that TIMP-1 might promote liver fibrosis by means other than its previously described anti-apoptotic effect on HSC. Moreover

  12. Endogenous MCM7 MicroRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

    PubMed Central

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L.; DiGiusto, David L.

    2012-01-01

    Abstract Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications. PMID:22834872

  13. Upregulation of microRNA-22 contributes to myocardial ischemia-reperfusion injury by interfering with the mitochondrial function.

    PubMed

    Du, Jian-Kui; Cong, Bin-Hai; Yu, Qing; Wang, He; Wang, Long; Wang, Chang-Nan; Tang, Xiao-Lu; Lu, Jian-Qiang; Zhu, Xiao-Yan; Ni, Xin

    2016-07-01

    Mitochondrial oxidative damage is critically involved in cardiac ischemia reperfusion (I/R) injury. MicroRNA-22 (miR-22) has been predicted to potentially target sirtuin-1 (Sirt1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), both of which are known to provide protection against mitochondrial oxidative injury. The present study aims to investigate whether miR-22 is involved in the regulation of cardiac I/R injury by regulation of mitochondrial function. We found that miR-22 level was significantly increased in rat hearts subjected to I/R injury, as compared with the sham group. Intra-myocardial injection of 20 ug miR-22 inhibitor reduced I/R injury as evidenced by significant decreases in cardiac infarct size, serum lactate dehydrogenase (LDH) and creatine kinase (CK) levels and the number of apoptotic cardiomyocytes. H9c2 cardiomyocytes exposed to hypoxia/reoxygenation (H/R) insult exhibited an increase in miR-22 expression, which was blocked by reactive oxygen species (ROS) scavenger and p53 inhibitor. In addition, miR-22 inhibitor attenuated, whereas miR-22 mimic aggravated H/R-induced injury in H9c2 cardiomyocytes. MiR-22 inhibitor per se had no significant effect on cardiac mitochondrial function. Mitochondria from rat receiving miR-22 inhibitor 48h before ischemia were found to have a significantly less mitochondrial superoxide production and greater mitochondrial membrane potential and ATP production as compared with rat receiving miR control. In H9c2 cardiomyocyte, it was found that miR-22 mimic aggravated, whilst miR-22 inhibitor significantly attenuated H/R-induced mitochondrial damage. By using real time PCR, western blot and dual-luciferase reporter gene analyses, we identified Sirt1 and PGC1α as miR-22 targets in cardiomyocytes. It was found that silencing of Sirt1 abolished the protective effect of miR-22 inhibitor against H/R-induced mitochondrial dysfunction and cell injury in cardiomyocytes. Taken together, our

  14. Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA.

    PubMed

    Wada, Shun-Ichi; Iwata, Masashi; Ozaki, Yuka; Ozaki, Takashi; Hayashi, Junsuke; Urata, Hidehito

    2016-09-15

    To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to αVβ3 integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the αVβ3 integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0μM PI/10nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA. PMID:27480031

  15. In vivo study of Dicer-2-mediated immune response of the small interfering RNA pathway upon systemic infections of virulent and avirulent viruses in Bombus terrestris.

    PubMed

    Niu, Jinzhi; Smagghe, Guy; De Coninck, Dieter I M; Van Nieuwerburgh, Filip; Deforce, Dieter; Meeus, Ivan

    2016-03-01

    Recent studies suggest a potent role of the small interfering RNA (siRNA) pathway in the control of bee viruses and its usefulness to tackle these viral diseases. However, the involvement of the siRNA pathway in the defense against different bee viruses is still poorly understood. Therefore, in this report, we comprehensively analyzed the response of the siRNA pathway in bumblebees of Bombus terrestris to systemic infections of the virulent Israeli acute paralysis virus (IAPV) and the avirulent slow bee paralysis virus (SBPV). Our results showed that IAPV and SBPV infections induced the expression of Dicer-2. IAPV infections also triggered the production of predominantly 22 nt-long virus-derived siRNAs (vsiRNAs). Intriguingly, these 22 nt-long vsiRNAs showed a high proportion of antigenomic IAPV sequences. Conversely, these predominantly 22 nt-long vsiRNAs of SBPV were not detected in SBPV infected bees. Furthermore, an "RNAi-of-RNAi" experiment on Dicer-2 did not result in altered genome copy numbers of IAPV (n = 17-18) and also not of SBPV (n = 11-12). Based on these results, we can speculate about the importance of the siRNA pathway in bumblebees for the antiviral response. During infection of IAPV, this pathway is probably recruited but it might be insufficient to control viral infection in our experimental setup. The host can control SBPV infection, but aside from the induction of Dicer-2 expression, no further evidence of the antiviral activity of the siRNA pathway was observed. This report may also enhance the current understanding of the siRNA pathway in the innate immunity of non-model insects upon different viral infections. PMID:26711439

  16. Inhibition of Dengue Virus Infections in Cell Cultures and in AG129 Mice by a Small Interfering RNA Targeting a Highly Conserved Sequence ▿

    PubMed Central

    Stein, David A.; Perry, Stuart T.; Buck, Michael D.; Oehmen, Christopher S.; Fischer, Matthew A.; Poore, Elizabeth; Smith, Jessica L.; Lancaster, Alissa M.; Hirsch, Alec J.; Slifka, Mark K.; Nelson, Jay A.; Shresta, Sujan; Früh, Klaus

    2011-01-01

    The dengue viruses (DENVs) exist as numerous genetic strains that are grouped into four antigenically distinct serotypes. DENV strains from each serotype can cause severe disease and threaten public health in tropical and subtropical regions worldwide. No licensed antiviral agent to treat DENV infections is currently available, and there is an acute need for the development of novel therapeutics. We found that a synthetic small interfering RNA (siRNA) (DC-3) targeting the highly conserved 5′ cyclization sequence (5′CS) region of the DENV genome reduced, by more than 100-fold, the titers of representative strains from each DENV serotype in vitro. To determine if DC-3 siRNA could inhibit DENV in vivo, an “in vivo-ready” version of DC-3 was synthesized and tested against DENV-2 by using a mouse model of antibody-dependent enhancement of infection (ADE)-induced disease. Compared with the rapid weight loss and 5-day average survival time of the control groups, mice receiving the DC-3 siRNA had an average survival time of 15 days and showed little weight loss for approximately 12 days. DC-3-treated mice also contained significantly less virus than control groups in several tissues at various time points postinfection. These results suggest that exogenously introduced siRNA combined with the endogenous RNA interference processing machinery has the capacity to prevent severe dengue disease. Overall, the data indicate that DC-3 siRNA represents a useful research reagent and has potential as a novel approach to therapeutic intervention against the genetically diverse dengue viruses. PMID:21795337

  17. Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

    PubMed

    Takahashi, Yuki; Nishikawa, Makiya; Kobayashi, Naoki; Takakura, Yoshinobu

    2005-07-20

    Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells. PMID:15936841

  18. Effects of brain IKKβ gene silencing by small interfering RNA on P-glycoprotein expression and brain damage in the rat kainic acid-induced seizure model.

    PubMed

    Yu, Nian; Liu, Hao; Zhang, Yan-Fang; Su, Ling-Ying; Liu, Xin-Hong; Li, Le-Chao; Hao, Jin-Bo; Huang, Xian-Jing; Di, Qing

    2014-01-01

    Multidrug resistance mediated by over-expression of P-glycoprotein (P-gp) in brain is an important mechanism accounting for the drug-therapy failure in epilepsy. Over-expression of P-gp in epilepsy rat brain may be regulated by inflammation and nuclear factor-kappa B (NF-κB) activation. Inhibitory κ B kinase subunit β (IKKβ) is an up-stream molecular controlling NF-κB activation. With the small interfering RNA (siRNA) technique and kainic acid (KA)-induced rat epileptic seizure model, the present study was aimed to further evaluate the role of NF-κB inhibition, via blocking IKKβ gene transcription, in the epileptic brain P-gp over-expression, seizure susceptibility, and post-seizure brain damage. siRNA targeting IKKβ was administered to rats via intracerebroventricular injection before seizure induction by KA microinjection; scrambled siRNA was used as control. Brain mRNA and protein levels of IKKβ and P-gp were detected by RT-PCR and immunohistochemistry. NF-κB activity was measured by electrophoretic mobility shift assay. Latency to grade III or V seizure onset was recorded, brain damage was evaluated by neuronal cell counting and epileptiform activity was monitored by electroencephalography. IKKβ siRNA pre-treatment inhibited NF-κB activation and abolished P-gp over-expression in KA-induced epileptic rat brain, accompanied by decreased seizure susceptibility. These findings suggested that epileptogenic-induced P-gp over-expression could be regulated by IKKβ through the NF-κB pathway. PMID:24040792

  19. Silencing expression of the catalytic subunit of DNA-dependent protein kinase by small interfering RNA sensitizes human cells for radiation-induced chromosome damage, cell killing, and mutation

    NASA Technical Reports Server (NTRS)

    Peng, Yuanlin; Zhang, Qinming; Nagasawa, Hatsumi; Okayasu, Ryuichi; Liber, Howard L.; Bedford, Joel S.

    2002-01-01

    Targeted gene silencing in mammalian cells by RNA interference (RNAi) using small interfering RNAs (siRNAs) was recently described by Elbashir et al. (S. M. Elbashir et al., Nature (Lond.), 411: 494-498, 2001). We have used this methodology in several human cell strains to reduce expression of the Prkdc (DNA-PKcs) gene coding for the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) that is involved in the nonhomologous end joining of DNA double-strand breaks. We have also demonstrated a radiosensitization for several phenotypic endpoints of radiation damage. In low-passage normal human fibroblasts, siRNA knock-down of DNA-PKcs resulted in a reduced capacity for restitution of radiation-induced interphase chromosome breaks as measured by premature chromosome condensation, an increased yield of acentric chromosome fragments at the first postirradiation mitosis, and an increased radiosensitivity for cell killing. For three strains of related human lymphoblasts, DNA-PKcs-targeted siRNA transfection resulted in little or no increase in radiosensitivity with respect to cell killing, a 1.5-fold decrease in induced mutant yield in TK6- and p53-null NH32 cells, but about a 2-fold increase in induced mutant yield in p53-mutant WTK1 cells at both the hypoxanthine quanine phosphoribosyl transferase (hprt) and the thymidine kinase loci.

  20. Small interfering RNA targeting ILK inhibits metastasis in human tongue cancer cells through repression of epithelial-to-mesenchymal transition

    SciTech Connect

    Xing, Yu; Qi, Jin; Deng, Shixiong; Wang, Cheng; Zhang, Luyu; Chen, Junxia

    2013-08-01

    Integrin-linked kinase (ILK) is a multifunctional serine/threonine kinase. Accumulating evidences suggest that ILK are involved in cell–matrix interactions, cell proliferation, invasion, migration, angiogenesis and Epithelial–mesenchymal transition (EMT). However, the underlying mechanisms remain largely unknown. EMT has been postulated as a prerequisite for metastasis. The reports have demonstrated that EMT was implicated in metastasis of oral squamous cell carcinomas. Therefore, here we further postulate that ILK might participate in EMT of tongue cancer. We showed that ILK siRNA inhibited EMT with low N-cadherin, Vimentin, Snail, Slug and Twist as well as high E-cadherin expression in vivo and in vitro. We found that knockdown of ILK inhibited cell proliferation, migration and invasion as well as changed cell morphology. We also demonstrated that ILK siRNA inhibited phosphorylation of downstream signaling targets Akt and GSK3β as well as reduced expression of MMP2 and MMP9. Furthermore, we found that the tongue tumor with high metastasis capability showed higher ILK, Vimentin, Snail, Slug and Twist as well as lower E-cadherin expression in clinical specimens. Finally, ILK siRNA led to the suppression for tumorigenesis and metastasis in vivo. Our findings suggest that ILK could be a novel diagnostic and therapeutic target for tongue cancer. Highlights: • ILK siRNA influences cell morphology, cell cycle, migration and invasion. • ILK siRNA affects the expression of proteins associated with EMT. • ILK expression is related to EMT in clinical human tongue tumors. • ILK siRNA inhibits metastasis of the tongue cancer cells through suppressing EMT.

  1. Self-assembled Micelle Interfering RNA for Effective and Safe Targeting of Dysregulated Genes in Pulmonary Fibrosis.

    PubMed

    Yoon, Pyoung Oh; Park, Jin Wook; Lee, Chang-Min; Kim, Sung Hwan; Kim, Han-Na; Ko, Youngho; Bae, Seon Joo; Yun, Sungil; Park, Jun Hong; Kwon, Taewoo; Kim, Woo Seok; Lee, Jiyoung; Lu, Qing; Kang, Hye-Ryun; Cho, Won-Kyung; Elias, Jack A; Yang, Joo-Sung; Park, Han-Oh; Lee, Kyuhong; Lee, Chun Geun

    2016-03-18

    The siRNA silencing approach has long been used as a method to regulate the expression of specific target genes in vitro and in vivo. However, the effectiveness of delivery and the nonspecific immune-stimulatory function of siRNA are the limiting factors for therapeutic applications of siRNAs. To overcome these limitations, we developed self-assembled micelle inhibitory RNA (SAMiRNA) nanoparticles made of individually biconjugated siRNAs with a hydrophilic polymer and lipid on their ends and characterized their stability, immune-stimulatory function, and in vivo silencing efficacy. SAMiRNAs form very stable nanoparticles with no significant degradation in size distribution and polydispersity index over 1 year. Overnight incubation of SAMiRNAs (3 μm) on murine peripheral blood mononuclear cells did not cause any significant elaboration of innate immune cytokines such as TNF-α, IL-12, or IL-6, whereas unmodified siRNAs or liposomes or liposome complexes significantly stimulated the expression of these cytokines. Last, the in vivo silencing efficacy of SAMiRNAs was evaluated by targeting amphiregulin and connective tissue growth factor in bleomycin or TGF-β transgenic animal models of pulmonary fibrosis. Intratracheal or intravenous delivery two or three times of amphiregulin or connective tissue growth factor SAMiRNAs significantly reduced the bleomycin- or TGF-β-stimulated collagen accumulation in the lung and substantially restored the lung function of TGF-β transgenic mice. This study demonstrates that SAMiRNA nanoparticle is a less toxic, stable siRNA silencing platform for efficient in vivo targeting of genes implicated in the pathogenesis of pulmonary fibrosis. PMID:26817844

  2. Silencing tumor necrosis factor-alpha in vitro from small interfering RNA-decorated titanium nanotube array can facilitate osteogenic differentiation of mesenchymal stem cells

    PubMed Central

    Wang, Zhenlin; Hu, Zhiqiang; Zhang, Dawei; Zhuo, Mengchuan; Cheng, Jiwei; Xu, Xingping; Xing, Yongming; Fan, Jie

    2016-01-01

    Titanium implants are known for their bone bonding ability. However, the osseointegration may be severely disturbed in the inflammation environment. In order to enhance osseointegration of the implant in an inflamed environment, the small interfering RNA (siRNA) targeting tumor necrosis factor alpha (TNF-α) was used to functionalize titanium surface for gene silencing. The chitosan–tripolyphosphate–hyaluronate complexes were used to formulate nanoparticles (NPs) with siRNA, which were adsorbed directly by the anodized titanium surface. The surface characterization was analyzed by scanning electron microscope, atomic force microscopy, as well as contact angle measurement. The fluorescence microscope was used to monitor the degradation of the layer. The coculture system was established with mesenchymal stem cells (MSCs) grown directly on functionalized titanium surface and RAW264.7 cells (preactivated by lipopolysaccharide) grown upside in a transwell chamber. The transfection and knockdown efficiency of TNF-α in RAW264.7 cells were determined by fluorescence microscope, quantitative polymerase chain reaction, and enzyme-linked immunosorbent assay. The cytoskeleton and osteogenic differentiation of MSCs were also analyzed. Regular vertical aligned nanotubes (~100 nm diameter and ~300 nm length) were generated after anodization of polished titanium. After loading with NPs, the nanotubes were filled and covered by a layer of amorphous particles. The surface topography changed and wettability decreased after covering with NPs. As expected, a burst degradation of the film was observed, which could provide sufficient NPs in the released supernatant and result in transfection and knockdown effects in RAW264.7 cells. The cytoskeleton arrangement of MSCs was elongated and the osteogenic differentiation was also significantly improved on NPs loading surface. In conclusion, the siRNA decorated titanium implant could simultaneously suppress inflammation and improve

  3. Genome-Wide Small Interfering RNA Screens Reveal VAMP3 as a Novel Host Factor Required for Uukuniemi Virus Late Penetration

    PubMed Central

    Meier, Roger; Franceschini, Andrea; Horvath, Peter; Tetard, Marilou; Mancini, Roberta; von Mering, Christian; Helenius, Ari

    2014-01-01

    ABSTRACT The Bunyaviridae constitute a large family of enveloped animal viruses, many of which are important emerging pathogens. How bunyaviruses enter and infect mammalian cells remains largely uncharacterized. We used two genome-wide silencing screens with distinct small interfering RNA (siRNA) libraries to investigate host proteins required during infection of human cells by the bunyavirus Uukuniemi virus (UUKV), a late-penetrating virus. Sequence analysis of the libraries revealed that many siRNAs in the screens inhibited infection by silencing not only the intended targets but additional genes in a microRNA (miRNA)-like manner. That the 7-nucleotide seed regions in the siRNAs can cause a perturbation in infection was confirmed by using synthetic miRNAs (miRs). One of the miRs tested, miR-142-3p, was shown to interfere with the intracellular trafficking of incoming viruses by regulating the v-SNARE VAMP3, a strong hit shared by both siRNA screens. Inactivation of VAMP3 by the tetanus toxin led to a block in infection. Using fluorescence-based techniques in fixed and live cells, we found that the viruses enter VAMP3+ endosomal vesicles 5 min after internalization and that colocalization was maximal 15 min thereafter. At this time, LAMP1 was associated with the VAMP3+ virus-containing endosomes. In cells depleted of VAMP3, viruses were mainly trapped in LAMP1-negative compartments. Together, our results indicated that UUKV relies on VAMP3 for penetration, providing an indication of added complexity in the trafficking of viruses through the endocytic network. IMPORTANCE Bunyaviruses represent a growing threat to humans and livestock globally. Unfortunately, relatively little is known about these emerging pathogens. We report here the first human genome-wide siRNA screens for a bunyavirus. The screens resulted in the identification of 562 host cell factors with a potential role in cell entry and virus replication. To demonstrate the robustness of our approach, we

  4. Design and validation of small interfering RNA on respiratory syncytial virus M2-2 gene: A potential approach in RNA interference on viral replication.

    PubMed

    Chin, V K; Atika Aziz, Nur A; Hudu, Shuaibu A; Harmal, Nabil S; Syahrilnizam, A; Jalilian, Farid A; Zamberi, S

    2016-10-01

    Human respiratory syncytial virus (RSV) is the leading cause of severe lower respiratory tract infection in infants and young children globally and is a significant pathogen of the elderly and immunocompromised. The M2-2 protein of respiratory syncytial virus (RSV) is particularly important in regulation of viral RNA transcription and replication that could be a potential anti-viral candidate against RSV infection. In this study, we designed and validated siRNAs that specifically target the RSV M2-2 gene. Four siRNAs targeting different regions of the M2-2 gene were designed using web tool. In-vitro evaluation of silencing effect was performed by using RSV infected Vero cell line. Viral M2-2 linked GFP recombinant plasmid was co-transfected with non-targeted siRNA, Pooled siRNA, siRNA 1, siRNA 2, siRNA 3 and siRNA 4 using synthetic cationic polymer. The silencing effect of M2-2 gene at the protein level was measured both qualitatively and quantitatively by using fluorescence microscopy and flow cytometry. Meanwhile, the silencing effect at the mRNA level was assessed by using RT-qPCR. This study showed that all four designed siRNAs can effectively and efficiently silence M2-2 gene. siRNA 2 showed the highest (98%) silencing effect on protein level and siRNA 4 with 83.1% at the mRNA level. The viral assay showed no significant cytopathic effects observed after 6days post-infection with siRNAs. In conclusion, this study showed the effectiveness of siRNA in silencing M2-2 gene both at the protein and mRNA level which could potentially be used as a novel therapeutic agent in the treatment of RSV infection. However, further study is warranted to investigate the silencing effect of M2-2 protein and inhibition of RSV infection. PMID:27432115

  5. Synergistic suppression of prostatic cancer cells by coexpression of both murine double minute 2 small interfering RNA and wild-type p53 gene in vitro and in vivo.

    PubMed

    Ji, Kun; Wang, Bo; Shao, Yue-Ting; Zhang, Ling; Liu, Ya-Nan; Shao, Chen; Li, Xiao-Jie; Li, Xin; Hu, Jia-di; Zhao, Xue-Jian; Xu, De-Qi; Li, Yang; Cai, Lu

    2011-07-01

    Our objective was to evaluate cell growth and death effects by inhibiting Murine Double Minute 2 (MDM2) expression in human prostate cancer cells overexpressing the wild-type (WT) p53 gene. Prostate PC-3 tumor cells were transfected with a plasmid containing either mdm2 small interfering (Si-mdm2) or the WT p53 gene (Pp53) alone, or both (Pmp53), using Lipofectamine in vitro and attenuated Salmonella enterica serovar Typhi vaccine strain Ty21a (Salmonella Typhi Ty21a) in vivo. Cell growth, apoptosis, and the expression of related genes and proteins were examined in vitro and in vivo by flow cytometry and Western blot assays. We demonstrated that human prostate tumors had increased expression of MDM2 and mutant p53 proteins. Transfection of the PC-3 cells with the Pmp53 plasmid in vitro offered significant inhibition of cell growth and an increase in apoptotic cell death compared with that of the Si-mdm2 or Pp53 group. These effects were associated with up-regulation of p21 and down-regulation of hypoxia-inducible factor 1α expression in Pmp53-transfected cells. To validate the in vitro findings, the nude mice implanted with PC-3 cells were treated with attenuated Salmonella Typhi Ty21a carrying the plasmids, which showed that the Pmp53 plasmid significantly inhibited the tumor growth rate in vivo compared with that of the Si-mdm2 or Pp53 plasmid alone. Tumor tissues from mice treated with the Pmp53 plasmid showed increased expression of p21 and decreased expression of hypoxia-inducible factor 1α proteins, with an increased apoptotic effect. These results suggest that knockdown of mdm2 expression by its specific small interfering RNA with overexpression of the WT p53 gene offers synergistic inhibition of prostate cancer cell growth in vitro and in vivo. PMID:21444629

  6. Shock-induced poration, cholesterol flip-flop and small interfering RNA transfection in a phospholipid membrane: Multimillion atom, microsecond molecular dynamics simulations

    NASA Astrophysics Data System (ADS)

    Choubey, Amit

    performing a 15 mus all-atom MD simulation of a DPPC-CHOL bilayer. We find that the CHOL flip-flop rates are on the sub microsecond timescale. These results are verified by performing various independent parallel replica (PR) simulations. Our PR simulations provide significant boost in sampling of the flip-flop events. We observe that the CHOL flip-flop can induce membrane order, regulate membrane-bending energy, and facilitate membrane relaxation. The rapid flip-flop rates reported here have important implications for the role of CHOL in mechanical properties of cell membranes, formation of domains, and maintaining CHOL concentration asymmetry in plasma membrane. Our PR approach can reach submillisecond time scales and bridge the gap between MD simulations and Nuclear Magnetic Resonance (NMR) experiments on CHOL flip-flop dynamics in membranes. The last project deals with transfection barriers encountered by a bare small interfering RNA (siRNA) in a phospholipid bilayer. SiRNA molecules play a pivotal role in therapeutic applications. A key limitation to the widespread implementation of siRNA-based therapeutics is the difficulty of delivering siRNA-based drugs to cells. We have examined structural and mechanical barriers to siRNA passage across a phospholipid bilayer using all-atom MD simulations. We find that the electrostatic interaction between the anionic siRNA and head groups of phospholipid molecules induces a phase transformation from the liquid crystalline to ripple phase. Steered MD simulations reveal that the siRNA transfection through the ripple phase requires a force of ˜ 1.5 nN.

  7. A Genome-wide Small Interfering RNA (siRNA) Screen Reveals Nuclear Factor-κB (NF-κB)-independent Regulators of NOD2-induced Interleukin-8 (IL-8) Secretion*

    PubMed Central

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-01-01

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  8. A genome-wide small interfering RNA (siRNA) screen reveals nuclear factor-κB (NF-κB)-independent regulators of NOD2-induced interleukin-8 (IL-8) secretion.

    PubMed

    Warner, Neil; Burberry, Aaron; Pliakas, Maria; McDonald, Christine; Núñez, Gabriel

    2014-10-10

    NOD2 encodes an intracellular multidomain pattern recognition receptor that is the strongest known genetic risk factor in the pathogenesis of Crohn disease (CD), a chronic relapsing inflammatory disorder of the intestinal tract. NOD2 functions as a sensor for bacterial cell wall components and activates proinflammatory and antimicrobial signaling pathways. Here, using a genome-wide small interfering RNA (siRNA) screen, we identify numerous genes that regulate secretion of the proinflammatory cytokine IL-8 in response to NOD2 activation. Moreover, many of the identified IL-8 regulators are linked by protein-protein interactions, revealing subnetworks of highly connected IL-8 regulators implicated in processes such as vesicle formation, mRNA stability, and protein ubiquitination and trafficking. A TNFα counterscreen to induce IL-8 secretion in an NOD2-independent manner reveals that the majority of the identified regulators affect IL-8 secretion irrespective of the initiating stimuli. Using immortalized macrophages, we validate the ubiquitin protease, USP8, and the endosomal sorting protein, VPS28, as negative regulators of NOD2-induced cytokine secretion. Interestingly, several genes that affect NOD2-induced IL-8 secretion are present in loci associated with CD risk by genome-wide association studies, supporting a role for the NOD2/IL-8 pathway, and not just NOD2, in the pathogenesis of CD. Overall, this screen provides a valuable resource in the advancement of our understanding of the genes that regulate the secretion of IL-8. PMID:25170077

  9. Exponential increases of RNA virus fitness during large population transmissions.

    PubMed Central

    Novella, I S; Duarte, E A; Elena, S F; Moya, A; Domingo, E; Holland, J J

    1995-01-01

    The great adaptability shown by RNA viruses is a consequence of their high mutation rates. Here we investigate the kinetics of virus fitness gains during repeated transfers of large virus populations in cell culture. Results always show that fitness increases exponentially. Low fitness clones exhibit regular increases observed as biphasic periods of exponential evolutionary improvement, while neutral clones show monophasic kinetics. These results are significant for RNA virus epidemiology, optimal handling of attenuated live virus vaccines, and routine laboratory procedures. PMID:7597039

  10. Epidermal growth factor increases the interaction between nucleolin and heterogeneous nuclear ribonucleoprotein K/poly(C) binding protein 1 complex to regulate the gastrin mRNA turnover.

    PubMed

    Lee, Pin-Tse; Liao, Pao-Chi; Chang, Wen-Chang; Tseng, Joseph T

    2007-12-01

    Gastrin, a gastrointestinal hormone responsible for gastric acid secretion, has been confirmed as a growth factor for gastrointestinal tract malignancies. High expression of gastrin mRNA was observed in pancreatic and colorectal cancer; however, the mechanism is unclear. Epidermal growth factor (EGF) was found to increase gastrin mRNA stability, indicating mRNA turnover regulation mechanism is involved in the control of gastrin mRNA expression. Using biotin-labeled RNA probe pull-down assay combined with mass spectrometry analysis, we identified the heterogeneous nuclear ribonucleoprotein K (hnRNP K) and poly(C) binding protein 1 (PCBP1) bound with the C-rich region in gastrin mRNA 3' untranslated region. Nucleolin bound with the AGCCCU motif and interacted with hnRNP K were also demonstrated. Under EGF treatment, we observed the amount of nucleolin interacting with hnRNP K and gastrin mRNA increased. Using small interfering RNA technology to define their functional roles, we found hnRNP K, PCBP1, and nucleolin were all responsible for stabilizing gastrin mRNA. Moreover, nucleolin plays a crucial role in mediating the increased gastrin mRNA stability induced by EGF signaling. Besides, we also observed hnRNP K/PCBP1 complex bound with the C-rich region in the gastrin mRNA increased nucleolin binding with gastrin mRNA. Finally, a novel binding model was proposed. PMID:17928403

  11. Effects of 5‑fluorouracil and class III phosphoinositide 3‑kinase small interfering RNA combination therapy on SGC7901 human gastric cancer cells.

    PubMed

    Zhu, Bao-Song; Sun, Jia-Lei; Gong, Wei; Zhang, Xing-Ding; Wu, Yong-You; Xing, Chun-Gen

    2015-03-01

    The aim of the present study was to investigate the effects of small interfering RNA‑mediated inhibition of Class III phosphoinositide 3‑kinase (PI3K) signal transduction on the proliferation, apoptosis and autophagy of SGC7901 gastric cancer cells. The present study also aimed to examine the contribution of autophagic inhibition to the antitumor effects of 5‑fluorouracil (5‑FU). A PI3K(III)‑RNA interference (i)‑green fluorescent protein (GFP) recombinant replication adenovirus (AD) and the negative control (NC)‑RNAi‑GFP control AD were constructed and infected into SGC7901 cells. A methyl thiazolyl tetrazolium assay was used to determine the growth rate of the SGC7901 cells. Immunofluorescent staining was used to detect microtubule‑associated protein 1 light chain 3 expression. The mitochondrial membrane potential was measured using the JC‑1 fluorescent probe. Autophagic expression was monitored with MDC staining and transmission electron microscopy. The results revealed that following combination treatment of the SGC7901 gastric cancer cells with 5‑FU + PI3K(III)‑RNAi‑AD, the optical density absorbance values at 24, 48 and 72 h were 0.17 ± 1.64, 0.13 ± 4.64 and 0.11 ± 3.56%, respectively, with cell viability inhibition ratios of 45.89 ± 6.67, 72.57 ± 9.48 and 87.51 ± 4.65%, respectively. As compared with the other treatment groups, the inhibition rate in the combined treatment group was significantly higher (P<0.05). The percentages of the cells with green fluorescence in the combined treatment group were 74.4 ± 3.86 (24 h), 82.3 ± 1.84 (48 h) and 92.5 ± 1.1% (72 h), which were larger than those of the other groups. The percentage of cells with green fluorescence became larger, which indicated that the mitochondrion membrane potential had been reduced to a greater extent. MDC staining revealed that the number of autophagic vacuoles in the cells (measured at 24, 48 and 72 h) decreased gradually with time, with more autophagic

  12. siRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway.

    PubMed

    Miyawaki-Shimizu, Kayo; Predescu, Dan; Shimizu, Jun; Broman, Michael; Predescu, Sanda; Malik, Asrar B

    2006-02-01

    Caveolin-1, the principal integral membrane protein of caveolae, has been implicated in regulating the structural integrity of caveolae, vesicular trafficking, and signal transduction. Although the functions of caveolin-1 are beginning to be explored in caveolin-1-/- mice, these results are confounded by unknown compensatory mechanisms and the development of pulmonary hypertension, cardiomyopathy, and lung fibrosis. To address the role of caveolin-1 in regulating lung vascular permeability, in the present study we used small interfering RNA (siRNA) to knock down caveolin-1 expression in mouse lung endothelia in vivo. Intravenous injection of siRNA against caveolin-1 mRNA incorporated in liposomes selectively reduced the expression of caveolin-1 by approximately 90% within 96 h of injection compared with wild-type mice. We observed the concomitant disappearance of caveolae in lung vessel endothelia and dilated interendothelial junctions (IEJs) as well as increased lung vascular permeability to albumin via IEJs. The reduced caveolin-1 expression also resulted in increased plasma nitric oxide concentration. The nitric oxide synthase inhibitor L-NAME, in part, blocked the increased vascular albumin permeability. These morphological and functional effects of caveolin-1 knockdown were reversible within 168 h after siRNA injection, corresponding to the restoration of caveolin-1 expression. Thus our results demonstrate the essential requirement of caveolin-1 in mediating the formation of caveolae in endothelial cells in vivo and in negatively regulating IEJ permeability. PMID:16183667

  13. Lettuce chlorosis virus P23 Suppresses RNA Silencing and Induces Local Necrosis with Increased Severity at Raised Temperatures.

    PubMed

    Kubota, Kenji; Ng, James C K

    2016-06-01

    RNA silencing functions as an antivirus defense strategy in plants, one that plant viruses counter by producing viral suppressors of RNA silencing (VSRs). VSRs have been identified in three members of the genus Crinivirus but they do not all share identical suppression mechanisms. Here, we used Agrobacterium co-infiltration assays to investigate the suppressor activity of proteins encoded by Lettuce chlorosis virus (LCV). Of 7 LCV proteins (1b, P23, HSP70 homolog, P60, CP, CPm, and P27) tested for the suppression of silencing of green fluorescent protein (GFP) expression in wild-type Nicotiana benthamiana plants, only P23 suppressed the onset of local silencing. Small-interfering (si)RNA accumulation was reduced in leaves co-infiltrated with P23, suggesting that P23 inhibited the accumulation or enhanced the degradation of siRNA. P23 also inhibited the cell-to-cell and systemic movement of RNA silencing in GFP-expressing transgenic N. benthamiana plants. Expression of P23 via agroinfiltration of N. benthamiana leaves induced local necrosis that increased in severity at elevated temperatures, a novelty given that a direct temperature effect on necrosis severity has not been reported for the other crinivirus VSRs. These results further affirm the sophistication of crinivirus VSRs in mediating the evasion of host's antiviral defenses and in symptom modulation. PMID:26828232

  14. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  15. Local Applications of Myostatin-siRNA with Atelocollagen Increase Skeletal Muscle Mass and Recovery of Muscle Function

    PubMed Central

    Kawakami, Emi; Kawai, Nobuhiko; Kinouchi, Nao; Mori, Hiroyo; Ohsawa, Yutaka; Ishimaru, Naozumi; Sunada, Yoshihide; Noji, Sumihare; Tanaka, Eiji

    2013-01-01

    Background Growing evidence suggests that small-interfering RNA (siRNA) can promote gene silencing in mammalian cells without induction of interferon synthesis or nonspecific gene suppression. Recently, a number of highly specific siRNAs targeted against disease-causing or disease-promoting genes have been developed. In this study, we evaluate the effectiveness of atelocollagen (ATCOL)-mediated application of siRNA targeting myostatin (Mst), a negative regulator of skeletal muscle growth, into skeletal muscles of muscular dystrophy model mice. Methods and Findings We injected a nanoparticle complex containing myostatin-siRNA and ATCOL (Mst-siRNA/ATCOL) into the masseter muscles of mutant caveolin-3 transgenic (mCAV-3Tg) mice, an animal model for muscular dystrophy. Scrambled (scr) -siRNA/ATCOL complex was injected into the contralateral muscles as a control. Two weeks after injection, the masseter muscles were dissected for histometric analyses. To investigate changes in masseter muscle activity by local administration of Mst-siRNA/ATCOL complex, mouse masseter electromyography (EMG) was measured throughout the experimental period via telemetry. After local application of the Mst-siRNA/ATCOL complex, masseter muscles were enlarged, while no significant change was observed on the contralateral side. Histological analysis showed that myofibrils of masseter muscles treated with the Mst-siRNA/ATCOL complex were significantly larger than those of the control side. Real-time PCR analysis revealed a significant downregulation of Mst expression in the treated masseters of mCAV-3Tg mice. In addition, expression of myogenic transcription factors was upregulated in the Mst-siRNA-treated masseter muscle, while expression of adipogenic transcription factors was significantly downregulated. EMG results indicate that masseter muscle activity in mCAV-3Tg mice was increased by local administration of the Mst-siRNA/ATCOL complex. Conclusion These data suggest local administration of

  16. MicroRNA overexpression increases cortical neuronal vulnerability to injury

    PubMed Central

    Truettner, Jessie S.; Motti, Dario; Dietrich, W. Dalton

    2013-01-01

    Previously we reported that several microRNAs (miRNA) are upregulated following experimentally induced traumatic brain injury (TBI) using both in vivo and in vitro approaches. Specific miRNAs were found to be sensitive to therapeutic hypothermia and may therefore be important targets for neuroprotective strategies. In this study we developed plasmid constructs that overexpress temperature sensitive miRNAs: miR-34a, miR-451, and miR-874. These constructs were transfected into cultured cortical neurons that were subjected to stretch injury using a cell injury controller device. Levels of expression of genes associated with stress, inflammation, apoptosis and transcriptional regulation were measured by qRT-PCR. mRNA levels of cytokines interleukin 1-β (IL1-β) and tumor necrosis factor alpha (TNF-α) as well as heat shock protein 70 (HSP70) and Caspase 11 were found to be increased up to 24 fold higher than controls in cells overexpressing these miRNAs. After moderate stretch injury, the expression of IL1-β, TNF-α, HSP70 and Caspase 11 all increased over control levels found in uninjured cells suggesting that overexpression of these miRNAs increases cellular vulnerability. miR-34a directly inhibits Bcl2 and XIAP, both anti-apoptotic proteins. The observed increase in Caspase 11 with over-expression of miR-34a indicates that miR-34a may be inducing apoptosis by reducing the levels of antiapoptotic proteins. miR-34a is predicted to inhibit Jun, which was seen to decrease in cells overexpressing this miRNA along with Fos. Over expression of several miRNAs found to be induced by TBI in vivo (miR-34a, miR-451 and miR-874) leads to increased vulnerability in transfected neurons. Therapeutic hypothermia blunts the expression of these miRNAs in vivo and antisense silencing could be a potential therapeutic approach to targeting the consequences of TBI. PMID:23948100

  17. Suppression of ITGB4 Gene Expression in PC-3 Cells with Short Interfering RNA Induces Changes in the Expression of β-Integrins Associated with RGD-Receptors.

    PubMed

    Knyazev, E N; Nyushko, K M; Alekseev, B Ya; Samatov, T R; Shkurnikov, M Yu

    2015-08-01

    We studied the effect of transfection of PC-3 prostate cancer cells with a plasmid encoding shRNA complimentary to a fragment of integrin β4 (ITGB4). The results attest to considerable changes in the transcriptome of transfected cells. For instance, compensatory changes in the expression of integrin family genes were found. PMID:26395630

  18. Inducible and reversible suppression of Npm1 gene expression using stably integrated small interfering RNA vector in mouse embryonic stem cells

    SciTech Connect

    Wang Beibei; Lu Rui; Wang Weicheng; Jin Ying . E-mail: yjin@sibs.ac.cn

    2006-09-08

    The tetracycline (Tc)-inducible small interference RNA (siRNA) is a powerful tool for studying gene function in mammalian cells. However, the system is infrequently utilized in embryonic stem (ES) cells. Here, we present First application of the Tc-inducible, stably integrated plasmid-based siRNA system in mouse ES cells to down-regulate expression of Npm1, an essential gene for embryonic development. The physiological role of Npm1 in ES cells has not been defined. Our data show that the knock-down of Npm1 expression by this siRNA system was not only highly efficient, but also Tc- dose- and induction time-dependent. Particularly, the down-regulation of Npm1 expression was reversible. Importantly, suppression of Npm1 expression in ES cells resulted in reduced cell proliferation. Taken together, this system allows for studying gene function in a highly controlled manner, otherwise difficult to achieve in ES cells. Moreover, our results demonstrate that Npm1 is essential for ES cell proliferation.

  19. Analysis of the small interfering RNA profiles of randomly inserted pTRM-TRI6 Fusarium graminearum mutants and their DON related phenotypes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Deoxynivalenol (DON) production by Fusarium graminearum requires activation of the trichothecene pathway in which TRI5 catalyzes the first step of trichothecene synthesis and TRI6 is a transcription factor activates the pathway. RNA interference (RNAi) has emerged as a useful fungal genetics tool f...

  20. Nucleolin mediates microRNA-directed CSF-1 mRNA deadenylation but increases translation of CSF-1 mRNA.

    PubMed

    Woo, Ho-Hyung; Baker, Terri; Laszlo, Csaba; Chambers, Setsuko K

    2013-06-01

    CSF-1 mRNA 3'UTR contains multiple unique motifs, including a common microRNA (miRNA) target in close proximity to a noncanonical G-quadruplex and AU-rich elements (AREs). Using a luciferase reporter system fused to CSF-1 mRNA 3'UTR, disruption of the miRNA target region, G-quadruplex, and AREs together dramatically increased reporter RNA levels, suggesting important roles for these cis-acting regulatory elements in the down-regulation of CSF-1 mRNA. We find that nucleolin, which binds both G-quadruplex and AREs, enhances deadenylation of CSF-1 mRNA, promoting CSF-1 mRNA decay, while having the capacity to increase translation of CSF-1 mRNA. Through interaction with the CSF-1 3'UTR miRNA common target, we find that miR-130a and miR-301a inhibit CSF-1 expression by enhancing mRNA decay. Silencing of nucleolin prevents the miRNA-directed mRNA decay, indicating a requirement for nucleolin in miRNA activity on CSF-1 mRNA. Downstream effects followed by miR-130a and miR-301a inhibition of directed cellular motility of ovarian cancer cells were found to be dependent on nucleolin. The paradoxical effects of nucleolin on miRNA-directed CSF-1 mRNA deadenylation and on translational activation were explored further. The nucleolin protein contains four acidic stretches, four RNA recognition motifs (RRMs), and nine RGG repeats. All three domains in nucleolin regulate CSF-1 mRNA and protein levels. RRMs increase CSF-1 mRNA, whereas the acidic and RGG domains decrease CSF-1 protein levels. This suggests that nucleolin has the capacity to differentially regulate both CSF-1 RNA and protein levels. Our finding that nucleolin interacts with Ago2 indirectly via RNA and with poly(A)-binding protein C (PABPC) directly suggests a nucleolin-Ago2-PABPC complex formation on mRNA. This complex is in keeping with our suggestion that nucleolin may work with PABPC as a double-edged sword on both mRNA deadenylation and translational activation. Our findings underscore the complexity of

  1. Acid-Sensitive Sheddable PEGylated PLGA Nanoparticles Increase the Delivery of TNF-α siRNA in Chronic Inflammation Sites.

    PubMed

    Aldayel, Abdulaziz M; Naguib, Youssef W; O'Mary, Hannah L; Li, Xu; Niu, Mengmeng; Ruwona, Tinashe B; Cui, Zhengrong

    2016-01-01

    There has been growing interest in utilizing small interfering RNA (siRNA) specific to pro-inflammatory cytokines, such as tumor necrosis factor-α ( TNF-α), in chronic inflammation therapy. However, delivery systems that can increase the distribution of the siRNA in chronic inflammation sites after intravenous administration are needed. Herein we report that innovative functionalization of the surface of siRNA-incorporated poly (lactic-co-glycolic) acid (PLGA) nanoparticles significantly increases the delivery of the siRNA in the chronic inflammation sites in a mouse model. The TNF-α siRNA incorporated PLGA nanoparticles were prepared by the standard double emulsion method, but using stearoyl-hydrazone-polyethylene glycol 2000, a unique acid-sensitive surface active agent, as the emulsifying agent, which renders (i) the nanoparticles PEGylated and (ii) the PEGylation sheddable in low pH environment such as that in chronic inflammation sites. In a mouse model of lipopolysaccharide-induced chronic inflammation, the acid-sensitive sheddable PEGylated PLGA nanoparticles showed significantly higher accumulation or distribution in chronic inflammation sites than PLGA nanoparticles prepared with an acid-insensitive emulsifying agent (i.e., stearoyl-amide-polyethylene glycol 2000) and significantly increased the distribution of the TNF-α siRNA incorporated into the nanoparticles in inflamed mouse foot. PMID:27434685

  2. Function and Evolution of a MicroRNA That Regulates a Ca2+-ATPase and Triggers the Formation of Phased Small Interfering RNAs in Tomato Reproductive Growth[W][OA

    PubMed Central

    Wang, Ying; Itaya, Asuka; Zhong, Xuehua; Wu, Yang; Zhang, Jianfeng; van der Knaap, Esther; Olmstead, Richard; Qi, Yijun; Ding, Biao

    2011-01-01

    MicroRNAs (miRNAs) regulate a wide variety of biological processes in most eukaryotes. We investigated the function and evolution of miR4376 in the family Solanaceae. We report that the 22-nucleotide miR4376 regulates the expression of an autoinhibited Ca2+-ATPase, tomato (Solanum lycopersicum) ACA10, which plays a critical role in tomato reproductive growth. Deep phylogenetic mapping suggested (1) an evolution course of MIR4376 loci and posttranscriptional processing of pre-miR4376 as a likely limiting step for the evolution of miR4376, (2) an independent phylogenetic origin of the miR4376 target site in ACA10 homologs, and (3) alternative splicing as a possible mechanism of eliminating such a target in some ACA10 homologs. Furthermore, miR4376 triggers the formation of phased small interfering RNAs (siRNAs) from Sl ACA10 and its Solanum tuberosum homolog. Together, our data provide experimental evidence of miRNA-regulated expression of universally important Ca2+-ATPases. The miR4376-regulated expression of ACA10 itself, and possibly also the associated formation of phased siRNAs, may function as a novel layer of molecular mechanisms underlying tomato reproductive growth. Finally, our data suggest that the stochastic emergence of a miRNA-target gene combination involves multiple molecular events at the genomic, transcriptional, and posttranscriptional levels that may vary drastically in even closely related species. PMID:21917547

  3. Measles Virus Defective Interfering RNAs Are Generated Frequently and Early in the Absence of C Protein and Can Be Destabilized by Adenosine Deaminase Acting on RNA-1-Like Hypermutations

    PubMed Central

    Pfaller, Christian K.; Mastorakos, George M.; Matchett, William E.; Ma, Xiao; Samuel, Charles E.

    2015-01-01

    ABSTRACT Defective interfering RNAs (DI-RNAs) of the viral genome can form during infections of negative-strand RNA viruses and outgrow full-length viral genomes, thereby modulating the severity and duration of infection. Here we document the frequent de novo generation of copy-back DI-RNAs from independent rescue events both for a vaccine measles virus (vac2) and for a wild-type measles virus (IC323) as early as passage 1 after virus rescue. Moreover, vaccine and wild-type C-protein-deficient (C-protein-knockout [CKO]) measles viruses generated about 10 times more DI-RNAs than parental virus, suggesting that C enhances the processivity of the viral polymerase. We obtained the nucleotide sequences of 65 individual DI-RNAs, identified breakpoints and reinitiation sites, and predicted their structural features. Several DI-RNAs possessed clusters of A-to-G or U-to-C transitions. Sequences flanking these mutation sites were characteristic of those favored by adenosine deaminase acting on RNA-1 (ADAR1), which catalyzes in double-stranded RNA the C-6 deamination of adenosine to produce inosine, which is recognized as guanosine, a process known as A-to-I RNA editing. In individual DI-RNAs the transitions were of the same type and occurred on both sides of the breakpoint. These patterns of mutations suggest that ADAR1 edits unencapsidated DI-RNAs that form double-strand RNA structures. Encapsidated DI-RNAs were incorporated into virus particles, which reduced the infectivity of virus stocks. The CKO phenotype was dominant: DI-RNAs derived from vac2 with a CKO suppressed the replication of vac2, as shown by coinfections of interferon-incompetent lymphatic cells with viruses expressing different fluorescent reporter proteins. In contrast, coinfection with a C-protein-expressing virus did not counteract the suppressive phenotype of DI-RNAs. IMPORTANCE Recombinant measles viruses (MVs) are in clinical trials as cancer therapeutics and as vectored vaccines for HIV-AIDS and

  4. Chitosan nanoparticles for siRNA delivery: optimizing formulation to increase stability and efficiency.

    PubMed

    Ragelle, H; Riva, R; Vandermeulen, G; Naeye, B; Pourcelle, V; Le Duff, C S; D'Haese, C; Nysten, B; Braeckmans, K; De Smedt, S C; Jérôme, C; Préat, V

    2014-02-28

    This study aims at developing chitosan-based nanoparticles suitable for an intravenous administration of small interfering RNA (siRNA) able to achieve (i) high gene silencing without cytotoxicity and (ii) stability in biological media including blood. Therefore, the influence of chitosan/tripolyphosphate ratio, chitosan physicochemical properties, PEGylation of chitosan as well as the addition of an endosomal disrupting agent and a negatively charged polymer was assessed. The gene silencing activity and cytotoxicity were evaluated on B16 melanoma cells expressing luciferase. We monitored the integrity and the size behavior of siRNA nanoparticles in human plasma using fluorescence fluctuation spectroscopy and single particle tracking respectively. The presence of PEGylated chitosan and poly(ethylene imine) was essential for high levels of gene silencing in vitro. Chitosan nanoparticles immediately released siRNA in plasma while the inclusion of hyaluronic acid and high amount of poly(ethylene glycol) in the formulation improved the stability of the particles. The developed formulations of PEGylated chitosan-based nanoparticles that achieve high gene silencing in vitro, low cytotoxicity and high stability in plasma could be promising for intravenous delivery of siRNA. PMID:24389132

  5. Induction of CML28-specific cytotoxic T cell responses using co-transfected dendritic cells with CML28 DNA vaccine and SOCS1 small interfering RNA expression vector

    SciTech Connect

    Zhou Hongsheng; Zhang Donghua . E-mail: hanson2008@gmail.com; Wang Yaya; Dai Ming; Zhang Lu; Liu Wenli; Liu Dan; Tan Huo; Huang Zhenqian

    2006-08-18

    CML28 is an attractive target for antigen-specific immunotherapy. SOCS1 represents an inhibitory control mechanism for DC antigen presentation and the magnitude of adaptive immunity. In this study, we evaluated the potential for inducing CML28-specific cytotoxic T lymphocytes (CTL) responses by dendritic cells (DCs)-based vaccination. We constructed a CML28 DNA vaccine and a SOCS1 siRNA vector and then cotransfect monocyte-derived DCs. Flow cytometry analysis showed gene silencing of SOCS1 resulted in higher expressions of costimulative moleculars in DCs. Mixed lymphocyte reaction (MLR) indicated downregulation of SOCS1 stronger capability to stimulate proliferation of responder cell in DCs. The CTL assay revealed transfected DCs effectively induced autologous CML28-specific CTL responses and the lytic activities induced by SOCS1-silenced DCs were significantly higher compared with those induced by SOCS1-expressing DCs. These results in our study indicates gene silencing of SOCS1 remarkably enhanced the cytotoxicity efficiency of CML28 DNA vaccine in DCs.

  6. Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.

    PubMed

    Benteyn, Daphné; Heirman, Carlo; Thielemans, Kris; Bonehill, Aude

    2016-01-01

    Dendritic cells (DCs) are the orchestrators of the immune system and are frequently used in clinical trials in order to boost the immune system in cancer patients. Among several available techniques for DC modification, mRNA electroporation is an interesting technique due to the favorable characteristics of mRNA. Antigen expression level and duration can be increased by multiple optimizations of an antigen-encoding mRNA template. Here, we describe different molecular modifications to a WT1-encoding mRNA construct in order to increase antigen expression and the subsequent introduction of mRNA into DCs. PMID:27236795

  7. Blocking miRNA Biogenesis in Adult Forebrain Neurons Enhances Seizure Susceptibility, Fear Memory, and Food Intake by Increasing Neuronal Responsiveness.

    PubMed

    Fiorenza, Anna; Lopez-Atalaya, Jose P; Rovira, Victor; Scandaglia, Marilyn; Geijo-Barrientos, Emilio; Barco, Angel

    2016-04-01

    The RNase Dicer is essential for the maturation of most microRNAs, a molecular system that plays an essential role in fine-tuning gene expression. To gain molecular insight into the role of Dicer and the microRNA system in brain function, we conducted 2 complementary RNA-seq screens in the hippocampus of inducible forebrain-restricted Dicer1 mutants aimed at identifying the microRNAs primarily affected by Dicer loss and their targets, respectively. Functional genomics analyses predicted the main biological processes and phenotypes associated with impaired microRNA maturation, including categories related to microRNA biology, signal transduction, seizures, and synaptic transmission and plasticity. Consistent with these predictions, we found that, soon after recombination, Dicer-deficient mice exhibited an exaggerated seizure response, enhanced induction of immediate early genes in response to different stimuli, stronger and more stable fear memory, hyperphagia, and increased excitability of CA1 pyramidal neurons. In the long term, we also observed slow and progressive excitotoxic neurodegeneration. Overall, our results indicate that interfering with microRNA biogenesis causes an increase in neuronal responsiveness and disrupts homeostatic mechanisms that protect the neuron against overactivation, which may explain both the initial and late phenotypes associated with the loss of Dicer in excitatory neurons. PMID:25595182

  8. Calpain expression in lymphoid cells. Increased mRNA and protein levels after cell activation.

    PubMed

    Deshpande, R V; Goust, J M; Chakrabarti, A K; Barbosa, E; Hogan, E L; Banik, N L

    1995-02-10

    Although calpain is ubiquitously present in human tissues and is thought to play a role in demyelination, its activity is very low in resting normal lymphocytes. To determine the nature of calpain expression at the mRNA and protein levels in human lymphoid cells, we studied human T lymphocytic, B lymphocytic, and monocytic lines as well as peripheral blood mononuclear cells. Stimulation of cells with the phorbol ester phorbol myristate acetate and the calcium ionophore A23187 resulted in increased calpain mRNA and protein expression. Calpain mRNA expression is also increased in human T cells stimulated with anti-CD3. A dissociation between the increases of RNA and protein suggested that calpain could be released from the cells; the subsequent experiments showed its presence in the extracellular environment. 5,6-Dichloro-1b-D-ribofuranosylbenzimidazole, a reversible inhibitor of mRNA synthesis, reduced calpain mRNA levels by 50-67% and protein levels by 72-91%. Its removal resulted in resumption of both calpain mRNA and protein synthesis. Cycloheximide, a translational inhibitor, reduced calpain protein levels by 77-81% and calpain mRNA levels by 96% in activated THP-1 cells. Interferon-gamma induced calpain mRNA and protein in U-937 and THP-1 cells. Dexamethasone increased mRNA expression in THP-1 cells. Our results indicate that activation of lymphoid cells results in de novo synthesis and secretion of calpain. PMID:7852311

  9. Coupling Aptamers to Short Interfering RNAs as Therapeutics

    PubMed Central

    Cerchia, Laura; Esposito, Carla Lucia; Camorani, Simona; Catuogno, Silvia; de Franciscis, Vittorio

    2011-01-01

    RNA-based approaches are among the most promising strategies aimed at developing safer and more effective therapeutics. RNA therapeutics include small non-coding miRNAs, small interfering RNA, RNA aptamers and more recently, small activating RNAs. However, major barriers exist to the use of RNAs as therapeutics such as resistance to nucleases present in biological fluids, poor chemical stability, need of specific cell targeted delivery and easy entry into the cell. Such issues have been addressed by several recent reports that show the possibility of introducing chemical modifications in small RNAs to stabilize the molecular conformation and increase by several fold their integrity, while still preserving the functional activity. Further, several aptamers have been developed as excellent candidates for the specific recognition of cell surface targets. In the last few years, by taking advantage of recent advances in the small RNA field, molecular bioconjugates have been designed that permit specific targeting and may act as cargoes for cell internalization of small RNAs acting on gene expression that will be discussed in this review.

  10. Low-Fidelity Polymerases of Alphaviruses Recombine at Higher Rates To Overproduce Defective Interfering Particles

    PubMed Central

    Poirier, Enzo Z.; Mounce, Bryan C.; Rozen-Gagnon, Kathryn; Hooikaas, Peter Jan; Stapleford, Kenneth A.; Moratorio, Gonzalo

    2015-01-01

    ABSTRACT Low-fidelity RNA-dependent RNA polymerases for many RNA virus mutators have been shown to confer attenuated phenotypes, presumably due to increased mutation rates. Additionally, for many RNA viruses, replication to high titers results in the production of defective interfering particles (DIs) that also attenuate infection. We hypothesized that fidelity, recombination, and DI production are tightly linked. We show that a Sindbis virus mutator replicating at a high multiplicity of infection manifests an earlier and greater accumulation of DIs than its wild-type counterpart. The isolated DIs interfere with the replication of full-length virus in a dose-dependent manner. Importantly, the ability of the mutator virus to overproduce DIs could be linked to an increased recombination frequency. These data confirm that RNA-dependent RNA polymerase fidelity and recombination are inversely correlated for this mutator. Our findings suggest that defective interference resulting from higher recombination rates may be more detrimental to RNA virus mutators than the increase in mutational burden. IMPORTANCE Replication, adaptation, and evolution of RNA viruses rely in large part on their low-fidelity RNA-dependent RNA polymerase. Viruses artificially modified in their polymerases to decrease fidelity (mutator viruses) are attenuated in vivo, demonstrating the important role of fidelity in viral fitness. However, attenuation was attributed solely to the modification of the viral mutation rate and the accumulation of detrimental point mutations. In this work, we described an additional phenotype of mutator viruses: an increased recombination rate leading to defective interfering particle (DI) overproduction. Because DIs are known for their inhibitory effect on viral replication, our work suggests that fidelity variants may be attenuated in vivo via several mechanisms. This has important implications in the development of fidelity variants as live attenuated vaccine strains

  11. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells

    PubMed Central

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  12. MicroRNA-21 Increases Proliferation and Cisplatin Sensitivity of Osteosarcoma-Derived Cells.

    PubMed

    Vanas, Vanita; Haigl, Barbara; Stockhammer, Verena; Sutterlüty-Fall, Hedwig

    2016-01-01

    Osteosarcoma is the most common primary bone tumor and poor prognosis for osteosarcoma patients is mainly due to chemotherapy resistance. MicroRNAs are important to maintain pathophysiological mechanisms of cancer and influence cell sensitivity to chemotherapy. In this study, we tested the functions of microRNA-21 for malignant features as well as for drug resistance of osteosarcoma. We used Northern blot to measure microRNA-21 levels in osteosarcoma-derived cell lines. MicroRNA-21 activity was modulated by either expressing a sponge to decrease its activity in an osteosarcoma-derived cell line expressing high levels of microRNA-21 or by introducing pri-microRNA-21 in a cell line with low endogenous levels. Cell migration was determined in a scratch assay and cell proliferation was measured by performing growth curve analysis. Sensitivity of the cells towards chemotherapeutics was investigated by performing cell viability assays and calculating the IC50 values. While cell migration was unaffected by modulated microRNA-21 levels, microRNA-21 inhibition slowed proliferation and exogenously expressed microRNA-21 promoted this process. Modulated microRNA-21 activity failed to effect sensitivity of osteosarcoma-derived cell lines to doxorubicin or methotrexate. Contrarily, reduction of microRNA-21 activity resulted in enhanced resistance towards cisplatin while ectopic expression of microRNA-21 showed the opposite effect. Increased microRNA-21 levels repressed the expression of Sprouty2 and ectopic expression of Sprouty2 was able to largely rescue the observed effects of microRNA-21 in osteosarcoma. In summary, our data indicate that in osteosarcoma microRNA-21 expression is an important component for regulation of cell proliferation and for determining sensitivity to cisplatin. PMID:27513462

  13. RNA-Based Methods Increase the Detection of Fecal Bacteria and Fecal Identifiers in Environmental Waters

    EPA Science Inventory

    We evaluated the use of qPCR RNA-based methods in the detection of fecal bacteria in environmental waters. We showed that RNA methods can increase the detection of fecal bacteria in multiple water matrices. The data suggest that this is a viable alternative for the detection of a...

  14. 5, 8, 11, 14-eicosatetraynoic acid suppresses CCL2/MCP-1 expression in IFN-γ-stimulated astrocytes by increasing MAPK phosphatase-1 mRNA stability

    PubMed Central

    2012-01-01

    Background The peroxisome proliferator-activated receptor (PPAR)-α activator, 5,8,11,14-eicosatetraynoic acid (ETYA), is an arachidonic acid analog. It is reported to inhibit up-regulation of pro-inflammatory genes; however, its underlying mechanism of action is largely unknown. In the present study, we focused on the inhibitory action of ETYA on the expression of the chemokine, CCL2/MCP-1, which plays a key role in the initiation and progression of inflammation. Methods To determine the effect of ETYA, primary cultured rat astrocytes and microglia were stimulated with IFN-γ in the presence of ETYA and then, expression of CCL2/MCP-1 and MAPK phosphatase (MKP-1) were determined using RT-PCR and ELISA. MKP-1 mRNA stability was evaluated by treating actinomycin D. The effect of MKP-1 and human antigen R (HuR) was analyzed by using specific siRNA transfection system. The localization of HuR was analyzed by immunocytochemistry and subcellular fractionation experiment. Results We found that ETYA suppressed CCL2/MCP-1 transcription and secretion of CCL2/MCP-1 protein through up-regulation of MKP-1mRNA levels, resulting in suppression of c-Jun N-terminal kinase (JNK) phosphorylation and activator protein 1 (AP1) activity in IFN-γ-stimulated brain glial cells. Moreover, these effects of ETYA were independent of PPAR-α. Experiments using actinomycin D revealed that the ETYA-induced increase in MKP-1 mRNA levels reflected an increase in transcript stability. Knockdown experiments using small interfering RNA demonstrated that this increase in MKP-1 mRNA stability depended on HuR, an RNA-binding protein known to promote enhanced mRNA stability. Furthermore, ETYA-induced, HuR-mediated mRNA stabilization resulted from HuR-MKP-1 nucleocytoplasmic translocation, which served to protect MKP-1 mRNA from the mRNA degradation machinery. Conclusion ETYA induces MKP-1 through HuR at the post-transcriptional level in a receptor-independent manner. The mechanism revealed here suggests

  15. Post-transcriptional regulation tends to attenuate the mRNA noise and to increase the mRNA gain

    NASA Astrophysics Data System (ADS)

    Shi, Changhong; Wang, Shuqiang; Zhou, Tianshou; Jiang, Yiguo

    2015-10-01

    Post-transcriptional regulation is ubiquitous in prokaryotic and eukaryotic cells, but how it impacts gene expression remains to be fully explored. Here, we analyze a simple gene model in which we assume that mRNAs are produced in a constitutive manner but are regulated post-transcriptionally by a decapping enzyme that switches between the active state and the inactive state. We derive the analytical mRNA distribution governed by a chemical master equation, which can be well used to analyze the mechanism of how post-transcription regulation influences the mRNA expression level including the mRNA noise. We demonstrate that the mean mRNA level in the stochastic case is always higher than that in the deterministic case due to the stochastic effect of the enzyme, but the size of the increased part depends mainly on the switching rates between two enzyme states. More interesting is that we find that in contrast to transcriptional regulation, post-transcriptional regulation tends to attenuate noise in mRNA. Our results provide insight into the role of post-transcriptional regulation in controlling the transcriptional noise.

  16. Defective Interfering Particles of Poliovirus I. Isolation and Physical Properties

    PubMed Central

    Cole, Charles N.; Smoler, Donna; Wimmer, Eckard; Baltimore, David

    1971-01-01

    A class of defective interfering (DI) poliovirus particles has been identified. The first was found as a contaminant of a viral stock; others have been isolated by serial passage at a high multiplicity of infection. The DI particles are less dense than standard virus and sediment more slowly. Their ribonucleic acid (RNA) sediments more slowly than standard RNA and has a higher electrophoretic mobility. Competition hybridization experiments with double-stranded viral RNA indicate that DI RNA is 80 to 90% of the length of standard RNA. The proteins of DI particles are indistinguishable from those of standard poliovirus. PMID:4329564

  17. Heat shock increases lifetime of a small RNA and induces its accumulation in cells.

    PubMed

    Tatosyan, Karina A; Kramerov, Dmitri A

    2016-08-01

    4.5SH and 4.5SI RNA are two abundant small non-coding RNAs specific for several related rodent families including Muridae. These RNAs have a number of common characteristics such as the short length (about 100nt), transcription by RNA polymerase III, and origin from Short Interspersed Elements (SINEs). However, their stabilities in cells substantially differ: the half-life of 4.5SH RNA is about 20min, while that of 4.5SI RNA is 22h. Here we studied the influence of cell stress such as heat shock or viral infection on these two RNAs. We found that the level of 4.5SI RNA did not change in stressed cells; whereas heat shock increased the abundance of 4.5SH RNA 3.2-10.5 times in different cell lines; and viral infection, 5 times. Due to the significant difference in the turnover rates of these two RNAs, a similar activation of their transcription by heat shock increases the level of the short-lived 4.5SH RNA and has minor effect on the level of the long-lived 4.5SI RNA. In addition, the accumulation of 4.5SH RNA results not only from the induction of its transcription but also from a substantial retardation of its decay. To our knowledge, it is the first example of a short-lived non-coding RNA whose elongated lifetime contributes significantly to its accumulation in stressed cells. PMID:27085482

  18. Epstein-Barr Virus MicroRNA Expression Increases Aggressiveness of Solid Malignancies

    PubMed Central

    Pandya, Deep; Mariani, Marisa; He, Shiquan; Andreoli, Mirko; Spennato, Manuela; Dowell-Martino, Candice; Fiedler, Paul; Ferlini, Cristiano

    2015-01-01

    The Cancer Genome Atlas (TCGA) microRNA (miRNA) initiative has revealed a pivotal role for miRNAs in cancer. Utilizing the TCGA raw data, we performed the first mapping of viral miRNA sequences within cancer and adjacent normal tissues. Results were integrated with TCGA RNA-seq to link the expression of viral miRNAs to the phenotype. Using clinical data and viral miRNA mapping results we also performed outcome analysis. Three lines of evidence lend credence to an active role of viral miRNAs in solid malignancies. First, expression of viral miRNA is consistently higher in cancerous compared to adjacent noncancerous tissues. Second, viral miRNA expression is associated with significantly worse clinical outcome among patients with early stage malignancy. These patients are also featured by increased expression of PD1/PD-L1, a pathway implicated in tumors escaping immune destruction. Finally, a particular cluster of EBV-miRNA (miR-BART2, miR-BART4, miR-BART5, miR-BART18, and miR-BART22) is associated with expression of cytokines known to inhibit host response to cancer. Quantification of specific viral miRNAs may help identify patients who are at risk of poor outcome. These patients may be candidates for novel therapeutic strategies incorporating antiviral agents and/or inhibitors of the PD-1/PD-L1 pathway. PMID:26375401

  19. MicroRNA Gene Polymorphisms and Environmental Factors Increase Patient Susceptibility to Hepatocellular Carcinoma

    PubMed Central

    Chu, Yin-Hung; Hsieh, Ming-Ju; Chiou, Hui-Ling; Liou, Yi-Sheng; Yang, Chen-Chieh; Yang, Shun-Fa; Kuo, Wu-Hsien

    2014-01-01

    Background Micro RNAs (miRNAs) are small RNA fragments that naturally exist in the human body. Through various physiological mechanisms, miRNAs can generate different functions for regulating RNA protein levels and balancing abnormalities. Abnormal miRNA expression has been reported to be highly related to several diseases and cancers. Single-nucleotide polymorphisms (SNPs) in miRNAs have been reported to increase patient susceptibility and affect patient prognosis and survival. We adopted a case-control research design to verify the relationship between miRNAs and hepatocellular carcinoma. Methodology/Principal Findings A total of 525 subjects, including 377 controls and 188 hepatocellular carcinoma patients, were selected. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and real-time PCR were used to analyze miRNA146a (rs2910164), miRNA149 (rs2292832), miRNA196 (rs11614913), and miRNA499 (rs3746444) genetic polymorphisms between the control group and the case group. The results indicate that people who carry the rs3746444 CT or CC genotypes may have a significantly increased susceptibility to hepatocellular carcinoma (adjusted odds ratio [AOR] = 2.84, 95% confidence interval [CI] = 1.88–4.30). In addition, when combined with environmental risk factors, such as smoking and alcohol consumption, interaction effects were observed between gene polymorphisms and environmental factors (odds ratio [OR] = 4.69, 95% CI = 2.52–8.70; AOR = 3.38, 95% CI = 1.68–6.80). Conclusions These results suggest that a significant association exists between miRNA499 SNPs and hepatocellular carcinoma. Gene-environment interactions of miRNA499 polymorphisms, smoking, and alcohol consumption might alter hepatocellular carcinoma susceptibility. PMID:24587132

  20. Adaptive antenna arrays for weak interfering signals

    NASA Technical Reports Server (NTRS)

    Gupta, I. J.

    1985-01-01

    The interference protection provided by adaptive antenna arrays to an Earth station or satellite receive antenna system is studied. The case where the interference is caused by the transmission from adjacent satellites or Earth stations whose signals inadverently enter the receiving system and interfere with the communication link is considered. Thus, the interfering signals are very weak. To increase the interference suppression, one can either decrease the thermal noise in the feedback loops or increase the gain of the auxiliary antennas in the interfering signal direction. Both methods are examined. It is shown that one may have to reduce the noise correlation to impractically low values and if directive auxiliary antennas are used, the auxiliary antenna size may have to be too large. One can, however, combine the two methods to achieve the specified interference suppression with reasonable requirements of noise decorrelation and auxiliary antenna size. Effects of the errors in the steering vector on the adaptive array performance are studied.

  1. T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats.

    PubMed

    Silva, F Goulart da; Giannocco, G; Luchessi, A D; Curi, R; Nunes, M T

    2010-04-12

    Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-I mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. PMID:20015464

  2. Prefrontal microRNA-221 Mediates Environmental Enrichment-Induced Increase of Locomotor Sensitivity to Nicotine

    PubMed Central

    Gomez, Adrian M.; Altomare, Diego; Sun, Wei-Lun; Midde, Narasimha M.; Ji, Hao; Shtutman, Michael; Turner, Jill R.; Creek, Kim E.

    2016-01-01

    Background: Environmental enrichment alters susceptibility in developing drug addiction. We have demonstrated that rats raised in an enriched condition are more sensitive than rats raised in an impoverished condition to nicotine-induced locomotor activity, and this is associated with alterations of phosphorylated extracellular signal-regulated kinase 1/2 within the prefrontal cortex. This study determined the impact of microRNA-221 in the prefrontal cortex on phosphorylated extracellular signal-regulated kinase 1/2 and the enriched environment-dependent behavioral changes in response to nicotine. Methods: A microRNA array was conducted to profile microRNA expression in the prefrontal cortex of enriched condition and impoverished condition rats in response to repeated nicotine (0.35mg/kg, s.c.) administration. microRNA-221 in the prefrontal cortex, nucleus accumbens, and striatum was further verified by quantitative real-time PCR. Lentiviral-mediated overexpression of microRNA-221 in PC12 cells and the medial prefrontal cortex was performed to determine the effects of microRNA-221 on nicotine-mediated phosphorylated extracellular signal-regulated kinase 1/2, phosphorylated cAMP-response element-binding protein, and locomotor activity. Results: microRNA-221 was profoundly upregulated in the prefrontal cortex but not in nucleus accumbens and striatum of enriched condition rats relative to impoverished condition rats following repeated administration of nicotine. Overexpression of lentiviral-microRNA-221 attenuated nicotine-induced increase in phosphorylated extracellular signal-regulated kinase 1/2 in PC12 cells. Lentiviral-microRNA-221 overexpression in the medial prefrontal cortex further increased locomotor activity in impoverished condition but not in enriched condition rats in response to repeated nicotine administration. Accordingly, lentiviral-microRNA-221 attenuated nicotine-induced increases in phosphorylated extracellular signal-regulated kinase 1/2 and

  3. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism.

    PubMed

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K; Lehtonen, Jukka Y A

    2016-04-20

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3'-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  4. Endoplasmic reticulum stress increases AT1R mRNA expression via TIA-1-dependent mechanism

    PubMed Central

    Backlund, Michael; Paukku, Kirsi; Kontula, Kimmo K.; Lehtonen, Jukka Y.A.

    2016-01-01

    As the formation of ribonucleoprotein complexes is a major mechanism of angiotensin II type 1 receptor (AT1R) regulation, we sought to identify novel AT1R mRNA binding proteins. By affinity purification and mass spectroscopy, we identified TIA-1. This interaction was confirmed by colocalization of AT1R mRNA and TIA-1 by FISH and immunofluorescence microscopy. In immunoprecipitates of endogenous TIA- 1, reverse transcription-PCR amplified AT1R mRNA. TIA-1 has two binding sites within AT1R 3′-UTR. The binding site proximal to the coding region is glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-dependent whereas the distal binding site is not. TIA-1 functions as a part of endoplasmic reticulum (ER) stress response leading to stress granule (SG) formation and translational silencing. We and others have shown that AT1R expression is increased by ER stress-inducing factors. In unstressed cells, TIA-1 binds to AT1R mRNA and decreases AT1R protein expression. Fluorescence microscopy shows that ER stress induced by thapsigargin leads to the transfer of TIA-1 to SGs. In FISH analysis AT1R mRNA remains in the cytoplasm and no longer colocalizes with TIA-1. Thus, release of TIA-1-mediated suppression by ER stress increases AT1R protein expression. In conclusion, AT1R mRNA is regulated by TIA-1 in a ER stress-dependent manner. PMID:26681690

  5. Dextromethorphan increases tyrosine hydroxylase mRNA in the mesencephalon of adolescent rats.

    PubMed

    Zhang, T Y; Jahng, J W; Kim, D G

    2001-08-24

    Dextromethorphan (DM), an antitussive widely available in over-the-counter, has been abused mostly in teenage groups at high doses. To examine effects of DM on the reward pathway, we injected a high dose of DM (40 mg/kg; intraperitoneally) into the adolescent rat and measured tyrosine hydroxylase (TH) mRNA by in situ hybridization in the ventral tegmental area (VTA) and the substantia nigra (SN). Remarkable increases in the level of TH mRNA were observed in the VTA and SN 2 h after DM injection. Stereotyped behavior and ataxia increased, and rearing decreased by DM administration. These results suggest that DM-induced increase in TH mRNA expression in mesencephalon contribute to the reinforcing property and the behavioral effects of DM. PMID:11502351

  6. Developmental expression of chicken antithrombin III is regulated by increased RNA abundance and intracellular processing.

    PubMed

    Amrani, D L; Rosenberg, J; Samad, F; Bergtrom, G; Banfield, D K

    1993-01-23

    We isolated and sequenced a 432 bp cDNA to cAT-III, that encoded 115 nucleotides of 5' untranslated sequence, a 17 amino acid long signal peptide and residues 1-88 of the mature protein, and used it to prepare a probe for measuring and correlating the developmental changes of steady-state cAT-III mRNA levels with known changes in antigen levels. Densitometric analysis of nuclease protection (n = 2), Northern blot (n = 4), and slot blots (n = 3) of total RNA from chick livers of 16-day-old embryos to 6-day-old chicks showed a 2.6 +/- 0.5-fold increase in steady-state cAT-III mRNA levels. Assay of functional mRNA levels by in vitro translation of poly(A)+ RNA and specific immunoprecipitation of 35S-Met-labelled cAT-III was comparable to RNA analysis (16-day-old embryos vs. 10-day-old hatchlings). We evaluated whether there were developmental differences in post-translational secretion which may also contribute to the regulation of the circulating level of this protein. Pulse-chase studies of freshly-isolated hepatocytes from 16-day-old embryos and 10-day-old hatchlings maintained in suspension demonstrated a approx. 5.0-5.5-fold increase in cAT-III levels at steady-state secretion. The above findings indicate that changes in circulating cAT-III levels during late embryonic development are primarily due to increased abundance of cAT-III mRNA. In addition, we postulate that post-translational intracellular processing may account for further differences in circulating protein levels. PMID:8424948

  7. Acidic Residues in the Hfq Chaperone Increase the Selectivity of sRNA Binding and Annealing.

    PubMed

    Panja, Subrata; Santiago-Frangos, Andrew; Schu, Daniel J; Gottesman, Susan; Woodson, Sarah A

    2015-11-01

    Hfq facilitates gene regulation by small non-coding RNAs (sRNAs), thereby affecting bacterial attributes such as biofilm formation and virulence. Escherichia coli Hfq recognizes specific U-rich and AAN motifs in sRNAs and target mRNAs, after which an arginine patch on the rim promotes base pairing between their complementary sequences. In the cell, Hfq must discriminate between many similar RNAs. Here, we report that acidic amino acids lining the sRNA binding channel between the inner pore and rim of the Hfq hexamer contribute to the selectivity of Hfq's chaperone activity. RNase footprinting, in vitro binding and stopped-flow fluorescence annealing assays showed that alanine substitution of D9, E18 or E37 strengthened RNA interactions with the rim of Hfq and increased annealing of non-specific or U-tailed RNA oligomers. Although the mutants were less able than wild-type Hfq to anneal sRNAs with wild-type rpoS mRNA, the D9A mutation bypassed recruitment of Hfq to an (AAN)4 motif in rpoS, both in vitro and in vivo. These results suggest that acidic residues normally modulate access of RNAs to the arginine patch. We propose that this selectivity limits indiscriminate target selection by E. coli Hfq and enforces binding modes that favor genuine sRNA and mRNA pairs. PMID:26196441

  8. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes.

    PubMed

    Tam, Oliver H; Aravin, Alexei A; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M; Hannon, Gregory J

    2008-05-22

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  9. Pseudogene-derived small interfering RNAs regulate gene expression in mouse oocytes

    PubMed Central

    Tam, Oliver H.; Aravin, Alexei A.; Stein, Paula; Girard, Angelique; Murchison, Elizabeth P.; Cheloufi, Sihem; Hodges, Emily; Anger, Martin; Sachidanandam, Ravi; Schultz, Richard M.; Hannon, Gregory J.

    2010-01-01

    Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways1. Here we show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. An inverted repeat pseudogene can also generate abundant small RNAs directly. A second class of endo-siRNAs may enforce repression of mobile genetic elements, acting together with Piwi-interacting RNAs. Loss of Dicer, a protein integral to small RNA production, increases expression of endo-siRNA targets, demonstrating their regulatory activity. Our findings indicate a function for pseudogenes in regulating gene expression by means of the RNA interference pathway and may, in part, explain the evolutionary pressure to conserve argonaute-mediated catalysis in mammals. PMID:18404147

  10. Small interfering RNAs targeting the rabies virus nucleoprotein gene.

    PubMed

    Yang, Yu-Jiao; Zhao, Ping-Sen; Zhang, Tao; Wang, Hua-Lei; Liang, Hong-Ru; Zhao, Li-Li; Wu, Hong-Xia; Wang, Tie-Cheng; Yang, Song-Tao; Xia, Xian-Zhu

    2012-10-01

    Rabies virus (RABV) infection continues to be a global threat to human and animal health, yet no curative therapy has been developed. RNA interference (RNAi) therapy, which silences expression of specific target genes, represents a promising approach for treating viral infections in mammalian hosts. We designed six small interfering (si)RNAs (N473, N580, N783, N796, N799 and N1227) that target the conserved region of the RABV challenge virus standard (CVS)-11 strain nucleoprotein (N) gene. Using a plasmid-based transient expression model, we demonstrated that N796, N580 and N799 were capable of significantly inhibiting viral replication in vitro and in vivo. These three siRNAs effectively suppressed RABV expression in infected baby hamster kidney-21 (BHK-21) cells, as evidenced by direct immunofluorescence assay, viral titer measurements, real-time PCR, and Western blotting. In addition, liposome-mediated siRNA expression plasmid delivery to RABV-infected mice significantly increased survival, compared to a non-liposome-mediated delivery method. Collectively, our results showed that the three siRNAs, N796, N580 and N799, targeting the N gene could potently inhibit RABV CVS-11 reproduction. These siRNAs have the potential to be developed into new and effective prophylactic anti-RABV drugs. PMID:22884777

  11. Internal 6-methyladenine residues increase the in vitro translation efficiency of dihydrofolate reductase messenger RNA.

    PubMed

    Heilman, K L; Leach, R A; Tuck, M T

    1996-07-01

    N6-Methyladenosine (m6A) is found internally in a number of mRNA molecules from higher eucaryotic cells. In these investigations, it was found that the presence of m6A residues increase the in vitro translation efficiency of capped T7 transcripts of mouse dihydrofolate reductase (DHFR) mRNA. Using an in vitro rabbit reticulocyte translation system, the formation of internal m6A residues in the DHFR transcripts resulted in a 1.5-fold increase in translated DHFR compared to transcripts void of internal m6A residues. Translation in a wheat germ system, however, resulted in no increase in translation efficiency upon m6A formation, suggesting that the mechanism may be species-specific. PMID:8925412

  12. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  13. Increased N6-methyladenosine in Human Sperm RNA as a Risk Factor for Asthenozoospermia

    PubMed Central

    Yang, Ying; Huang, Wei; Huang, Jing-Tao; Shen, Fan; Xiong, Jun; Yuan, Er-Feng; Qin, Shan-shan; Zhang, Ming; Feng, Yu-Qi; Yuan, Bi-Feng; Liu, Song-Mei

    2016-01-01

    Male infertility is a worldwide medical problem. Asthenozoospermia is a common cause of infertility. Epigenetic modifications of DNA and histones have been shown to influence human infertility, but no research has explored whether N6-methyladenosine (m6A) level in RNA is associated with asthenozoospermia. Here, we collected a total of 52 semen samples, including 20 asthenozoospermia patients and 32 healthy controls. An LC-ESI-MS/MS method was used to detect m6A contents in sperm RNA, and real-time PCR was performed to determine the mRNA expression of demethylase (FTO, ALKBH5), methyltransferase (METTL3, METTL14, WTAP) and an m6A-selective-binding protein (YTHDF2). We found that m6A content (p = 0.033) and the mRNA expression of METTL3 (p = 0.016) and METTL14 (p = 0.025) in asthenozoospermia patients were significantly higher than those of controls. Increased m6A content was a risk factor for asthenozoospermia (odds ratio (OR) 3.229, 95% confidence interval (CI) 1.178 – 8.853, p = 0.023). Moreover, m6A content was correlated with the expression of METTL3 (r = 0.303, p = 0.032) and with sperm motility (progressive motility: r = −0.288, p = 0.038; non-progressive motility: r = −0.293, p = 0.037; immotility: r = 0.387, p = 0.005). Our data suggest that increased m6A content is a risk factor for asthenozoospermia and affects sperm motility. Methyltransferases, particularly METTL3, play key roles in increasing m6A contents in sperm RNA. PMID:27072590

  14. Acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA without increasing proteolysis in skeletal muscle

    PubMed Central

    Vary, Thomas C.; Frost, Robert A.; Lang, Charles H.

    2008-01-01

    Acute alcohol intoxication decreases muscle protein synthesis, but there is a paucity of data on the ability of alcohol to regulate muscle protein degradation. Furthermore, various types of atrophic stimuli appear to regulate ubiquitin-proteasome-dependent proteolysis by increasing the muscle-specific E3 ligases atrogin-1 and MuRF1 (i.e., “atrogenes”). Therefore, the present study was designed to test the hypothesis that acute alcohol intoxication increases atrogene expression leading to an elevated rate of muscle protein breakdown. In male rats, the intraperitoneal injection of alcohol dose- and time-dependently increased atrogin-1 and MuRF1 mRNA in gastrocnemius, the latter of which was most pronounced. A comparable change was absent in the soleus and heart. The ability of in vivo-administered ethanol to increase atrogene expression was independent of the route of alcohol administration (intraperitoneal vs. oral), as well as of nutritional status (fed vs. fasted) and gender (male vs. female). The increase in atrogin-1 and MuRF1 was independent of alcohol metabolism, and the overproduction of endogenous glucocorticoids and could not be prevented by maintaining the circulating concentration of insulin-like growth factor-I. Despite marked changes in atrogene expression, acute alcohol in vivo did not alter the release of either 3-methylhistidine (MH) or tyrosine from the isolated perfused hindlimb, suggesting that the rate of muscle proteolysis remains unchanged. Moreover, alcohol did not increase the directly determined rate of protein degradation in isolated epitrochlearis muscles or cultured myocytes. Finally, no increase in atrogene expression or 3-MH release was detected in muscle from rats fed an alcohol-containing diet. Our results indicate that although acute alcohol intoxication increases atrogin-1 and MuRF1 mRNA preferentially in fast-twitch skeletal muscle, this change was not associated with increased rates of muscle proteolysis. Therefore, the loss

  15. RNA Viruses: RNA Roles in Pathogenesis, Coreplication and Viral Load

    PubMed Central

    Poltronieri, Palmiro; Sun, Binlian; Mallardo, Massimo

    2015-01-01

    The review intends to present and recapitulate the current knowledge on the roles and importance of regulatory RNAs, such as microRNAs and small interfering RNAs, RNA binding proteins and enzymes processing RNAs or activated by RNAs, in cells infected by RNA viruses. The review focuses on how non-coding RNAs are involved in RNA virus replication, pathogenesis and host response, especially in retroviruses HIV, with examples of the mechanisms of action, transcriptional regulation, and promotion of increased stability of their targets or their degradation. PMID:27047253

  16. Increased DNA and RNA damage by oxidation in patients with bipolar I disorder.

    PubMed

    Jacoby, A S; Vinberg, M; Poulsen, H E; Kessing, L V; Munkholm, K

    2016-01-01

    The mechanisms underlying bipolar disorder (BD) and the associated medical burden are unclear. Damage generated by oxidation of nucleosides may be implicated in BD pathophysiology; however, evidence from in vivo studies is limited and the extent of state-related alterations is unclear. This prospective study investigated for we believe the first time the damage generated by oxidation of DNA and RNA strictly in patients with type I BD in a manic or mixed state and subsequent episodes and remission compared with healthy control subjects. Urinary excretion of 8-oxo-deoxyguanosine (8-oxodG) and 8-oxo-guanosine (8-oxoGuo), valid markers of whole-body DNA and RNA damage by oxidation, respectively, was measured in 54 patients with BD I and in 35 healthy control subjects using a modified ultraperformance liquid chromatography and mass spectrometry assay. Repeated measurements were evaluated in various affective phases during a 6- to 12-month period and compared with repeated measurements in healthy control subjects. Independent of lifestyle and demographic variables, a 34% (P<0.0001) increase in RNA damage by oxidation across all affective states, including euthymia, was found in patients with BD I compared with healthy control subjects. Increases in DNA and RNA oxidation of 18% (P<0.0001) and 8% (P=0.02), respectively, were found in manic/hypomanic states compared with euthymia, and levels of 8-oxodG decreased 15% (P<0.0001) from a manic or mixed episode to remission. The results indicate a role for DNA and RNA damage by oxidation in BD pathophysiology and a potential for urinary 8-oxodG and 8-oxoGuo to function as biological markers of diagnosis, state and treatment response in BD. PMID:27505230

  17. Metformin-mediated increase in DICER1 regulates microRNA expression and cellular senescence.

    PubMed

    Noren Hooten, Nicole; Martin-Montalvo, Alejandro; Dluzen, Douglas F; Zhang, Yongqing; Bernier, Michel; Zonderman, Alan B; Becker, Kevin G; Gorospe, Myriam; de Cabo, Rafael; Evans, Michele K

    2016-06-01

    Metformin, an oral hypoglycemic agent, has been used for decades to treat type 2 diabetes mellitus. Recent studies indicate that mice treated with metformin live longer and have fewer manifestations of age-related chronic disease. However, the molecular mechanisms underlying this phenotype are unknown. Here, we show that metformin treatment increases the levels of the microRNA-processing protein DICER1 in mice and in humans with diabetes mellitus. Our results indicate that metformin upregulates DICER1 through a post-transcriptional mechanism involving the RNA-binding protein AUF1. Treatment with metformin altered the subcellular localization of AUF1, disrupting its interaction with DICER1 mRNA and rendering DICER1 mRNA stable, allowing DICER1 to accumulate. Consistent with the role of DICER1 in the biogenesis of microRNAs, we found differential patterns of microRNA expression in mice treated with metformin or caloric restriction, two proven life-extending interventions. Interestingly, several microRNAs previously associated with senescence and aging, including miR-20a, miR-34a, miR-130a, miR-106b, miR-125, and let-7c, were found elevated. In agreement with these findings, treatment with metformin decreased cellular senescence in several senescence models in a DICER1-dependent manner. Metformin lowered p16 and p21 protein levels and the abundance of inflammatory cytokines and oncogenes that are hallmarks of the senescence-associated secretory phenotype (SASP). These data lead us to hypothesize that changes in DICER1 levels may be important for organismal aging and to propose that interventions that upregulate DICER1 expression (e.g., metformin) may offer new pharmacotherapeutic approaches for age-related disease. PMID:26990999

  18. Increased RNA-Induced Silencing Complex (RISC) Activity Contributes to Hepatocellular Carcinoma

    PubMed Central

    Yoo, Byoung Kwon; Santhekadur, Prasanna K.; Gredler, Rachel; Chen, Dong; Emdad, Luni; Bhutia, Sujit; Pannell, Lewis; Fisher, Paul B.; Sarkar, Devanand

    2011-01-01

    There is virtually no effective treatment for advanced hepatocellular carcinoma (HCC) and novel targets need to be identified to develop effective treatment. We recently documented that the oncogene Astrocyte elevated gene-1 (AEG-1) plays a seminal role in hepatocarcinogenesis. Employing yeast two-hybrid assay and co-immunoprecipitation followed by mass spectrometry we identified Staphylococcal nuclease domain containing 1 (SND1), a nuclease in the RNA-induced silencing complex (RISC) facilitating RNAi-mediated gene silencing, as an AEG-1 interacting protein. Co-immunoprecipitation and co-localization studies confirmed that AEG-1 is also a component of RISC and both AEG-1 and SND1 are required for optimum RISC activity facilitating siRNA and miRNA-mediated silencing of luciferase reporter gene. In 109 human HCC samples SND1 was overexpressed in ∼74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to immortal normal hepatocytes. Increased RISC activity, conferred by AEG-1 or SND1, resulted in increased degradation of tumor suppressor mRNAs that are target of oncomiRs. Inhibition of enzymatic activity of SND1 significantly inhibited proliferation of human HCC cells. As a corollary, stable overexpression of SND1 augmented and siRNA-mediated inhibition of SND1 abrogated growth of human HCC cells in vitro and in vivo thus revealing a potential role of SND1 in hepatocarcinogenesis. Conclusion We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC. PMID:21520169

  19. Hypoxia-upregulated microRNA-630 targets Dicer, leading to increased tumor progression.

    PubMed

    Rupaimoole, R; Ivan, C; Yang, D; Gharpure, K M; Wu, S Y; Pecot, C V; Previs, R A; Nagaraja, A S; Armaiz-Pena, G N; McGuire, M; Pradeep, S; Mangala, L S; Rodriguez-Aguayo, C; Huang, L; Bar-Eli, M; Zhang, W; Lopez-Berestein, G; Calin, G A; Sood, A K

    2016-08-18

    MicroRNAs (miRNAs) are small RNA molecules that affect cellular processes by controlling gene expression. Recent studies have shown that hypoxia downregulates Drosha and Dicer, key enzymes in miRNA biogenesis, causing a decreased pool of miRNAs in cancer and resulting in increased tumor growth and metastasis. Here we demonstrate a previously unrecognized mechanism by which hypoxia downregulates Dicer. We found that miR-630, which is upregulated under hypoxic conditions, targets and downregulates Dicer expression. In an orthotopic mouse model of ovarian cancer, delivery of miR-630 using 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) nanoliposomes resulted in increased tumor growth and metastasis, and decreased Dicer expression. Treatment with the combination of anti-miR-630 and anti-vascular endothelial growth factor antibody in mice resulted in rescue of Dicer expression and significantly decreased tumor growth and metastasis. These results indicate that targeting miR-630 is a promising approach to overcome Dicer deregulation in cancer. As demonstrated in the study, use of DOPC nanoliposomes for anti-miR delivery serves as a better alternative approach to cell line-based overexpression of sense or antisense miRNAs, while avoiding potential in vitro selection effects. Findings from this study provide a new understanding of miRNA biogenesis downregulation observed under hypoxia and suggest therapeutic avenues to target this dysregulation in cancer. PMID:26725326

  20. Hypoxia-upregulated microRNA-630 targets Dicer, leading to increased tumor progression

    PubMed Central

    Rupaimoole, Rajesha; Ivan, Cristina; Yang, Da; Gharpure, Kshipra M.; Wu, Sherry Y.; Pecot, Chad V.; Previs, Rebecca A.; Nagaraja, Archana S.; Armaiz-Pena, Guillermo N; McGuire, Michael; Pradeep, Sunila; Mangala, Lingegowda S.; Rodriguez-Aguayo, Cristian; Huang, Li; Bar-Eli, Menashe; Zhang, Wei; Lopez-Berestein, Gabriel; Calin, George A.; Sood, Anil K.

    2015-01-01

    MicroRNAs (miRNAs) are small RNA molecules that affect cellular processes by controlling gene expression. Recent studies have shown that hypoxia downregulates Drosha and Dicer, key enzymes in miRNA biogenesis, causing a decreased pool of miRNAs in cancer, and resulting in increased tumor growth and metastasis. Here, we demonstrate a previously unrecognized mechanism by which hypoxia downregulates Dicer. We found that miR-630, which is upregulated under hypoxic conditions, targets and downregulates Dicer expression. In an orthotopic mouse model of ovarian cancer, delivery of miR-630 using DOPC nanoliposomes resulted in increased tumor growth and metastasis and decreased Dicer expression. Treatment with the combination of anti-miR-630 and anti-vascular endothelial growth factor antibody in mice resulted in rescue of Dicer expression and significantly decreased tumor growth and metastasis. These results indicate that targeting miR-630 is a promising approach to overcome Dicer deregulation in cancer. As demonstrated in the study, use of DOPC nanoliposomes for anti-miR delivery serves as a better alternative approach to cell line based overexpression of sense or anti-sense miRNAs, while avoiding potential in vitro selection effects. Findings from this study provide a new understanding of miRNA biogenesis downregulation observed under hypoxia and suggest therapeutic avenues to target this dysregulation in cancer. PMID:26725326

  1. Acute exercise induces biphasic increase in respiratory mRNA in skeletal muscle

    SciTech Connect

    Ikeda, Shin-ichi; Kizaki, Takako; Haga, Shukoh; Ohno, Hideki; Takemasa, Tohru

    2008-04-04

    Peroxisome proliferator-activated receptor {gamma} coactivator-1{alpha} (PGC-1{alpha}) promotes the expression of oxidative enzymes in skeletal muscle. We hypothesized that activation of the p38 MAPK (mitogen-activated protein kinase) in response to exercise was associated with exercise-induced PGC-1{alpha} and respiratory enzymes expression and aimed to demonstrate this under the physiological level. We subjected mice to a single bout of treadmill running and found that the exercise induced a biphasic increase in the expression of respiratory enzymes mRNA. The second phase of the increase was accompanied by an increase in PGC-1{alpha} protein, but the other was not. Administration of SB203580 (SB), an inhibitor of p38 MAPK, suppressed the increase in PGC-1{alpha} expression and respiratory enzymes mRNA in both phases. These data suggest that p38 MAPK is associated with the exercise-induced expression of PGC-1{alpha} and biphasic increase in respiratory enzyme mRNAs in mouse skeletal muscle under physiological conditions.

  2. Identification of a Male-Specific RNA Binding Protein That Regulates Sex-Specific Splicing of Bmdsx by Increasing RNA Binding Activity of BmPSI▿ §

    PubMed Central

    Suzuki, Masataka G.; Imanishi, Shigeo; Dohmae, Naoshi; Asanuma, Miwako; Matsumoto, Shogo

    2010-01-01

    Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI. PMID:20956562

  3. Calmodulin antagonists increase the amount of mRNA for the low-density-lipoprotein receptor in skin fibroblasts.

    PubMed Central

    Eckardt, H; Filipovic, I; Hasilik, A; Buddecke, E

    1988-01-01

    The effects of calmodulin antagonists on the amount of LDL receptor (LDL-R) mRNA in cultured human fibroblasts was examined by hybridization with a fragment of LDL-R cDNA. In a 'Northern' blot the fragment hybridized to a 5.3-kilobase RNA, as expected for LDL-R mRNA. The concentration of this RNA was increased in preparations from cells that were treated with trifluoperazine or W-7 [N-(6-aminohexyl)-5-chloronaphthalene-1-sulphonamide]. The selectivity of the increase was established by using a probe for beta-actin mRNA. In dot-blot hybridization it was observed that the calmodulin antagonists cause 2-4-fold relative increase in the amount of LDL-R mRNA. Images Fig. 1. Fig. 2. Fig. 3. PMID:3421929

  4. Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

    PubMed Central

    Strezoska, Žaklina; Licon, Abel; Haimes, Josh; Spayd, Katie Jansen; Patel, Kruti M.; Sullivan, Kevin; Jastrzebski, Katarzyna; Simpson, Kaylene J.; Leake, Devin; van Brabant Smith, Anja; Vermeulen, Annaleen

    2012-01-01

    RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of ∼10 000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction. PMID:22870320

  5. Exercise training does not increase muscle FNDC5 protein or mRNA expression in pigs

    PubMed Central

    Fain, John N.; Company, Joseph M.; Booth, Frank W.; Laughlin, M. Harold; Padilla, Jaume; Jenkins, Nathan T.; Bahouth, Suleiman W.; Sacks, Harold S.

    2013-01-01

    Background Exercise training elevates circulating irisin and induces the expression of the FNDC5 gene in skeletal muscles of mice. Our objective was to determine whether exercise training also increases FNDC5 protein or mRNA expression in the skeletal muscles of pigs as well as plasma irisin. Methods Castrated male pigs of the Rapacz familial hypercholesterolemic (FHM) strain and normal (Yucatan miniature) pigs were sacrificed after 16–20 weeks of exercise training. Samples of cardiac muscle, deltoid and triceps brachii muscle, subcutaneous and epicardial fat were obtained and FNDC5 mRNA, along with that of 6 other genes, was measured in all tissues of FHM pigs by reverse transcription polymerase chain reaction. FNDC protein in deltoid and triceps brachii was determined by Western blotting in both FHM and normal pigs. Citrate synthase activity was measured in the muscle samples of all pigs as an index of exercise training. Irisin was measured by an ELISA assay. Results There was no statistically significant effect of exercise training on FNDC5 gene expression in epicardial or subcutaneous fat, deltoid muscle, triceps brachii muscle or heart muscle. Exercise-training elevated circulating levels of irisin in the FHM pigs and citrate synthase activity in deltoid and triceps brachii muscle. A similar increase in citrate synthase activity was seen in muscle extracts of exercise-trained normal pigs but there was no alteration in circulating irisin. Conclusion Exercise training in pigs does not increase FNDC5 mRNA or protein in the deltoid or triceps brachii of FHM or normal pigs while increasing circulating irisin only in the FHM pigs. These data indicate that the response to exercise training in normal pigs is not comparable to that seen in mice. PMID:23831442

  6. Increased expression of the long noncoding RNA CRNDE-h indicates a poor prognosis in colorectal cancer, and is positively correlated with IRX5 mRNA expression

    PubMed Central

    Liu, Tong; Zhang, Xin; Yang, Yong-mei; Du, Lu-tao; Wang, Chuan-xin

    2016-01-01

    Background The long noncoding RNA (lncRNA) colorectal neoplasia differentially expressed – h (CRNDE-h) plays important roles in the early stages of human development and cancer progression. We investigated the expression and clinical significance of lncRNA CRNDE-h in colorectal cancer (CRC). Methods The expression level of lncRNA CRNDE-h was analyzed in 142 CRC tissues and 142 paired adjacent nontumorous tissues, along with 21 inflammatory bowel diseases, 69 hyperplastic polyp, and 73 colorectal adenoma samples, using quantitative real-time polymerase chain reaction. The association between lncRNA CRNDE-h, and Iroquois homeobox protein 5 (IRX5) mRNA was examined in the same 142 CRC tissues. Results We found that lncRNA CRNDE-h level was elevated in the CRC and adenoma groups compared with the other groups (all at P<0.001). In CRC, upregulation of lncRNA CRNDE-h was significantly correlated with large tumor size, positive regional lymph node metastasis, and distant metastasis (all at P<0.05). Area under the curve for lncRNA CRNDE-h showed diagnostic capability for distinguishing CRC from other groups. Patients with CRC with high lncRNA CRNDE-h expression level had poorer overall survival than those with low lncRNA CRNDE-h expression (log-rank test, P<0.001). Further, multivariable Cox regression analysis suggested that increased expression of lncRNA CRNDE-h was an independent prognostic indicator for CRC (hazard ratio [HR]=2.173; 95% confidence interval [CI], 1.282–3.684, P=0.004). Furthermore, lncRNA CRNDE-h expression was positively correlated with IRX5 mRNA in CRC tissues. Conclusions Our data offers convincing evidence for the first time that lncRNA CRNDE-h is associated with adverse clinical characteristics and poor prognosis, which suggests that it might play an important role in CRC development and progression and might have clinical potential as a useful prognostic predictor. PMID:27042112

  7. Increased microRNA-34c abundance in Alzheimer's disease circulating blood plasma

    PubMed Central

    Bhatnagar, Shephali; Chertkow, Howard; Schipper, Hyman M.; Yuan, Zongfei; Shetty, Vikranth; Jenkins, Samantha; Jones, Timothy; Wang, Eugenia

    2014-01-01

    Circulating microRNAs, present either in the cellular component, peripheral blood mononuclear cells (PBMC), or in cell-free plasma, have emerged as biomarkers for age-dependent systemic, disease-associated changes in many organs. Previously, we have shown that microRNA (miR)-34a is increased in circulating PBMC of Alzheimer's disease (AD) patients. In the present study, we show that this microRNA's sister, miR-34c, exhibits even greater increase in both cellular and plasma components of AD circulating blood samples, compared to normal age-matched controls. Statistical analysis shows the accuracy of levels of miR-34c assayed by receiver operating characteristic (ROC) analysis: the area under the curve is 0.99 (p < 0.0001) and the 95% confidence level extends from 0.97 to 1. Pearson correlation between miR-34c levels and mild and moderate AD, as defined by the mini-mental state examination (MMSE), shows an r-value of −0.7, suggesting a relatively strong inverse relationship between the two parameters. These data show that plasma levels of microRNA 34c are much more prominent in AD than those of its sister, miR-34a, or than its own level in PBMC. Transfection studies show that miR-34c, as does its sister miR-34a, represses the expression of several selected genes involved in cell survival and oxidative defense pathways, such as Bcl2, SIRT1, and others, in cultured cells. Taken together, our results indicate that increased levels of miR-34c in both PBMC and plasma may reflect changes in circulating blood samples in AD patients, compared to age-matched normal controls. PMID:24550773

  8. Increased microRNA-155 expression in the serum and peripheral monocytes in chronic HCV infection

    PubMed Central

    2012-01-01

    Background Hepatitis C Virus (HCV), a single stranded RNA virus, affects millions of people worldwide and leads to chronic infection characterized by chronic inflammation in the liver and in peripheral immune cells. Chronic liver inflammation leads to progressive liver damage. MicroRNAs (miRNA) regulate inflammation (miR-155, -146a and -125b) as well as hepatocyte function (miR-122). Methods Here we hypothesized that microRNAs are dysregulated in chronic HCV infection. We examined miRNAs in the circulation and in peripheral monocytes of patients with chronic HCV infection to evaluate if specific miRNA expression correlated with HCV infection. Results We found that monocytes from chronic HCV infected treatment-naïve (cHCV) but not treatment responder patients showed increased expression of miR-155, a positive regulator of TNFα, and had increased TNFα production compared to monocytes of normal controls. After LPS stimulation, miR-155 levels were higher in monocytes from cHCV patients compared to controls. MiR-125b, which has negative regulatory effects on inflammation, was decreased in cHCV monocytes compared to controls. Stimulation of normal monocytes with TLR4 and TLR8 ligands or HCV core, NS3 and NS5 recombinant proteins induced a robust increase in both miR-155 expression and TNFα production identifying potential mechanisms for in vivo induction of miR-155. Furthermore, we found increased serum miR-155 levels in HCV patients compared to controls. Serum miR-125b and miR-146a levels were also increased in HCV patients. Serum levels of miR-122 were elevated in cHCV patients and correlated with increased ALT and AST levels and serum miR-155 levels. Conclusion In conclusion, our novel data demonstrate that miR-155, a positive regulator of inflammation, is upregulated both in monocytes and in the serum of patients with chronic HCV infection. Our study suggests that HCV core, NS3, and NS5 proteins or TLR4 and TLR8 ligands can mediate increased miR-155 and TNF

  9. Thyroid sialyltransferase mRNA level and activity are increased in Graves' disease.

    PubMed

    Kiljański, Jacek; Ambroziak, Michał; Pachucki, Janusz; Jazdzewski, Krystian; Wiechno, Wieslaw; Stachlewska, Elzbieta; Górnicka, Barbara; Bogdańska, Magdalena; Nauman, Janusz; Bartoszewicz, Zbigniew

    2005-07-01

    Sialylation of cell components is an important immunomodulating mechanism affecting cell response to hormones and adhesion molecules. To study alterations in sialic acid metabolism in Graves' disease (GD) we measured the following parameters in various human thyroid tissues: lipid-bound sialic acid (LBSA) content, ganglioside profile, total sialyltransferase activity, and the two major sialyltransferase mRNAs for sialyltransferase-1 (ST6Gal I) and for sialyltransferase-4A (ST3Gal I). Fragments of toxic thyroid nodules (TN), nontoxic thyroid nodules (NN) and nontumorous tissue from patients with nodular goiter or thyroid cancer were used as a control (C). The LBSA content and sialyltransferase activity were the highest in the GD group (164 +/- 4.44 versus 120 +/- 2.00 nmoL/g, p = 0.005 and 1625 +/- 283.5 versus 324 +/- 54.2 cpm/mg of protein, p < 0.005 compared to control group C). Ganglioside profile in the GD group was similar to that in control tissues. Sialyltransferase- 1 mRNA and sialyltransferase-4A mRNA levels were significantly higher in the GD group than in the control group (12.52 +/- 6.90 versus 2.54 +/- 1.24 arbitrary units, p < 0.005 and 2,49 +/- 1.16 versus 1.23 +/- 0.46 arbitrary units, p < 0.05, respectively). There was a positive correlation between the increased sialyltransferase-1 mRNA level and the TSH-receptor antibody titer determined by the TRAK test. These results indicate that sialyltransferases expression and activity are increased in GD. Exact mechanism of this upregulation remains unknown, though one of possible explanations is the activation of the thyrotropin (TSH) receptor. PMID:16053379

  10. MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer.

    PubMed

    Chang, Chia-Wei; Yu, Jyh-Cherng; Hsieh, Yi-Hsien; Yao, Chung-Chin; Chao, Jui-I; Chen, Po-Ming; Hsieh, Hsiao-Yen; Hsiung, Chia-Ni; Chu, Hou-Wei; Shen, Chen-Yang; Cheng, Chun-Wen

    2016-03-29

    The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3'-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer. PMID:26918943

  11. MicroRNA-30a increases tight junction protein expression to suppress the epithelial-mesenchymal transition and metastasis by targeting Slug in breast cancer

    PubMed Central

    Chang, Chia-Wei; Yu, Jyh-Cherng; Hsieh, Yi-Hsien; Yao, Chung-Chin; Chao, Jui-I; Chen, Po-Ming; Hsieh, Hsiao-Yen; Hsiung, Chia-Ni; Chu, Hou-Wei; Shen, Chen-Yang; Cheng, Chun-Wen

    2016-01-01

    The epithelial-to-mesenchymal (EMT) transition is a prerequisite for conferring metastatic potential during tumor progression. microRNA-30a (miR-30a) expression was significantly lower in aggressive breast cancer cell lines compared with non-invasive breast cancer and non-malignant mammary epithelial cell lines. In contrast, miR-30a overexpression reversed the mesenchymal appearance of cancer cells to result in a cobblestone-like epithelial phenotype. We identified Slug, one of the master regulators of EMT, as a target of miR-30a using in silico prediction. Reporter assays indicated that miR-30a could bind to the 3′-untranslted region of Slug mRNA. Furthermore, we linked miR-30a to increased expression of claudins, a family of tight junction transmembrane proteins. An interaction between Slug and E-box in the claudin promoter sequences was reduced upon miR-30a overexpression, further leading to reduction of filopodia formation and decreased invasiveness/metastasis capabilities of breast cancer cells. Consistently, delivery of miR-30a in xenografted mice decreased tumor invasion and migration. In patients with breast cancer, a significantly elevated risk of the miR-30alow/CLDN2low/FSCNhigh genotype was observed, linking to a phenotypic manifestation of larger tumor size, lymph node metastasis, and advanced tumor stage among patients. In conclusion, the miR-30a/Slug axis inhibits mesenchymal tumor development by interfering with metastatic cancer cell programming and may be a potential target for therapy in breast cancer. PMID:26918943

  12. Spinal muscular atrophy phenotype is ameliorated in human motor neurons by SMN increase via different novel RNA therapeutic approaches.

    PubMed

    Nizzardo, Monica; Simone, Chiara; Dametti, Sara; Salani, Sabrina; Ulzi, Gianna; Pagliarani, Serena; Rizzo, Federica; Frattini, Emanuele; Pagani, Franco; Bresolin, Nereo; Comi, Giacomo; Corti, Stefania

    2015-01-01

    Spinal muscular atrophy (SMA) is a primary genetic cause of infant mortality due to mutations in the Survival Motor Neuron (SMN) 1 gene. No cure is available. Antisense oligonucleotides (ASOs) aimed at increasing SMN levels from the paralogous SMN2 gene represent a possible therapeutic strategy. Here, we tested in SMA human induced pluripotent stem cells (iPSCs) and iPSC-differentiated motor neurons, three different RNA approaches based on morpholino antisense targeting of the ISSN-1, exon-specific U1 small nuclear RNA (ExSpeU1), and Transcription Activator-Like Effector-Transcription Factor (TALE-TF). All strategies act modulating SMN2 RNA: ASO affects exon 7 splicing, TALE-TF increase SMN2 RNA acting on the promoter, while ExSpeU1 improves pre-mRNA processing. These approaches induced up-regulation of full-length SMN mRNA and differentially affected the Delta-7 isoform: ASO reduced this isoform, while ExSpeU1 and TALE-TF increased it. All approaches upregulate the SMN protein and significantly improve the in vitro SMA motor neurons survival. Thus, these findings demonstrate that therapeutic tools that act on SMN2 RNA are able to rescue the SMA disease phenotype. Our data confirm the feasibility of SMA iPSCs as in vitro disease models and we propose novel RNA approaches as potential therapeutic strategies for treating SMA and other genetic neurological disorders. PMID:26123042

  13. Permissive effect of dexamethasone on the increase of proenkephalin mRNA induced by depolarization of chromaffin cells

    SciTech Connect

    Naranjo, J.R.; Mocchetti, I.; Schwartz, J.P.; Costa, E.

    1986-03-01

    In cultured bovine chromaffin cells, changes in the dynamic state of enkephalin stores elicited experimentally were studied by measuring cellular proenkephalin mRNA, as well as enkephalin precursors and authentic enkephalin content of cells and culture media. In parallel, tyrosine hydroxylase mRNA and catecholamine cell content were also determined. Low concentrations (0.5-100 pM) of dexamethasone increased the cell contents of proenkephalin mRNA and enkephalin-containing peptides. High concentrations of the hormone(1 ..mu..M) were required to increase the cell contents of tyrosine hydroxylase mRNA and catecholamines. Depolarization of the cells with 10 ..mu..M veratridine resulted in a depletion of enkephalin and catecholamine stores after 24 hr. The enkephalin, but not the catecholamine, content was restored by 48 hr. An increase in proenkephalin mRNA content might account for the recovery; this increase was curtailed by tetrodotoxin and enhanced by 10 pM dexamethasone. Tyrosine hydroxylase mRNA content was not significantly modified by depolarization, even in the presence of 1 ..mu..M dexamethasone. Aldosterone, progesterone, testosterone, or estradiol (1 ..mu..M) failed to change proenkephalin mRNA. Hence, dexamethasone appears to exert a specific permissive action on the stimulation of the proenkephalin gene elicited by depolarization. Though the catecholamines and enkephalins are localized in the same chromaffin granules and are coreleased by depolarization, the genes coding for the processes that are rate limiting in the production of these neuromodulators can be differentially regulated.

  14. Herpes simplex virus virion stimulatory protein mRNA leader contains sequence elements which increase both virus-induced transcription and mRNA stability.

    PubMed

    Blair, E D; Blair, C C; Wagner, E K

    1987-08-01

    To investigate the role of 5' noncoding leader sequence of herpes simplex virus type 1 (HSV-1) mRNA in infected cells, the promoter for the 65,000-dalton virion stimulatory protein (VSP), a beta-gamma polypeptide, was introduced into plasmids bearing the chloramphenicol acetyltransferase (cat) gene together with various lengths of adjacent viral leader sequences. Plasmids containing longer lengths of leader sequence gave rise to significantly higher levels of CAT enzyme in transfected cells superinfected with HSV-1. RNase T2 protection assays of CAT mRNA showed that transcription was initiated from an authentic viral cap site in all VSP-CAT constructs and that CAT mRNA levels corresponded to CAT enzyme levels. Use of cis-linked simian virus 40 enhancer sequences demonstrated that the effect was virus specific. Constructs containing 12 and 48 base pairs of the VSP mRNA leader gave HSV infection-induced CAT activities intermediate between those of the leaderless construct and the VSP-(+77)-CAT construct. Actinomycin D chase experiments demonstrated that the longest leader sequences increased hybrid CAT mRNA stability at least twofold in infected cells. Cotransfection experiments with a cosmid bearing four virus-specified transcription factors (ICP4, ICP0, ICP27, and VSP-65K) showed that sequences from -3 to +77, with respect to the viral mRNA cap site, also contained signals responsive to transcriptional activation. PMID:3037112

  15. Positive AMPA receptor modulation rapidly stimulates BDNF release and increases dendritic mRNA translation.

    PubMed

    Jourdi, Hussam; Hsu, Yu-Tien; Zhou, Miou; Qin, Qingyu; Bi, Xiaoning; Baudry, Michel

    2009-07-01

    Brain-derived neurotrophic factor (BDNF) stimulates local dendritic mRNA translation and is involved in formation and consolidation of memory. 2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan-10-one (CX614), one of the best-studied positive AMPA receptor modulators (also known as ampakines), increases BDNF mRNA and protein and facilitates long-term potentiation (LTP) induction. Several other ampakines also improve performance in various behavioral and learning tasks. Since local dendritic protein synthesis has been implicated in LTP stabilization and in memory consolidation, this study investigated whether CX614 could influence synaptic plasticity by upregulating dendritic protein translation. CX614 treatment of primary neuronal cultures and acute hippocampal slices rapidly activated the translation machinery and increased local dendritic protein synthesis. CX614-induced activation of translation was blocked by K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], CNQX, APV, and TTX, and was inhibited in the presence of an extracellular BDNF scavenger, TrkB-Fc. The acute effect of CX614 on translation was mediated by increased BDNF release as demonstrated with a BDNF scavenging assay using TrkB-Fc during CX614 treatment of cultured primary neurons and was blocked by nifedipine, ryanodine, and lack of extracellular Ca(2+) in acute hippocampal slices. Finally, CX614, like BDNF, rapidly increased dendritic translation of an exogenous translation reporter. Together, our results demonstrate that positive modulation of AMPA receptors rapidly stimulates dendritic translation, an effect mediated by BDNF secretion and TrkB receptor activation. They also suggest that increased BDNF secretion and stimulation of local protein synthesis contribute to the effects of ampakines on synaptic plasticity. PMID:19587275

  16. Haem is necessary for a continued increase in ferrochelatase mRNA in murine erythroleukaemia cells during erythroid differentiation.

    PubMed

    Fukuda, Y; Fujita, H; Taketani, S; Sassa, S

    1993-12-01

    The level of mRNA encoding ferrochelatase (FeC) was examined in two murine erythroleukaemia (MEL) clones, DS and DR, a DMSO-sensitive, and a DMSO-resistant clone, respectively. DS cells undergo erythroid differentiation by DMSO treatment with a marked increase in haem synthesis, while DR cells fail to do so due to the lack of the erythroid-specific delta-aminolaevulinate synthase (ALAS-E). Both DS and DR cells showed an increase in the level of FeC mRNA within 18 h of DMSO treatment. The level of FeC mRNA in DR cells was then decreased, while that in DS cells continued to increase for 72 h. Treatment with haemin significantly increased FeC mRNA in DR cells. When cells were treated with both DMSO and haemin, the level of FeC mRNA in DR cells increased to a level comparable to that in DS cells. These findings suggest that the failure to maintain increased FeC mRNA DR cells after DMSO treatment may be due to a deficiency of haem in these cells. PMID:7918029

  17. Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43

    PubMed Central

    Koyama, Akihide; Sugai, Akihiro; Kato, Taisuke; Ishihara, Tomohiko; Shiga, Atsushi; Toyoshima, Yasuko; Koyama, Misaki; Konno, Takuya; Hirokawa, Sachiko; Yokoseki, Akio; Nishizawa, Masatoyo; Kakita, Akiyoshi; Takahashi, Hitoshi; Onodera, Osamu

    2016-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder. In motor neurons of ALS, TAR DNA binding protein-43 (TDP-43), a nuclear protein encoded by TARDBP, is absent from the nucleus and forms cytoplasmic inclusions. TDP-43 auto-regulates the amount by regulating the TARDBP mRNA, which has three polyadenylation signals (PASs) and three additional alternative introns within the last exon. However, it is still unclear how the autoregulatory mechanism works and how the status of autoregulation in ALS motor neurons without nuclear TDP-43 is. Here we show that TDP-43 inhibits the selection of the most proximal PAS and induces splicing of multiple alternative introns in TARDBP mRNA to decrease the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. When TDP-43 is depleted, the TARDBP mRNA uses the most proximal PAS and is increased in the cytoplasm. Finally, we have demonstrated that in ALS motor neurons—especially neurons with mislocalized TDP-43—the amount of TARDBP mRNA is increased in the cytoplasm. Our observations indicate that nuclear TDP-43 contributes to the autoregulation and suggests that the absence of nuclear TDP-43 induces an abnormal autoregulation and increases the amount of TARDBP mRNA. The vicious cycle might accelerate the disease progression of ALS. PMID:27257061

  18. Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide-repeat protein deposition.

    PubMed

    Mori, Kohji; Nihei, Yoshihiro; Arzberger, Thomas; Zhou, Qihui; Mackenzie, Ian R; Hermann, Andreas; Hanisch, Frank; Kamp, Frits; Nuscher, Brigitte; Orozco, Denise; Edbauer, Dieter; Haass, Christian

    2016-09-01

    Intronic hexanucleotide (G4C2) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat-dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G4C2 repeat RNA We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci. PMID:27461252

  19. Increased cytoplasmic TARDBP mRNA in affected spinal motor neurons in ALS caused by abnormal autoregulation of TDP-43.

    PubMed

    Koyama, Akihide; Sugai, Akihiro; Kato, Taisuke; Ishihara, Tomohiko; Shiga, Atsushi; Toyoshima, Yasuko; Koyama, Misaki; Konno, Takuya; Hirokawa, Sachiko; Yokoseki, Akio; Nishizawa, Masatoyo; Kakita, Akiyoshi; Takahashi, Hitoshi; Onodera, Osamu

    2016-07-01

    Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disorder. In motor neurons of ALS, TAR DNA binding protein-43 (TDP-43), a nuclear protein encoded by TARDBP, is absent from the nucleus and forms cytoplasmic inclusions. TDP-43 auto-regulates the amount by regulating the TARDBP mRNA, which has three polyadenylation signals (PASs) and three additional alternative introns within the last exon. However, it is still unclear how the autoregulatory mechanism works and how the status of autoregulation in ALS motor neurons without nuclear TDP-43 is. Here we show that TDP-43 inhibits the selection of the most proximal PAS and induces splicing of multiple alternative introns in TARDBP mRNA to decrease the amount of cytoplasmic TARDBP mRNA by nonsense-mediated mRNA decay. When TDP-43 is depleted, the TARDBP mRNA uses the most proximal PAS and is increased in the cytoplasm. Finally, we have demonstrated that in ALS motor neurons-especially neurons with mislocalized TDP-43-the amount of TARDBP mRNA is increased in the cytoplasm. Our observations indicate that nuclear TDP-43 contributes to the autoregulation and suggests that the absence of nuclear TDP-43 induces an abnormal autoregulation and increases the amount of TARDBP mRNA. The vicious cycle might accelerate the disease progression of ALS. PMID:27257061

  20. Increased neutrophil adherence and adhesion molecule mRNA expression in endothelial cells during selenium deficiency.

    PubMed

    Maddox, J F; Aherne, K M; Reddy, C C; Sordillo, L M

    1999-05-01

    Leukocyte aggregation and activation on endothelial cells (EC) are important preliminary events in leukocyte migration into tissue and subsequent inflammation. Thus, an increase in leukocyte adherence has the potential to affect inflammatory disease outcome. Selenium (Se) is an integral part of the antioxidant enzyme glutathione peroxidase (GSH-Px) and plays an important role in the maintenance of the redox state of a cell. Se supplementation in the bovine has been shown to improve the outcome of acute mastitis caused by coliform bacteria, in part by enhancing the speed of neutrophil migration into the affected mammary gland. However, the mechanisms by which Se modulates neutrophil migration have not been elucidated. Therefore, an in vitro model of Se deficiency in primary bovine mammary artery EC was used to examine the impact of Se status on the adhesive properties of EC. The effect of Se on functional activities was examined by measuring neutrophil adherence to Se-deficient and Se-supplemented EC. Se-deficient EC showed significantly enhanced neutrophil adherence when stimulated with tumor necrosis factor alpha (TNF-alpha) for 4 or 24 h, interleukin-1 for 12 h, or H2O2 for 20 min (P < 0.05). To determine the mechanisms underlying these changes in neutrophil adherence, the expression of EC adhesion molecules, ICAM-1, E-selectin, and P-selectin were examined at the molecular level by a competitive reverse transcription-polymerase chain reaction. Results revealed higher mRNA expression for E-selectin and ICAM-1 in Se-deficient EC stimulated with TNF-alpha for 3 and 6 h, and greater expression of P-selectin mRNA in Se-supplemented EC with 3-h TNF-alpha stimulation. These studies provide new information to establish the role of Se nutrition in the initiation of leukocyte adherence to endothelium. PMID:10331495

  1. Increased P-glycoprotein messenger RNA stability in rat liver tumors in vivo.

    PubMed

    Lee, C H; Bradley, G; Ling, V

    1998-10-01

    P-glycoproteins (Pgp) are comprised of a small family of plasma membrane proteins whose abundance in cultured cells is often associated with the multidrug resistance phenotype. Overexpression of Pgp has been observed in many types of human cancers, but the molecular basis for this overexpression has not been established. We have used primary monolayer cultures of adult rat hepatocytes and a stepwise model of rat liver carcinogenesis to study the regulation of Pgp gene expression. We observed a marked overexpression of Pgp, specifically the class II Pgp, in both systems. In addition, we observed that a number of unrelated genes including alpha-tubulin, beta-actin, gamma-actin, cytokeratin 8, cytokeratin 18, and c-myc are overexpressed in cultured hepatocytes, and they are also overexpressed during liver carcinogenesis and in transplantable tumors. Nuclear run-on assays showed no increase in the transcriptional activity of Pgp genes in transplantable liver tumors compared to normal liver. Studies of in vivo mRNA stability, however, revealed that all three Pgp mRNAs were relatively stable in transplantable liver tumors (t(1/2) > 12 h), in contrast to what was found in normal liver (t(1/2) < 2 h). In addition, mRNA for several other genes, including alpha-tubulin, c-myc, and cyclin D1, all appear to be stabilized in the tumors. These findings suggest that the overexpression of Pgp genes in rat liver tumors may be the result of a mechanism involving stabilization of a diverse group of mRNAs. PMID:9731740

  2. Increased B-type-natriuretic peptide promotes myocardial cell apoptosis via the B-type-natriuretic peptide/long non-coding RNA LSINCT5/caspase-1/interleukin 1β signaling pathway

    PubMed Central

    ZHANG, XIAN; SHA, MINGLEI; YAO, YUTING; DA, JIA; JING, DADAO

    2015-01-01

    Chronic heart failure (CHF) is the final stage of various heart diseases, and is increasingly recognized as a major health problem in the elderly. Previous studies demonstrated that B-type-natriuretic peptide (BNP) is an established biomarker of CHF. Furthermore, BNP also regulates cell proliferation, differentiation and apoptosis. Recent evidence has revealed that BNP affects myocardial cell apoptosis during myocardial ischemia-reperfusion injury. Long non-coding RNAs (lncRNAs) are emerging as novel molecular compounds involved in gene regulation, and have important roles in numerous human diseases. However, the mechanism underlying the BNP and lncRNA-induced regulation of myocardial cell apoptosis remains to be elucidated. The present study reported that lncRNA LSINCT5, upregulated by BNP, is able to regulate myocardial cell apoptosis via the activation of the caspase-1/interleukin (IL)-1β signaling pathway. BNP-induced apoptosis of HCM cells was observed using flow cytometry, and involved caspase-1. In addition, expression profiling using a human lncRNA polymerase chain reaction array revealed that LSINCT5 was highly expressed in BNP-treated myocardial cells, as compared with untreated cells. The role of lncRNA LSINCT5 in HCM cell apoptosis was also investigated. The results of the present study indicated that LSINCT5 silencing by small interfering RNA inhibits caspase-1/IL-1β signaling, and suppresses apoptosis in BNP-treated HCM cells. Therefore, high expression levels of BNP promote the apoptosis of myocardial cells through the lncRNA LSINCT5 mediator, which activates the caspase-1/IL-1β signaling pathway. These findings uncovered a novel pathogenic mechanism, and provided a potential therapeutic target for CHF. PMID:26323562

  3. Increased keratinocyte proliferation initiated through downregulation of desmoplakin by RNA interference

    SciTech Connect

    Wan Hong . E-mail: hong.wan@cancer.org.uk; South, Andrew P.; Hart, Ian R.

    2007-07-01

    The intercellular adhesive junction desmosomes are essential for the maintenance of tissue structure and integrity in skin. Desmoplakin (Dp) is a major obligate plaque protein which plays a fundamental role in anchoring intermediate filaments to desmosomal cadherins. Evidence from hereditary human disease caused by mutations in the gene encoding Dp, e.g. Dp haploinsufficiency, suggests that alterations in Dp expression result not only in the disruption of tissue structure and integrity but also could evoke changes in keratinocyte proliferation. We have used transient RNA interference (RNAi) to downregulate Dp specifically in HaCaT keratinocytes. We showed that this Dp downregulation also caused reduced expression of several other desmosomal proteins. Increased cell proliferation and enhanced G{sub 1}-to-S-phase entry in the cell cycle, as monitored by colonial cellular density and BrdU incorporation, were seen in Dp RNAi-treated cells. These proliferative changes were associated with elevated phospho-ERK1/2 and phospho-Akt levels. Furthermore, this increase in phospho-ERK/1/2 and phospho-Akt levels was sustained in Dp RNAi-treated cells at confluence whereas in control cells there was a significant reduction in phosphorylation of ERK1/2. This study indicates that Dp may participate in the regulation of keratinocyte cell proliferation by, in part at least, regulating cell cycle progression.

  4. Acute intermittent morphine increases preprodynorphin and kappa opioid receptor mRNA levels in the rat brain.

    PubMed

    Wang, X M; Zhou, Y; Spangler, R; Ho, A; Han, J S; Kreek, M J

    1999-03-20

    We determined the effects of morphine on mRNA levels for the opioid ligands preprodynorphin (PPD) and preproenkephalin (PPE) and the kappa opioid receptor (KOR). Rats received six injections of morphine (6.25 mg/kg/injection) every 2 h, and were sacrificed 30 min later. mRNA levels were measured in brain tissue after removal of the cortex, cerebellum and brainstem. There were increases in PPD and KOR mRNA levels (P<0.05 and P<0.005, respectively), with no alteration of PPE. These alterations in the kappa/dynorphin system may counter morphine-induced effects on the brain. PMID:10095091

  5. 5-Azacytidine and RNA secondary structure increase the retrovirus mutation rate.

    PubMed Central

    Pathak, V K; Temin, H M

    1992-01-01

    A broad spectrum of mutations occurs at a high rate during a single round of retrovirus replication (V.K. Pathak and H. M. Temin, Proc. Natl. Acad. Sci. USA 87:6019-6023, 1990). We have now determined that this high rate of spontaneous mutation can be further increased by 5-azacytidine (AZC) treatment or by regions of potential RNA secondary structure. We found a 13-fold increase in the mutation rate after AZC treatment of retrovirus-producing cells and target cells. The AZC-induced substitutions were located at the same target sites as previously identified spontaneous substitutions. The concordance of the AZC-induced and spontaneous substitutions indicates the presence of reverse transcription "pause sites," where the growing point is error prone. An analysis of nucleotides that neighbored substitutions revealed that transversions occur primarily by transient template misalignment, whereas transitions occur primarily by misincorporation. We also introduced a 34-bp potential stem-loop structure as an in-frame insertion within a lacZ alpha gene that was inserted in the long terminal repeat (LTR) U3 region and determined whether this potential secondary structure increased the rate of retrovirus mutations. We found a threefold increase in the retrovirus mutation rate. Fifty-seven of 96 mutations were deletions associated with the potential stem-loop. We also determined that these deletion mutations occurred primarily during minus-strand DNA synthesis by comparing the frequencies of mutations in recovered provirus plasmids containing both LTRs and in provirus plasmids containing only one LTR. PMID:1373201

  6. The group A Streptococcus small regulatory RNA FasX enhances streptokinase activity by increasing the stability of the ska mRNA transcript

    PubMed Central

    Ramirez-Peña, Esmeralda; Treviño, Jeanette; Liu, Zhuyun; Perez, Nataly; Sumby, Paul

    2010-01-01

    Summary Small RNA molecules play key regulatory roles in many bacterial species. However, little mechanistic data exists for the action of small regulatory RNAs (sRNAs) in the human pathogen group A Streptococcus (GAS). Here, we analyzed the relationship between a putative GAS sRNA and production of the secreted virulence factor streptokinase (SKA). SKA promotes GAS dissemination by activating conversion of host plasminogen into the fibrin-degrading protease plasmin. Homologues of the putative sRNA-encoding gene fibronectin/fibrinogen-binding/hemolytic-activity/streptokinase-regulator-X (fasX) were identified in four different pyogenic streptococcal species. However, despite 79% fasX nucleotide identity, a fasX allele from the animal pathogen Streptococcus zooepidemicus failed to complement a GAS fasX mutant. Using a series of precisely-constructed fasX alleles we discovered that FasX is a bona-fide sRNA that post-transcriptionally regulates SKA production in GAS. By base-pairing to the 5’ end of ska mRNA, FasX enhances ska transcript stability, resulting in a ~10-fold increase in SKA activity. Our data provide new insights into the mechanisms used by sRNAs to activate target mRNAs, and enhances our understanding of the regulation of a key GAS virulence factor. PMID:21143309

  7. Delivery of Small Interfering RNAs to Cells via Exosomes.

    PubMed

    Wahlgren, Jessica; Statello, Luisa; Skogberg, Gabriel; Telemo, Esbjörn; Valadi, Hadi

    2016-01-01

    Exosomes are small membrane bound vesicles between 30 and 100 nm in diameter of endocytic origin that are secreted into the extracellular environment by many different cell types. Exosomes play a role in intercellular communication by transferring proteins, lipids, and RNAs to recipient cells.Exosomes from human cells could be used as vectors to provide cells with therapeutic RNAs. Here we describe how exogenous small interfering RNAs may successfully be introduced into various kinds of human exosomes using electroporation and subsequently delivered to recipient cells. Methods used to confirm the presence of siRNA inside exosomes and cells are presented, such as flow cytometry, confocal microscopy, and Northern blot. PMID:26472446

  8. Increases of CCK mRNA and peptide in different brain areas following acute and chronic administration of morphine.

    PubMed

    Ding, X Z; Bayer, B M

    1993-10-15

    The present study examined whether either acute or chronic administration of morphine resulted in changes in the content of CCK mRNA and CCK immunoactive peptide in selective areas of the rat brain and spinal cord. Two hours after a single injection of morphine (10 mg/kg, s.c.), CCK mRNA significantly increased in the hypothalamus (0.8-fold) and spinal cord (2-fold) relative to the CCK mRNA content in saline-injected controls. No significant differences in CCK mRNA were observed in the frontal cortex, hippocampus, midbrain or brainstem. There were no significant alterations in CCK immunoreactivity in any brain regions and spinal cord after the acute treatment with morphine. Upon repeated morphine administration, the content of CCK mRNA in both the hypothalamus and the spinal cord was further elevated by at least 3-fold. A significant increase of CCK mRNA content in brain stem (2.8-fold) was also observed following chronic morphine administration. In contrast to the acute exposure to morphine, chronic administration resulted in significant increases in CCK immunoactive peptide in hypothalamus (2.6-fold), spinal cord (2.1-fold) and brainstem (1.6-fold), but not in the other brain areas. These results demonstrate that morphine, especially following repeated administrations, stimulates endogenous CCK biosynthesis in selective brain regions. PMID:8242392

  9. Lack of the Delta Subunit of RNA Polymerase Increases Virulence Related Traits of Streptococcus mutans

    PubMed Central

    Xue, Xiaoli; Sztajer, Helena; Buddruhs, Nora; Petersen, Jörn; Rohde, Manfred; Talay, Susanne R.; Wagner-Döbler, Irene

    2011-01-01

    The delta subunit of the RNA polymerase, RpoE, maintains the transcriptional specificity in Gram-positive bacteria. Lack of RpoE results in massive changes in the transcriptome of the human dental caries pathogen Streptococcus mutans. In this study, we analyzed traits of the ΔrpoE mutant which are important for biofilm formation and interaction with oral microorganisms and human cells and performed a global phenotypic analysis of its physiological functions. The ΔrpoE mutant showed higher self-aggregation compared to the wild type and coaggregated with other oral bacteria and Candida albicans. It formed a biofilm with a different matrix structure and an altered surface attachment. The amount of the cell surface antigens I/II SpaP and the glucosyltransferase GtfB was reduced. The ΔrpoE mutant displayed significantly stronger adhesion to human extracellular matrix components, especially to fibronectin, than the wild type. Its adhesion to human epithelial cells HEp-2 was reduced, probably due to the highly aggregated cell mass. The analysis of 1248 physiological traits using phenotype microarrays showed that the ΔrpoE mutant metabolized a wider spectrum of carbon sources than the wild type and had acquired resistance to antibiotics and inhibitory compounds with various modes of action. The reduced antigenicity, increased aggregation, adherence to fibronection, broader substrate spectrum and increased resistance to antibiotics of the ΔrpoE mutant reveal the physiological potential of S. mutans and show that some of its virulence related traits are increased. PMID:21625504

  10. Heat shock protein 90 stabilizes nucleolin to increase mRNA stability in mitosis.

    PubMed

    Wang, Shao-An; Li, Hao-Yi; Hsu, Tsung-I; Chen, Shu-Hui; Wu, Chin-Jen; Chang, Wen-Chang; Hung, Jan-Jong

    2011-12-23

    Most studies on heat shock protein 90 (Hsp90) have focused on the involvement of Hsp90 in the interphase, whereas the role of this protein in the nucleus during mitosis remains largely unclear. In this study, we found that the level of the acetylated form of Hsp90 decreased dramatically during mitosis, which indicates more chaperone activity during mitosis. We thus probed proteins that interacted with Hsp90 by liquid chromatography/mass spectrometry (LC/MS) and found that nucleolin was one of those interacting proteins during mitosis. The nucleolin level decreased upon geldanamycin treatment, and Hsp90 maintained the cyclin-dependent kinase 1 (CDK1) activity to phosphorylate nucleolin at Thr-641/707. Mutation of Thr-641/707 resulted in the destabilization of nucleolin in mitosis. We globally screened the level of mitotic mRNAs and found that 229 mRNAs decreased during mitosis in the presence of geldanamycin. Furthermore, a bioinformatics tool and an RNA immunoprecipitation assay found that 16 mRNAs, including cadherin and Bcl-xl, were stabilized through the recruitment of nucleolin to the 3'-untranslated regions (3'-UTRs) of those genes. Overall, strong correlations exist between the up-regulation of Hsp90, nucleolin, and the mRNAs related to tumorigenesis of the lung. Our findings thus indicate that nucleolin stabilized by Hsp90 contributes to the lung tumorigenesis by increasing the level of many tumor-related mRNAs during mitosis. PMID:21998300

  11. Increased serum hepcidin-25 level and increased tumor expression of hepcidin mRNA are associated with metastasis of renal cell carcinoma

    PubMed Central

    2009-01-01

    Background Hepcidin has an important role in iron metabolism. We investigated whether hepcidin was involved in renal cell carcinoma (RCC). Methods We measured serum hepcidin-25 levels in 32 patients by liquid chromatograpy (LC)-mass spectrometry (MS)/MS, and assessed hepcidin mRNA expression in paired tumor and non-tumor tissue samples from the surgical specimens of 53 consecutive patients with RCC by real-time reverse transcription polymerase chain reaction. Results The serum hepcidin-25 level was higher in patients with metastatic RCC than nonmetastatic RCC (P < 0.0001), and was positively correlated with the serum interleukin-6 and C-reactive protein levels (P < 0.001). Expression of hepcidin mRNA was lower in tumor tissues than in non-tumor tissues (P < 0.0001). The serum hepcidin-25 level was not correlated with the expression of hepcidin mRNA in the corresponding tumor tissue specimens from 32 patients. Hepcidin mRNA expression in tumor tissue was correlated with metastatic potential, but not with histological differentiation or tumor stage. Kaplan-Meier analysis showed that over expression of hepcidin mRNA was related to shorter overall survival in RCC patients. Univariate analysis (Cox proportional hazards model) showed that the hepcidin mRNA level was an independent prognostic factor for overall survival. Conclusion Our findings suggest that a high serum hepcidin-25 level may indicate the progression of RCC, and that upregulation of hepcidin mRNA expression in tumor tissue may be related to increased metastatic potential. PMID:19656379

  12. Ethanol blocks the cold-induced increase in thyrotropin-releasing hormone mRNA in paraventricular nuclei but not the cold-induced increase in thyrotropin.

    PubMed

    Zoeller, R T; Rudeen, P K

    1992-05-01

    The effects of a single intraperitoneal injection of ethanol (3 g/kg b.wt.) on the hypothalamic-pituitary-thyroid system was explored as a possible explanation of the hypothermic effect of ethanol. Serum thyroid hormones were significantly reduced by ethanol injection, but ethanol did not affect the cold-induced increase in serum thyroid hormones or thyroid-stimulating hormone (TSH). Since cold-exposure stimulates serum levels of TSH and thyroid hormones by stimulating thyroid-releasing hormone (TRH) release from neurons of the PVN, these findings demonstrate that ethanol did not block pituitary response to TRH or thyroid response to TSH. Paradoxically, ethanol increased cellular levels of TRH mRNA in the paraventricular nucleus (PVN), and blocked the cold-induced increase in TRH mRNA, suggesting that ethanol uncouples the regulation of TRH gene expression from the regulation of TRH release specifically in neurons of the PVN. Measurements of the effects of ethanol on TRH mRNA in thalamus, and beta-actin, vasopressin, somatostatin and corticotropin-releasing hormone (CRH) mRNAs in the PVN in addition to TRH mRNA revealed very specific effects of ethanol on the TRH neuronal system. PMID:1352612

  13. The number of preproghrelin mRNA expressing cells is increased in mice with activity-based anorexia.

    PubMed

    François, Marie; Barde, Swapnali; Achamrah, Najate; Breton, Jonathan; do Rego, Jean-Claude; Coëffier, Moïse; Hökfelt, Tomas; Déchelotte, Pierre; Fetissov, Sergueï O

    2015-06-01

    Plasma levels of ghrelin, an orexigenic peptide, are increased during conditions of chronic starvation, such as in patients with anorexia nervosa. However, it is not known whether such increase can be related to the number of preproghrelin mRNA-expressing cells in the stomach, and if chronic starvation may activate a tentative central ghrelin production. In this work, in situ hybridization technique was used to analyze the presence and number of preproghrelin mRNA-expressing cells in the stomach and the hypothalamus of mice with activity-based anorexia (ABA) induced by the combination of running wheel activity with progressive, during 10 days, feeding-time restriction (FTR) and compared with sedentary FTR, ABA pair-fed (PF) and ad libitum-fed control mice. All food-restricted mice lost more than 20% of body weight. Body weight loss was similar in ABA and PF mice, but it was more pronounced than in FTR mice. Food intake was also lower in ABA than in FTR mice. Preproghrelin mRNA-expressing cells in the stomach were increased proportionally to the body weight loss in all food-restricted groups with the highest number in ABA mice. No preproghrelin mRNA-producing cells were detectable in the hypothalamus of either control or food-restricted mice. Thus, the increased number of gastric preproghrelin mRNA-producing cells during chronic starvation proportionally to the body weight loss and reduced food intake may underlie increased plasma ghrelin. Hyperactivity-induced anorexia appears to further increase the number of preproghrelin mRNA-producing cells in the stomach. No evidence was found for ghrelin expression in the hypothalamus, not even in any of the present experimental models. PMID:25963530

  14. Adverse early life experience and social stress during adulthood interact to increase serotonin transporter mRNA expression

    PubMed Central

    Gardner, Katherine L.; Hale, Matthew W.; Lightman, Stafford L.; Plotsky, Paul M.; Lowry, Christopher A.

    2009-01-01

    Anxiety disorders, depression and animal models of vulnerability to a depression-like syndrome have been associated with dysregulation of serotonergic systems in the brain. To evaluate the effects of early life experience, adverse experiences during adulthood, and potential interactions between these factors on serotonin transporter (slc6a4) mRNA expression, we investigated in rats the effects of maternal separation (180 min/day from days 2–14 of life; MS180), neonatal handing (15 min/day from days 2–14 of life; MS15), or normal animal facility rearing control conditions (AFR) with or without subsequent exposure to adult social defeat on slc6a4 mRNA expression in the dorsal raphe nucleus (DR) and caudal linear nucleus. At the level of specific subdivisions of the DR, there were no differences in slc6a4 mRNA expression between MS15 and AFR rats. Among rats exposed to a novel cage control condition, increased slc6a4 mRNA expression was observed in the dorsal part of the DR in MS180 rats, relative to AFR control rats. In contrast, MS180 rats exposed to social defeat as adults had increased slc6a4 mRNA expression throughout the DR compared to both MS15 and AFR controls. Social defeat increased slc6a4 mRNA expression, but only in MS180 rats and only in the “lateral wings” of the DR. Overall these data demonstrate that early life experience and stressful experience during adulthood interact to determine slc6a4 mRNA expression. These data support the hypothesis that early life experience and major stressful life events contribute to dysregulation of serotonergic systems in stress-related neuropsychiatric disorders. PMID:19781533

  15. Mechanical stretch of human uterine smooth muscle cells increases IL-8 mRNA expression and peptide synthesis.

    PubMed

    Loudon, J A Z; Sooranna, S R; Bennett, P R; Johnson, M R

    2004-12-01

    Labour is associated with increased synthesis of interleukin-8 (IL-8) by the fetal membranes and myometrium, which leads to an inflammatory infiltrate. Stretch has been shown to increase the expression of contraction-associated proteins in animal models of labour and in human myocytes in vitro. In this study, we tested the hypothesis that mechanical stretch of human myometrial cells increases IL-8 messenger ribonucleic acid (mRNA) expression. We isolated myocytes from non-pregnant women undergoing hysterectomy and pregnant women undergoing Caesarean section before and after the onset of labour. Myocytes in culture were subjected to stretch of varying intensity (6-16%) and duration (1 or 6 h) using the Flexercell system. IL-8 mRNA expression was lowest in myocytes from pregnant women not in labour, intermediate in those from non-pregnant women and greatest in those from pregnant women in labour. Stretch increased IL-8 mRNA expression independent of reproductive state. The stretch-induced increase in IL-8 mRNA expression was associated with higher IL-8 levels in the culture supernatant and enhanced promoter activity. These data suggest that stretch contributes to the increase in myometrial IL-8 synthesis associated with the onset of labour in humans. PMID:15489245

  16. Long noncoding RNA Saf and splicing factor 45 increase soluble Fas and resistance to apoptosis

    PubMed Central

    Riberdy, Janice M.; Persons, Derek A.; Wilber, Andrew

    2016-01-01

    In multicellular organisms, cell growth and differentiation is controlled in part by programmed cell death or apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Neoplastic cells are frequently resistant to Fas-mediated apoptosis, evade Fas signals through down regulation of Fas and produce soluble Fas proteins that bind FasL thereby blocking apoptosis. Soluble Fas (sFas) is an alternative splice product of Fas pre-mRNA, commonly created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔEx6). Long non-coding RNAs (lncRNAs) interact with other RNAs, DNA, and proteins to regulate gene expression. One lncRNA, Fas-antisense or Saf, was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. We show that Saf is localized in the nucleus where it interacts with Fas receptor pre-mRNA and human splicing factor 45 (SPF45) to facilitate alternative splicing and exclusion of exon 6. The product is a soluble Fas protein that protects cells against FasL-induced apoptosis. Collectively, these studies reveal a novel mechanism to modulate this critical cell death program by an lncRNA and its protein partner. PMID:26885613

  17. Long noncoding RNA Saf and splicing factor 45 increase soluble Fas and resistance to apoptosis.

    PubMed

    Villamizar, Olga; Chambers, Christopher B; Riberdy, Janice M; Persons, Derek A; Wilber, Andrew

    2016-03-22

    In multicellular organisms, cell growth and differentiation is controlled in part by programmed cell death or apoptosis. One major apoptotic pathway is triggered by Fas receptor (Fas)-Fas ligand (FasL) interaction. Neoplastic cells are frequently resistant to Fas-mediated apoptosis, evade Fas signals through down regulation of Fas and produce soluble Fas proteins that bind FasL thereby blocking apoptosis. Soluble Fas (sFas) is an alternative splice product of Fas pre-mRNA, commonly created by exclusion of transmembrane spanning sequences encoded within exon 6 (FasΔEx6). Long non-coding RNAs (lncRNAs) interact with other RNAs, DNA, and proteins to regulate gene expression. One lncRNA, Fas-antisense or Saf, was shown to participate in alternative splicing of Fas pre-mRNA through unknown mechanisms. We show that Saf is localized in the nucleus where it interacts with Fas receptor pre-mRNA and human splicing factor 45 (SPF45) to facilitate alternative splicing and exclusion of exon 6. The product is a soluble Fas protein that protects cells against FasL-induced apoptosis. Collectively, these studies reveal a novel mechanism to modulate this critical cell death program by an lncRNA and its protein partner. PMID:26885613

  18. Chimeric DNA-RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo.

    PubMed Central

    Taylor, N R; Kaplan, B E; Swiderski, P; Li, H; Rossi, J J

    1992-01-01

    Subsequent to the discovery that RNA can have site specific cleavage activity, there has been a great deal of interest in the design and testing of trans-acting catalytic RNAs as both surrogate genetic tools and as therapeutic agents. We have been developing catalytic RNAs or ribozymes with target specificity for HIV-1 RNA and have been exploring chemical synthesis as one method for their production. To this end, we have chemically synthesized and experimentally analyzed chimeric catalysts consisting of DNA in the non-enzymatic portions, and RNA in the enzymatic core of hammerhead type ribozymes. Substitutions of DNA for RNA in the various stems of a hammerhead ribozyme have been analyzed in vitro for kinetic efficiency. One of the chimeric ribozymes used in this study, which harbors 24 bases of DNA capable of base-pairing interactions with an HIV-1 gag target, but maintains RNA in the catalytic center and in stem-loop II, has a sixfold greater kcat value than the all RNA counterpart. This increased activity appears to be the direct result of enhanced product dissociation. Interestingly, a chimeric ribozyme in which stem-loop II (which divides the catalytic core) is comprised of DNA, exhibited a marked reduction in cleavage activity, suggesting that DNA in this region of the ribozyme can impart a negative effect on the catalytic function of the ribozyme. DNA-RNA chimeric ribozymes transfected by cationic liposomes into human T-lymphocytes are more stable than their all-RNA counterparts. Enhanced catalytic turnover and stability in the absence of a significant effect on Km make chimeric ribozymes favorable candidates for therapeutic agents. Images PMID:1408757

  19. Chronic neonatal nicotine exposure increases mRNA expression of neurotrophic factors in the postnatal rat hippocampus.

    PubMed

    Son, Jong-Hyun; Winzer-Serhan, Ursula H

    2009-06-30

    Nicotine, the psychoactive ingredient in tobacco, can be neuroprotective but the mechanism is unknown. In the adult hippocampus, chronic nicotine can increase expression of growth factors which could contribute to nicotine's neuroprotective effects. During development, nicotine could also increase expression of neurotrophic factors. Therefore, we determined whether chronic neonatal nicotine (CNN) exposure increased mRNA expression levels of brain-derived neurotrophic factor (BDNF), nerve-growth factor (NGF), neurotrophin-3 (NT-3), fibroblast growth factor-2 (FGF-2), and insulin-like growth factor-1 (IGF-1). Nicotine (6 mg/kg/day in milk formula) or milk formula (controls) were delivered in three daily doses via oral gastric intubation to rat pups from postnatal day (P)1 to P8, and then sacrificed. Brains were processed for in situ hybridization using specific (35)S-labeled cRNA probes. At P8, CNN had a significant stimulant treatment effect on the expression of BDNF, FGF-2, NT-3 and IGF-1 [p<0.01], but not NGF. Specifically, BDNF mRNA expression, detected in CA1, CA3 stratum (s.) pyramidal and granule cell layer of the dentate gyrus (DG), was increased by 27.4%, 23.26% and 27.3%, respectively. FGF-2 mRNA expression, detected in neurons and astrocytes in CA1 s. radiatum, CA2 and CA3 s. pyramidale, and molecular layer of the DG, was increased by 34.0%, 8.9%, 31.0% and 23.1%, respectively. NT-3 mRNA expression in CA2 s. pyramidale was increased by 80.0%, and CNN increased the number of IGF-1-expressing cells in CA1 (18.0%), CA3 (20.9%) and DG (17.7%). Thus, nicotine exposure during early postnatal development differentially up-regulated expression of neurotrophic factor mRNAs in the hippocampus, which could increase neurotrophic tone and alter developmental processes. PMID:19410565

  20. Sulindac increases the expression of APC mRNA in malignant colonic epithelial cells: an in vitro study.

    PubMed Central

    Schnitzler, M; Dwight, T; Robinson, B G

    1996-01-01

    BACKGROUND--Sulindac is a non-steroidal anti-inflammatory drug which induces regression of colonic polyps in patients with familial adenomatous polyposis. Animal and in vitro studies have shown that both the sulphide metabolite of sulindac, which is able to inhibit cyclo-oxygenase, and the sulphone metabolite, which lacks this ability, are able to inhibit the growth of colonic carcinoma cells. The exact mechanism by which these effects occurs is not known. AIMS--To examine the effect of sulindac sulphide and sulindac sulphone on the expression of APC messenger RNA (mRNA), and on the proliferation of colonic carcinoma cells in vitro. METHODS--The colonic carcinoma cell line LIM 1215 was treated with sulindac sulphide and sulindac sulphone (10 microM or 100 microM) for 24 hours. Total RNA was extracted and APC mRNA was quantitated using competitive reverse transcription polymerase chain reaction. Measurements of cell number, cell proliferation, and prostaglandin E2 concentrations were also made. RESULTS--A significant increase in APC mRNA was observed after treatment with 10 microM of both sulindac sulphide and sulindac sulphone (control: 37.2 (19.7); 10 microM sulindac sulphide: 129 (112.8); 10 microM sulindac sulphone: 207.7 (102.9) pg/(g total RNA) (p < 0.05). Prostaglandin E2 concentrations were significantly reduced after treatment with sulindac sulphide, but not after sulindac sulphone. Both agents produced a dose dependent reduction in cell numbers and cell proliferation, which was more noticeable after treatment with sulindac sulphide. CONCLUSIONS--Both sulindac sulphide and sulindac sulphone inhibit the growth of carcinoma cells in vitro and cause an increase in APC mRNA. The effect of these agents on colonic carcinogenesis is not mediated entirely by means of an inhibition of prostaglandin biosynthesis. Images Figure 1 PMID:8707116

  1. Increase in plasma leptin and Lep mRNA concentrations by food intake is dependent on insulin.

    PubMed

    Patel, B K; Koenig, J I; Kaplan, L M; Hooi, S C

    1998-05-01

    Obese (Lep) gene expression and leptin secretion are regulated by changes in food intake. However, the mechanism by which leptin concentrations are altered by fasting and feeding is unclear. Since these changes occur in parallel with changes in plasma insulin, it is possible that the changes observed are mediated by insulin. To test this hypothesis, we studied the role of insulin in the regulation of Lep gene expression in epididymal fat and leptin secretion during feeding. As shown previously, fasted animals showed significant reductions in Lep mRNA, plasma leptin, and plasma insulin concentrations. Conversely, feeding increased plasma insulin, Lep mRNA, and plasma leptin. In streptozotocin (STZ)-treated animals, plasma insulin concentrations were low. This was associated with low Lep mRNA and plasma leptin concentrations. Changes in food intake, whether fasting or feeding, did not significantly alter plasma insulin levels in STZ-treated animals. Under these circumstances, Lep mRNA and plasma leptin concentrations also remained low. Our results demonstrate that the decrease in Lep mRNA and plasma leptin during fasting and the increase with feeding are dependent on changes in the plasma insulin concentration. PMID:9591754

  2. Brain-derived peptides increase blood-brain barrier GLUT1 glucose transporter gene expression via mRNA stabilization.

    PubMed

    Boado, R J

    1998-10-23

    The present investigation studied the effect of the brain-derived peptide preparation Cerebrolysin (CI, EBEWE, Austria) on the turnover rate and gene expression of the blood-brain barrier (BBB) GLUT1 glucose transporter mRNA. Studies were performed in brain endothelial cultured cells transfected with the human (h) GLUT1 transcript. In control cells, the full length 2.8 Kb hGLUT1 mRNA was rapidly degraded following transfection, and the abundance of this transcript at 4 and 6 h was comparable to background mRNA levels seen in cells transfected without hGLUT1 mRNA. On the contrary, the decay of the hGLUT1 mRNA was stabilized in CI-treated cells resulting in a marked reduction in the fractional turnover rate (72.4 and 4.0%/h, control and CI, respectively). In parallel experiments, CI induced a significant increase in the levels of immunoreactive GLUT1 protein measured by enzyme-linked immunosorbent assay (ELISA). In conclusion, data presented here demonstrate that factors in CI increase BBB-GLUT1 transcript stability, and that this is associated with an induction of BBB-GLUT1 gene expression in brain endothelial cultured cells. PMID:9832194

  3. Interactions between PTB RRMs induce slow motions and increase RNA binding affinity

    PubMed Central

    Maynard, Caroline M

    2010-01-01

    Polypyrimidine tract binding protein (PTB) participates in a variety of functions in eukaryotic cells, including alternative splicing, mRNA stabilization, and internal ribosomal entry site (IRES) mediated translation initiation. Its mechanism of RNA recognition is determined in part by the novel geometry of its two C-terminal RNA Recognition Motifs (RRM3 and RRM4), which interact with each other to form a stable complex (PTB1:34). This complex itself is unusual among RRMs, suggesting that it performs a specific function for the protein. In order to understand the advantage it provides to PTB, the fundamental properties of PTB1:34 are examined here as a comparative study of the complex and its two constituent RRMs. Both RRM3 and RRM4 adopt folded structures that NMR data show to be similar to their structure in PRB1:34. The RNA binding properties of the domains differ dramatically. The affinity of each separate RRM for polypyrimidine tracts is far weaker than that of PTB1:34, and simply mixing the two RRMs does not create an equivalent binding platform. 15N-NMR relaxation experiments show that PTB1:34 has slow, microsecond motions throughout both RRMs including the interdomain linker. This is in contrast to the individual domains, RRM3 and RRM4, where only a few backbone amides are flexible on this timescale. The slow backbone dynamics of PTB1:34, induced by packing of RRM3 and RRM4, could be essential for high affinity binding to a flexible polypyrimidine tract RNA and also provide entropic compensation for its own formation. PMID:20080103

  4. Lactobacillus acidophilus Increases the Anti-apoptotic Micro RNA-21 and Decreases the Pro-inflammatory Micro RNA-155 in the LPS-Treated Human Endothelial Cells.

    PubMed

    Kalani, Mehdi; Hodjati, Hossein; Sajedi Khanian, Mahdi; Doroudchi, Mehrnoosh

    2016-06-01

    Given the anti-inflammatory and protective role of probiotics in atherosclerosis and the regulatory role of micro RNA (miRNA) in endothelial cell (dys) functions, this study aimed to investigate the effect of Lactobacillus acidophilus (La) on cellular death and the expression of miRNA-21, 92a, 155, and 663 in human umbilical vein endothelial cell (HUVEC) induced by Escherichia coli lipopolysaccharide (Ec-LPS). LPS-treated and untreated HUVECs were cultured in the presence of different La conditions such as La-conditioned media (LaCM), La water extract (LaWE), La culture-filtered (LaFS) and unfiltered supernatants (LaUFS). After 24 h, apoptosis, necrosis and the levels of the mentioned miRNAs were measured using flow cytometry and real-time PCR methods, respectively. LaCM decreased apoptosis, necrosis and inflammatory miR-155 and conversely increased anti-apoptotic miR-21 in Ec-LPS-treated HUVECs. Association analysis revealed negative correlations between necrosis and the levels of miR-21, miR-92a, and miR-155. The beneficial effects of L. acidophilus on the ECs death and expression of atherosclerosis related miRNAs in these cells imply a new aspect of its regulation in cardiovascular diseases rather than previously described ones and suggest this probiotic bacterium as a candidate in the preventative therapy of atherosclerosis. PMID:27107761

  5. Systemic oxidative DNA and RNA damage are not increased during early phases of psychosis: A case control study.

    PubMed

    Nordholm, Dorte; Poulsen, Henrik Enghusen; Hjorthøj, Carsten; Randers, Lasse; Nielsen, Mette Ø; Wulff, Sanne; Krakauer, Kristine; Nørbak-Emig, Henrik; Henriksen, Trine; Glenthøj, Birte; Nordentoft, Merete

    2016-07-30

    It has been suggested that patients with schizophrenia develop higher levels of oxidative stress, which may contribute to deteriorating mental illness. In order to examine oxidative stress in the early stages of severe mental illness, we examined the levels of systemic Deoxyribonucleic Acid (DNA) and Ribonucleic Acid (RNA) oxidation, 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine, perceived stress and recent life events in patients at ultra high-risk (UHR) of developing psychosis, in antipsychotic naïve patients with first-episode schizophrenia (FES), and in healthy controls. We included 41 UHR patients, 35 FES patients, and 29 healthy controls. There was no difference in the level of DNA/RNA oxidative damage between UHR patients and FES patients compared with healthy controls. We found no association between levels of DNA/RNA oxidative damage and perceived stress/life events. Based on the results, we suggest that DNA and RNA oxidative markers are not increased during the early stages of illness, but further longitudinal studies in first-episode psychosis should be carried out to examine whether DNA and RNA oxidative damage are potential markers of severe illness. PMID:27183105

  6. Chronic anemic hypoxemia increases plasma glucagon and hepatic PCK1 mRNA in late-gestation fetal sheep.

    PubMed

    Culpepper, Christine; Wesolowski, Stephanie R; Benjamin, Joshua; Bruce, Jennifer L; Brown, Laura D; Jonker, Sonnet S; Wilkening, Randall B; Hay, William W; Rozance, Paul J

    2016-07-01

    Hepatic glucose production (HGP) normally begins just prior to birth. Prolonged fetal hypoglycemia, intrauterine growth restriction, and acute hypoxemia produce an early activation of fetal HGP. To test the hypothesis that prolonged hypoxemia increases factors which regulate HGP, studies were performed in fetuses that were bled to anemic conditions (anemic: n = 11) for 8.9 ± 0.4 days and compared with control fetuses (n = 7). Fetal arterial hematocrit and oxygen content were 32% and 50% lower, respectively, in anemic vs. controls (P < 0.005). Arterial plasma glucose was 15% higher in the anemic group (P < 0.05). Hepatic mRNA expression of phosphonenolpyruvate carboxykinase (PCK1) was twofold higher in the anemic group (P < 0.05). Arterial plasma glucagon concentrations were 70% higher in anemic fetuses compared with controls (P < 0.05), and they were positively associated with hepatic PCK1 mRNA expression (P < 0.05). Arterial plasma cortisol concentrations increased 90% in the anemic fetuses (P < 0.05), but fetal cortisol concentrations were not correlated with hepatic PCK1 mRNA expression. Hepatic glycogen content was 30% lower in anemic vs. control fetuses (P < 0.05) and was inversely correlated with fetal arterial plasma glucagon concentrations. In isolated primary fetal sheep hepatocytes, incubation in low oxygen (3%) increased PCK1 mRNA threefold compared with incubation in normal oxygen (21%). Together, these results demonstrate that glucagon and PCK1 may potentiate fetal HGP during chronic fetal anemic hypoxemia. PMID:27170658

  7. Small interfering ribonucleic acid induces liquid-to-ripple phase transformation in a phospholipid membrane

    SciTech Connect

    Choubey, Amit; Nomura, Ken-ichi; Kalia, Rajiv K.; Nakano, Aiichiro; Vashishta, Priya

    2014-09-15

    Small interfering ribonucleic acid (siRNA) molecules play a pivotal role in silencing gene expression via the RNA interference mechanism. A key limitation to the widespread implementation of siRNA therapeutics is the difficulty of delivering siRNA-based drugs to cells. Here, we examine changes in the structure and dynamics of a dipalmitoylphosphatidylcholine bilayer in the presence of a siRNA molecule and mechanical barriers to siRNA transfection in the bilayer. Our all-atom molecular dynamics simulation shows that siRNA induces a liquid crystalline-to-ripple phase transformation in the bilayer. The ripple phase consists of a major region of non-interdigitated and a minor region of interdigitated lipid molecules with an intervening kink. In the ripple phase, hydrocarbon chains of lipid molecules have large compressive stresses, which present a considerable barrier to siRNA transfection.

  8. Nanostructures created by interfered femtosecond laser

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Chang, Yun-Ching; Yao, Jimmy; Luo, Claire; Yin, Shizhuo; Ruffin, Paul; Brantley, Christina; Edwards, Eugene

    2011-10-01

    The method by applying the interfered femtosecond laser to create nanostructured copper (Cu) surface has been studied. The nanostructure created by direct laser irradiation is also realized for comparison. Results show that more uniform and finer nanostructures with sphere shape and feature size around 100 nm can be induced by the interfered laser illumination comparing with the direct laser illumination. This offers an alternative fabrication approach that the feature size and the shape of the laser induced metallic nanostructures can be highly controlled, which can extremely improve its performance in related application such as the colorized metal, catalyst, SERS substrate, and etc.

  9. Elevated Intracellular Calcium Increases Ferritin H Expression Through an NFAT-Independent Posttranscriptional Mechanism Involving mRNA Stabilization

    PubMed Central

    MacKenzie, Elizabeth L.; Tsuji, Yoshiaki

    2009-01-01

    An increase in intracellular Ca2+ is one of the initiating events in T cell activation. A calcium-mediated signaling cascade in T cells involves activation of calcineurin and the dephosphorylation and translocation of Nuclear Factor of Activated T-cells (NFAT), resulting in the transcriptional activation of target genes such as IL-2. In the present study, we found that increased intracellular calcium leads to induction of the antioxidant protein ferritin H. We previously reported that the ferritin H gene is transcriptionally activated under oxidative stress conditions through an antioxidant responsive element (ARE). The facts that the ferritin H ARE contains a composite AP1 site, and that NFAT collaborates with AP1 transcription factors, led us to test whether calcium-activated NFAT is involved in the ferritin H induction through the ARE. Treatment of Jurkat T cells with the calcium ionophore, ionomycin, increased ferritin H mRNA and protein expression. Though NFAT translocated to the nucleus and bound a consensus NFAT sequence located in the IL-2 promoter following ionomycin treatment, it did not activate ferritin H transcription despite the presence of a putative NFAT binding sequence in the ferritin H ARE. In addition, the calcineurin inhibitor cyclosporin A treatment blocked ionomycin-mediated NFAT nuclear translocation but failed to abrogate the increase in ferritin H mRNA. Analysis of mRNA stability following actinomycin D treatment revealed that ionomycin prolongs ferritin H mRNA half-life. Taken together, these results suggest that ionomycin-mediated induction of ferritin H may occur in an NFAT-independent manner but through posttranscriptional stabilization of the ferritin H mRNA. PMID:18076382

  10. Cholesterol-based cationic liposome increases dsRNA protection of yellow head virus infection in Penaeus vannamei.

    PubMed

    Sanitt, Poohrawind; Apiratikul, Nuttapon; Niyomtham, Nattisa; Yingyongnarongkul, Boon-Ek; Assavalapsakul, Wanchai; Panyim, Sakol; Udomkit, Apinunt

    2016-06-20

    Protection of shrimp from yellow head virus (YHV) infection has been demonstrated by injection and oral delivery of dsRNA-YHV protease gene (dsYHV) or shrimp endogenous gene (dsRab7). However, to achieve complete viral suppression and to prolong dsRNA activity, the development of an effective dsRNA delivery system is required. In this study, four cationic liposomes were synthesized and tested for their ability to increase dsRNA efficiency. The results demonstrated that entrapping dsYHV in a cholesterol-based cationic liposome gave the best protection against YHV infection when compared with other cationic lipids. The cholesterol-based cationic liposome-dsYHV (Chol-dsYHV) complex conferred YHV protection in a dose-dependent manner. Injection with Chol-dsYHV at 0.05μg dsYHV/g shrimp could give comparable level of YHV protection to the injection with 1.25μg naked dsYHV/g shrimp. The shrimp injected with Chol- dsYHV at 1.25μg dsRNA/g shrimp showed only 50% mortality at 60days post injection whereas the naked dsYHV at the same concentration gave 90% mortality. Thus, the liposome-entrapped dsYHV could lower an effective dsRNA concentration in viral protection and prolong dsRNA activity. In addition, encapsulating dsRab7 in the cholesterol-based cationic liposome could protect the dsRab7 from enzymatic digestion, and continuous feeding the shrimp with the diet formulated with the liposome-entrapped dsRab7 for 4days in the total of 960μg dsRab7/g shrimp could enhance YHV protection efficiency compared with the naked dsRab7. Our studies reveal that cholesterol-based cationic liposome is a promising dsRNA carrier to enhance dsRNA efficiency in both injection and oral delivery systems. PMID:27140871