Small Double-stranded RNA Research Articles

Current Advances in Small Activating RNAs for Gene Therapy: Principles, Applications and Challenges.

Small activating RNAs (saRNAs) are small double-stranded RNAs that could mediate the target-specific gene expression by targeting selected sequences in gene promoters at both the transcriptional and epigenetic levels. This phenomenon of gene manipulation is known as RNA activation (RNAa), which opens up a new pathway for RNA-based gene therapeutics in contrast to RNA interference. Although the exact molecular mechanism of RNAa mediated by saRNAs still remains foggy, some studies have provided the possible ones to explain it. Furthermore, mounting evidence exhibit that saRNAs not only provide a new approach to study gene function and manipulate transcriptional activity, but also promise a great potential for clinical therapy against various diseases, especially cancer. Cancer-associated genes could be up-regulated by saRNAs to modulate cell cycle and proliferation, induce cell senescence and apoptosis, inhibit cancer cell invasion and migration, and reverse chemotherapy resistance. Herein, we summarize the known mechanisms of saRNAs on up-regulating specific gene expression and focus on the potential applications of saRNAs in gene therapy. In addition, some concerns about mechanisms and challenges for delivery of saRNAs are involved in this review. The precise mechanisms of saRNAs need to be further illustrated and some novel delivery systems for saRNAs are expected to be developed for clinical applications.

Upregulation of E‑cadherin expression mediated by a novel dsRNA suppresses the growth and metastasis of bladder cancer cells by inhibiting β-catenin/TCF target genes.

Low expression levels of E-cadherin are correlated with poor prognosis in patients with bladder cancer (BCa). A small activating RNA (saRNA) targeting a specific promoter region can activate gene expression. In the present study, two small double-stranded RNAs (dsRNAs) targeting the promoter region of human E-cadherin were designed and synthesized, and the regulatory role of saRNAs in E-cadherin expression was investigated. The results of reverse transcription-quantitative polymerase chain reaction and western blotting demonstrated that transfection of dsEcad-346 into the BCa cell lines T24 and 5637 significantly activated E-cadherin expression. Furthermore, transfection of dsEcad-346 and miR-373 induced cell cycle arrest in G0/G1 phase, promoted apoptosis and significantly inhibited migration and invasion of BCa cells. Results of immunofluorescence and western blotting indicated that β-catenin was redistributed from the nucleus to the cell membrane following transfection of dsEcad-346 and miR-373. Additionally, the expression of β-catenin/T-cell factor complex (TCF) target genes (c-MYC, matrix metallopeptidase 2, cyclin D1) was suppressed following transfection of BCa cells with saRNA. Silencing of E-cadherin expression blocked the inhibitory effect of dsEcad-346 and miR-373 on BCa cells. In conclusion, a novel designed dsEcad-346 can activate the expression of E-cadherin in BCa cells. saRNA-mediated activation of E-cadherin expression inhibited the growth and metastasis of BCa cells by promoting the redistribution of β-catenin from nucleus to cell membrane and inhibiting the β-catenin/TCF target genes.

Activation of KLF4 expression by small activating RNA promotes migration and invasion in colorectal epithelial cells.

RNA activation mediated by small double-stranded RNAs targeting promoter sequence named small activating RNAs (saRNAs) is one of the mechanisms for gene activation. Artificial regulation of gene expression through RNA activation does not affect the alteration of the genomic DNA sequences or exogenous plasmid DNA, therefore it is a relative manageable approach for gene perturbation. KLF4 is a member of zinc-finger transcription factors and its functions in colorectal cells are still controversial. In order to elucidate the functions of KLF4, we synthesized saRNAs that target the promoter regions of KLF4 and transfected into varied colorectal epithelial cell lines. We found the KLF4 gene expression is specifically increased in the human normal epithelial cell NCM460 and colorectal epithelial cancer cell Caco-2 and HCT116, but not in other human colorectal epithelial cell lines. In addition, we observed that saRNAs induced overexpression of KLF4 could promote cell migration/invasion in NCM460 and HCT116 cell lines. This effect is mediated partly by inducing EMT and facilitating nuclear translocation of β-catenin.

Anti-adenoviral Artificial MicroRNAs Expressed from AAV9 Vectors Inhibit Human Adenovirus Infection in Immunosuppressed Syrian Hamsters.

Infections of immunocompromised patients with human adenoviruses (hAd) can develop into life-threatening conditions, whereas drugs with anti-adenoviral efficiency are not clinically approved and have limited efficacy. Small double-stranded RNAs that induce RNAi represent a new class of promising anti-adenoviral therapeutics. However, as yet, their efficiency to treat hAd5 infections has only been investigated in vitro. In this study, we analyzed artificial microRNAs (amiRs) delivered by self-complementary adeno-associated virus (scAAV) vectors for treatment of hAd5 infections in immunosuppressed Syrian hamsters. In vitro evaluation of amiRs targeting the E1A, pTP, IVa2, and hexon genes of hAd5 revealed that two scAAV vectors containing three copies of amiR-pTP and three copies of amiR-E1A, or six copies of amiR-pTP, efficiently inhibited hAd5 replication and improved the viability of hAd5-infected cells. Prophylactic application of amiR-pTP/amiR-E1A- and amiR-pTP-expressing scAAV9 vectors, respectively, to immunosuppressed Syrian hamsters resulted in the reduction of hAd5 levels in the liver of up to two orders of magnitude and in reduction of liver damage. Concomitant application of the vectors also resulted in a decrease of hepatic hAd5 infection. No side effects were observed. These data demonstrate anti-adenoviral RNAi as a promising new approach to combat hAd5 infection.

Comparison of different methods for the establishment of RNA silencing in plants

RNA interference (RNAi) occurs naturally in plants and is involved in developmental gene regulation as well as in transposon silencing and defence against viruses. RNAi is initiated by small double-stranded RNAs (dsRNAs) of different origins, eventually leading to the silencing of genes with complementary sequences. Besides its use as a basic tool to study gene function, RNAi has widely been exploited for the generation of pathogen resistant crops and for modifying plant metabolism. For the elucidation of gene functions, silencing-inducing dsRNA is often delivered transiently by means of agroinfiltration. This method is independent of plant regeneration and allows rapid testing of multiple silencing constructs. To get more insight into the applicability of transient gene silencing, we compared a variety of different transient and stable approaches to induce silencing of the β-glucuronidase (gus) transgene by the expression of gus hairpin (hpGus) transcripts. While stable expression of the hairpin always resulted in specific gene silencing, transiently expressed hpGus transcripts could only trigger silencing when the hairpin construct was introduced simultaneously with or prior to the target gene.

Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells.

It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.

Small activating RNA upregulates NIS expression: promising potential for hepatocellular carcinoma endoradiotherapy.

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide. Currently, the clinical strategies available for the treatment of HCC remain insufficient for the poor prognosis. Sodium/iodide symporter (NIS)-based radioiodine therapy is proposed as a promising therapeutic strategy for the treatment of HCC. However, it is difficult for HCC cells to trap iodine for the lower expression of NIS. Small activating RNA (saRNA) is a newly identified small double-stranded RNA (dsRNA) that can induce endogenous gene expression by targeting promoter sequences. Here, we designed an saRNA (saRNA-482) that targeted the NIS promoter sequences. In the cultured HepG2 cells and Hep3B cells, the expressions of NIS were upregulated after transfection of saRNA-482. In addition, the uptake of 125I increased in the cultured HepG2 and Hep3B cells transfected with saRNA-482. Furthermore, the cell viabilities were significantly inhibited in the saRNA-482-transfected HepG2 and Hep3B cells after 131I treatment. Meanwhile, the apoptosis of saRNA-482-transfected HepG2 and Hep3B cells significantly increased after 131I treatment. The results suggest that RNA activation-mediated upregulation of NIS may have an endoradiotherapeutic potential in the treatment of HCC.

Transcriptional regulation of E-cadherin by small activating RNA: A new double-stranded RNA.

Recent studies have reported that chemically synthesized small activating RNA (saRNA) targeting the promoter regions of a gene can activate its expression in different cell lines. This technique can be a powerful therapeutic method for diseases caused by complete inactivation or reduced expression of specific genes. E-cadherin is a typical tumor suppressor gene. Loss of E-cadherin mediates the transition from benign lesions to invasive, metastatic cancer. In this study, several 21-nt small double-stranded RNAs (dsRNAs) targeting the promoter regions of human E-cadherin were designed and synthesized and the features of their function were investigated to study the regulatory role of dsRNA on E-cadherin expression. A new saRNA (dsEcad‑661) that can enhance E-cadherin expression by targeting non-coding regulatory regions in gene promoters was identified. Using dsRNA with modified base quantity and cholesterol-conjugated dsRNA, we found the antisense strand may be the guide strand of saRNA in the upregulation of E-cadherin. These findings provide several important pieces of evidence that may improve understanding of the function of saRNA and may promote its development for clinical application.

Effect of synthetic small double-stranded RNA on the development of bladder cancer by activating P21 expression

To investigate the effects of a synthetic small double-stranded RNA (dsRNA) dsP21-555 on the development of bladder cancer cell lines T24 and EJ. According to the different treatments, bladder cancer cells were divided into three groups: negative control group (transfected with dsControl), positive control group (transfected with candidate microRNA, i.e. miR-370) and experimental group (transfected with dsP21-555). Real-time fluorescent quantitative polymerase chain reaction (qPCR) was conducted to detect the expressions of p21 mRNA and cyclin-dependent kinases 4/6 (CDK4/6) mRNA; Western blot was operated to verify the expression of P21 and CDK4/6 proteins. Cell cycle distribution was measured by flow cytometry after transfection. Cell proliferation assay was performed to evaluate the proliferative capacity of transfected cells. Colony formation assay was carried out to analyze the proliferative ability of single cancer cells. qPCR showed that, compared with the negative control group, dsP21-555 up-regulated the expressions of p21 mRNA by 2.46 times (P<0.01) in T24 cells and 2.60 times (P<0.01) in EJ cells; compared with the positive control group, the expression of p21 mRNA was no significantly different in the experimental group (P>0.05). Compared with the dsControl group, dsP21-555 suppressed the expressions of CDK4 mRNA by 43% (P<0.01) in T24 and 54% (P<0.01) in EJ cells, the expression of CDK6 mRNA by 39% (P<0.01) in T24 and 36% (P<0.01) in EJ cells; the differences in the expression of CDK4 and CDK6 mRNAs between the miR-370 and dsP21-555 groups were not statistically significant (P> 0.05). Western blot verified the differences of p21 and CDK4/6 genes expression among groups. Flow cytometry revealed that the G0/G1 phase cells significantly increased while S and G2/M phase cells decreased in the miR-370 and the dsP21-555 groups, compared with the dsControl group. Cell proliferation assay showed that, compared with the dsControl group, the proliferative capacities of cells transfected with miR-370 or dsP21-555 decreased significantly (both P<0.05), but the difference in proliferative capacities between the miR-370 and the dsP21-555 groups was no statistically significant (P>0.05). Colony formation assay showed that the numbers of colonies formed in the miR-370 and the dsP21-555 groups were both smaller than that in the dsControl group. dsP21-555 may activate the expression of P21 protein by RNA activation, thereby significantly inhibit the growth of bladder cancer cells.

Upregulation of PAWR by small activating RNAs induces cell apoptosis in human prostate cancer cells.

RNA activation (RNAa) is a promising discovery whereby expression of a particular gene can be induced by targeting its promoter using small double-stranded RNAs (dsRNAs) also termed small activating RNAs (saRNAs). We previously reported that several small dsRNAs targeting the PRKC apoptosis WT1 regulator (PAWR) promoter can upregulate PAWR gene expression effectively in human cancer cells. The present study was conducted to evaluate the antitumor potential of PAWR gene induction by these saRNAs in prostate cancer cells. Promisingly, we found that upregulation of PAWR by saRNA inhibited the growth of prostate cancer cells by inducing cell apoptosis which was related to inactivation of the NF-κB and Akt pathways. The decreased anti‑apoptotic protein Bcl-2 and activation of the caspase cascade and poly(ADP-ribose) polymerase (PARP) also supported the efficacy of the treatment. Overall, these data suggest that activation of PAWR by saRNA may have a therapeutic benefit for prostate and other types of cancer.

Conditional RNAi Using the Lentiviral GLTR System.

RNA interference (RNAi) has become an essential technology for functional gene analysis. Its success depends on the effective expression of target gene-specific RNAi-inducing small double-stranded interfering RNA molecules (siRNAs). Here, were describe the use of a recently developed lentiviral RNAi system that allows the rapid generation of stable cell lines with inducible RNAi based on conditional expression of double-stranded short hairpin RNA (shRNA). These lentiviral vectors can be generated rapidly using the GATEWAY recombination cloning technology. Conditional cell lines can be established by using either a two-vector system in which the regulator is encoded by a separate vector or by a one-vector system. The available different lentiviral vectors for conditional shRNA expression cassette delivery co-express additional genes that allow (1) the use of fluorescent proteins for color-coded combinatorial RNAi or monitoring RNAi induction (pGLTR-FP), (2) selection of transduced cells (pGLTR-S), and (3) the generation of conditional cell lines using a one-vector system (pGLTR-X).

Targeted p21WAF1/CIP1 activation by miR-1236 inhibits cell proliferation and correlates with favorable survival in renal cell carcinoma

BackgroundmicroRNA’s function to silence gene expression by targeting 3′UTR regions has been widely studied. And microRNAs, similar to small double-stranded RNAs, have also been implicated to gene activation triggered by promoter-targeted, which is known as RNA activation (RNAa). p21WAF1/CIP1 (p21), as a key negative regulator of cell proliferation, is frequently down-regulated in various cancers, making it an ideal target for RNAa-based activation to restore its tumor suppressor function in renal cell carcinoma (RCC). MethodsReal-time polymerase chain reaction was used to identify the expression of miR-1236 and p21 in RCC cell lines and clinical specimens. Protein expression and signaling pathway modulation were validated through Western blot analysis, whereas p21, direct target of miR-1236, was validated by using chromatin immunoprecipitation assay. The Kaplan-Meier method and the log-rank test were used to calculate overall survival. The CellTiter 96® AQueous One Solution Cell Proliferation Assay, colony formation assay, and 5-ethynyl-2′-deoxyuridine assays were used to evaluate the effect of miR-1236 on RCC cell proliferation. ResultsIn this study, we found that miR-1236 and p21 were both significantly down-regulated in RCC tissues and cell lines. Combined low expression of both miR-1236 and p21 emerged as an independent prognostic factor for poor clinical survival in 45 patients with RCC. Chromatin immunoprecipitation assay in the human RCC cell lines 786-O and ACHN showed that miR-1236 directly targeted the p21 promoter and that its activation may provide a plausible link between the positive correlation of miR-1236 and p21 in RCC specimens. Functional experiments showed that increased miR-1236 expression inhibited cell proliferation, and decreased CDK4/6 and cyclin D1 expression. Furthermore, knockdown of p21 using small interfering RNA reversed the antiproliferation function of miR-1236, whereas silencing the p21 expression attenuated the function of miR-1236 in RCC cells. ConclusionOur findings provide insight into the specific RNAa of miR-1236–targeted p21 promoter activation in suppressing RCC cell proliferation. Considering the poor prognostic outcomes associated with reduced miR-1236 and p21 expression, our data imply that these factors likely play important antitumor effects in RCC progression.

Abstract 3107: MicroRNA-1289 is a novel tumor suppressive microRNA in human oral squamous cell carcinoma

Abstract MicroRNAs (miRNAs) are small non-coding double-stranded RNA with sizes of approximately 22 nucleotides, and those inhibit protein translation by binding the 3′-untranslated region of target mRNAs. Each miRNA generally can regulate multiple mRNAs and each mRNA can be inhibited by a number of miRNAs. MiRNAs can behave as not only oncogenes but also tumor suppressive (TS) genes in human malignancies. In fact, a number of TS miRNAs (TS-miRs) were found in human many types of cancer, such as let-7, miR-1, miR-34, miR-145, miR-200, miR-206 and more. In this study, we have attempted to identify a novel TS-miR in human oral squamous cell carcinoma (OSCC) using function-based screening. First, we transfected human OSCC cells (GFP-SAS) with approximately 1,000 synthetic mimicking human mature miRNAs at the concentration of 20 nM complexed with Lipofectamine RNAiMAX. After transfection for 72 hours, the growth of these cells was evaluated by WST-8 assay. As a result, miR-1289 had the most potent growth inhibitory effect against GFP-SAS cells. The growth rate of these cells transfected with miR-1289 was reduced by 86.4%. MiR-1289 also remarkably inhibited the growth of other human OSCC cells (Ca9-22, HSC2, HSC3 and primary cultured cells). Subsequently, we assessed the growth inhibitory effect of miR-1289 in vivo using a mouse model. GFP-SAS cells (2 × 106) complexed with matrigel in 100 μl aliquots were injected subcutaneously in nude mice. After tumor formation, we administered 100 ng of miRNA with 0.5% atelocollagen complexes (100 μl) around tumor every 3 days for a total of 5 injections. MiR-1289/atelocollagen complex administration group significantly reduced the size of subcutaneously xenografted GFP-SAS tumors compared with the control group. Next, we investigated the expression of miR-1289 in OSCC tissues by real-time quantitative RT-PCR. The expression levels of miR-1289 were significant decreased in OSCC tissues compared to adjacent normal oral mucosa tissues. Finally, target genes of miR-1289 were explored by microarray and Ingenuity Pathway Analysis (IPA). Microarray analysis revealed that the expression levels of 51 genes were commonly down-regulated in OSCC cells (GFP-SAS, Ca9-22 and HSC2) by transfection with miR-1289. Among these genes, 15 genes were identified as target genes of miR-1289 by IPA. These results suggest that miR-1289 functions as a novel TS-miR in OSCC, and may be a useful therapeutic tool for the patients with OSCC. Citation Format: Kazuki Iwamoto, Koh-ichi Nakashiro, Hiroshi Tnaka, Norihiko Tokuzen, Hiroyuki Hamakawa. MicroRNA-1289 is a novel tumor suppressive microRNA in human oral squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3107. doi:10.1158/1538-7445.AM2015-3107

RNase L Cleavage Products Promote Switch from Autophagy to Apoptosis by Caspase-Mediated Cleavage of Beclin-1.

Autophagy and apoptosis share regulatory molecules enabling crosstalk in pathways that affect cellular homeostasis including response to viral infections and survival of tumor cells. Ribonuclease L (RNase L) is an antiviral endonuclease that is activated in virus-infected cells and cleaves viral and cellular single-stranded RNAs to produce small double-stranded RNAs with roles in amplifying host responses. Activation of RNase L induces autophagy and apoptosis in many cell types. However, the mechanism by which RNase L mediates crosstalk between these two pathways remains unclear. Here we show that small dsRNAs produced by RNase L promote a switch from autophagy to apoptosis by caspase-mediated cleavage of Beclin-1, terminating autophagy. The caspase 3-cleaved C-terminal fragment of Beclin-1 enhances apoptosis by translocating to the mitochondria along with proapoptotic protein, Bax, and inducing release of cytochrome C to the cytosol. Cleavage of Beclin-1 determines switch to apoptosis since expression of caspase-resistant Beclin-1 inhibits apoptosis and sustains autophagy. Moreover, inhibiting RNase L-induced autophagy promotes cell death and inhibiting apoptosis prolongs autophagy in a cross-inhibitory mechanism. Our results demonstrate a novel role of RNase L generated small RNAs in cross-talk between autophagy and apoptosis that impacts the fate of cells during viral infections and cancer.

Small RNAs inhibit bladder cancer by up-regulating tumor suppressor genes

RNA activation (RNAa) is a newly discovered mechanism in which non-coding RNAs like small double-stranded RNAs (dsRNAs) or micro RNAs (miRNAs) induce sequence-specific gene activation by targeting promoter. Although its underlying mechanism remains unclear, we and others have demonstrated that Ago protein, RNA polymerase II (RNA Pol II) and heterogeneous nuclear ribonucleoprotein A2/B1 (hnRNPA2/B1) are required for RNAa. In addition, RNAa is conserved in mammalian cells. Increasing evidences indicated that dsRNAs or miRNAs can induce tumor suppressing genes expression and hold great capacity to inhibit bladder cancer cells. RNAa provides a novel method for gene manipulation and offers an exciting potential for therapeutic modality against bladder cancers. In this review, we will focus on the research advances in exploiting the mechanism of RNAa and its applications in bladder cancer therapeutics.

Immunotherapy of hepatocellular carcinoma with small double-stranded RNA

BackgroundHepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with limited therapeutic options. Since HCC has been shown to be immunogenic, immunotherapy is considered a promising therapeutic approach. Small interfering RNAs (siRNAs), depending on their structure and sequence, can trigger the innate immune system, which can potentially enhance the adaptive anticancer immune response in the tumor-bearing subjects. Immunostimulatory properties of nucleic acids can be applied to develop adjuvants for HCC treatment.MethodsThe transplantable HCC G-29 tumor in male CBA/LacSto (CBA) mice was used to study the effects of immunostimulatory RNA on tumor growth. Tumor size, metastases area in different organs of mice and mouse survival rate were analyzed. Furthermore the mouse serum IFN-α levels were measured using ELISA.ResultsIn the present study, we found that a 19-bp RNA duplex (ImmunoStimulattory RNA or isRNA) with 3-nt overhangs at the 3′-ends of specific sequence displays immunostimulatory, antitumor, and antimetastatic activities in mice bearing HCC G-29. Our results demonstrate that isRNA strongly increases the level of interferon-α (IFN-α) by up to 25-fold relative to the level in mice injected with Lipofectamine alone (Mock), and to a lesser extent increases the level of proinflammatory cytokine interleukin-6 (IL-6) (by up to 5.5-fold relative to the Mock level), in mice blood serum. We showed that isRNA reliably (P < 0.05) inhibits primary tumor growth in mice compared to the mock group. Furthermore, injections of isRNA significantly enhanced necrotic processes in the center of the primary tumor, and decreased by twofold the width of the undifferentiated peripheral zone and the number of mitotic cells in this zone. The results showed that isRNA efficiently reduces the area of metastases in the liver, kidneys, and heart of CBA/LacSto mice with HCC.ConclusionsThe obtained results clearly demonstrate immunostimulatory and antimetastatic properties of the isRNAs in mice with HCC. Consequently, this short double-stranded RNA can be considered as a potential adjuvant for the therapy of HCC.

Competition between HIV-1-encoded RRE RNA and miRNA-TRBP interactions alters RNA interference activity and gene expression

Aim RNA interference (RNAi) is a mechanism by which small double-stranded RNAs called micro(mi) or small interfering(si) RNAs bind messenger RNAs (mRNAs) to inhibit their expression. The mechanism involves the RNA-induced silencing complex (RISC) composed of Dicer, TRBP and Ago2 proteins in which TRBP loads miRNAs into the active complex [1]. Several mammalian viruses interfere with RNAi activity. Changes in miRNA and mRNA expression have been observed in patients infected by HIV-1, but the mechanisms are not understood. To explain part of the relationship between RNAi and HIV-1, we investigated the ability and the mechanism of the HIV-1-encoded RNA Rev-Response Element (RRE) to suppress RNAi.

Upregulation of RECK gene expression by small double-stranded RNA targeting the promoter region

Recent studies have demonstrated that small double-stranded RNAs (dsRNAs) complementary to the promoter region of target genes enhance the expression of those genes following transfection into cells. Here we show that expression of the matrix metalloproteinase (MMP) inhibitor RECK is activated in the cultured tumor cell lines by transfection with dsRNA complementary to the promoter of the RECK gene, leading to suppression of the expression of MMPs and it inhibited tumor cell invasion. These results support the suggestion that dsRNA complementary to the promoter region of tumor suppressor genes would have potential as a novel antitumor agent.

Small RNAs: the qualified candidates for gene manipulation in diverse clinical pathologies.

In recent years, RNA interference (RNAi) has become a tool of choice to analyze and target the in vitro and in vivo function of mammalian genes. RNAi down-regulates gene expression by inducing enzyme-dependent degradation of targeted mRNA. This can be achieved using small double-stranded RNAs (dsRNAs), including small interfering RNAs (siRNAs), micro RNAs, and piwi RNAs. These active, small dsRNAs can regulate endogenous genes in both somatic and germ cells such that they can defend the genome from invasive nucleic acids. Extension of the concept of RNAi to preclinical studies indicates its probable application in the treatment of cancers, viral infections, arterial stenosis, and genetic disorders. Exciting results from ongoing clinical trials have raised the expectations of the scientific community for the use of RNAi in "real cures". Rational pharmaceutical design of these molecules can further open a Pandora's box of newer therapeutic options. However, efficient delivery of these small dsRNAs to target tissues or cells and their unanticipated nonspecific effects comprise a few important issues that still need to be addressed. In this review, we offer an overview of different small dsRNAs, their biogenesis, and their applicability in therapeutics. Current delivery strategies and formulation techniques to achieve the desired transfection capability and RNAi products in clinical trials are also discussed.

Small Double-Stranded RNA Mediates the Anti-Cancer Effects of p21WAF1/ClP1Transcriptional Activation in a Human Glioma Cell Line

PurposeThis study was conducted to investigate the small double-stranded RNA (dsRNA) mediated anti-tumor effects of p21WAF1/ClP1 (p21) transcriptional activation in vitro in the human glioma SHG-44 cell line.Materials and MethodsHuman glioma SHG-44 cells were transfected with dsRNA using LipofectAMINE 2000 transfection reagent. Real-time PCR and Western blot analysis were conducted to detect p21 and survivin mRNA and protein levels, respectively. Cell proliferation was examined by MTT assay. Cell cycle distribution and apoptosis were detected by flow-cytometric analysis.ResultsWe found that dsRNA targeting p21 promoter (dsP21) significantly induced the expression of p21 at transcription and protein levels, and reduced the expression of survivin. AS well, dsP21 transcription significantly inhibited human glioma SHG-44 cell proliferation. Analysis of cell cycle distribution revealed that dsP21 transfection increased accumulation of cells in the G0/G1 phase and reduced accumulation of cells in the S phase. Further analysis revealed that dsP21 transcription led to an increase in both early and late stages of apoptosis in human glioma SHG-44 cells.ConclusionIn the present study, P21 activation by RNA-induced gene activation (RNAa) induced anti-tumor activity in vitro in a human glioma SHG-44 cell line. The results suggested that RNAa could be used for human glioma treatment by targeted activation of tumor suppressor genes.