RNA干扰在疾病治疗方面的应用研究

RNA干扰是由双链RNA引起的序列特异的基因沉默现象。由于RNA干扰能在细胞组织及动物模型中沉默疾病相关基因,因此,RNA干扰也是各种疾病治疗的有效手段。在哺乳动物细胞内诱导RNA干扰可以通过导入小干扰RNA（siRNA）,或是以质粒、病毒为载体表达短的发夹RNA（shRNA）而实现。本文介绍了RNA干扰在疾病治疗方面的应用,并就其面临的挑战进行讨论。     

针对leader序列的siRNA能够有效抑制SARS冠状病毒多个mRNA的表达

小干扰RNAs(siRNAs)能够有效降解具有互补序列的RNA.在SARS-CoV的基因组RNA和所有亚基因组RNA的5′端均有一段共同的leader序列,而且该leader序列在不同的病毒分离物中高度保守,因此leader序列可作为一个用于抑制SARS-CoV复制的有效靶点.研究表明,针对leader序列化学合成的siRNA和DNA载体表达的shRNA都可以有效抑制SARS-CoV mRNA的表达.Leader序列特异的siRNA或shRNA不仅可以有效抑制leader与报告基因EGFP融合基因的表达,而且还可以有效抑制leader与刺突蛋白(spikeprotein)、膜蛋白(membrane protein)和核衣壳蛋白(nucleocapsid protein)基因的融合转录产物的表达.结果表明,针对leader序列的RNA干扰可以发展成为一种抗SARS-CoV治疗的有效策略.     

siRNA Targeting the 2Apro Genomic Region Prevents Enterovirus 71 Replication In Vitro

Enterovirus 71 (EV71) is the most important etiological agent of hand, foot, and mouth disease (HFMD) in young children, which is associated with severe neurological complications and has caused significant mortalities in recent HFMD outbreaks in Asia. However, there is no effective antiviral therapy against EV71. In this study, RNA interference (RNAi) was used as an antiviral strategy to inhibit EV71 replication. Three small interfering RNAs (siRNAs) targeting the 2A^pro region of the EV71 genome were designed and synthesized. All the siRNAs were transfected individually into rhabdomyosarcoma (RD) cells, which were then infected with strain EV71-2006-52-9. The cytopathic effects (CPEs) in the infected RD cells, cell viability, viral titer, and viral RNA and protein expression were examined to evaluate the specific viral inhibition by the siRNAs. The results of cytopathogenicity and MTT tests indicated that the RD cells transfected with the three siRNAs showed slight CPEs and significantly high viability. The 50% tissue culture infective dose (TCID₅₀) values demonstrated that the viral titer of the groups treated with three siRNAs were lower than those of the control groups. qRT–PCR and western blotting revealed that the levels of viral RNA and protein in the RD cells treated with the three siRNAs were lower than those in the controls. When RD cells transfected with siRNAs were also infected with strain EV71-2008-43-16, the expression of the VP1 protein was significantly inhibited. The levels of interferon α (IFN-α) and IFN-β did not differ significantly in any group. These results suggest that siRNAs targeting the 2A^pro region of the EV71 genome exerted antiviral effects in vitro.     

miR-545 promoted enterovirus 71 replication via directly targeting phosphatase and tensin homolog and tumor necrosis factor receptor-associated factor 6

Enterovirus 71 (EV71) is a small, nonenveloped icosahedral RNA virus and is the predominant causative pathogen of hand-foot-and-mouth disease. Recently, microRNAs (miRNAs) are reported to play important roles in the pathogenesis of EV71 replication. This study investigated the role of miR-545 in the EV71 replication and explored the underlying molecular mechanisms. We showed that miR-545 was upregulated in the EV71-infected human embryonic kidney (HEK) 293 cells and rhabdomyosarcoma (RD) cells. Overexpression of miR-545 promoted the viral replication of EV71 and attenuated the inhibitory effects of EV71 on cell viability in HEK293 and RD cells; while knockdown of miR-545 significantly suppressed the EV71 replication in these two cell lines. Bioinformatics analysis and luciferase reporter assay showed that miR-545 directly targeted the 3′untranslated region of phosphatase and tensin homolog (PTEN) and tumor necrosis factor receptor-associated factor 6 (TRAF6) in HEK293 cells. Furthermore, miR-545 negatively regulated the messenger RNA (mRNA) and protein expression of PTEN and TRAF6. The mRNA and protein expression of PTEN and TRAF6 was also suppressed by EV71 infection, which was attenuated by miR-545 knockdown in HEK293 cells. Overexpression of PTEN and TRAF6 both suppressed the EV71 replication in HKE293 cells, and also attenuated the enhanced effects of miR-545 overexpression on the EV71 replication in HEK293 cells. Collectively, our study for the first time showed that miR-545 had an enhanced effect on the EV71 replication in HEK293 and RD cells. Further mechanistic results indicated that miR-545 promoted EV71 replication at least partly via targeting PTEN and TRAF6.     

Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of the viral genome

RNA interference (RNAi) is a phenomenon in which small interfering RNA (siRNA), an RNA duplex 21 to 23 nucleotides (nt) long, or short hairpin RNA (shRNA) resembling siRNA, mediates degradation of the target RNA molecule in a sequence-specific manner. RNAi is now expected to be a useful therapeutic strategy for hepatitis C virus (HCV) infection. In the present study we compared the efficacy of a number of shRNAs directed against different target regions of the HCV genome, such as 5'-untranslated region (5'UTR) (nt 286 to 304), Core (nt 371 to 389), NS3-1 (nt 2052 to 2060), NS3-2 (nt 2104 to 2122), and NS5B (nt 7326 to 7344), all of which except for NS5B are conserved among most, if not all, HCV subtype 1b (HCV-1b) isolates in Japan. We utilized two methods to express shRNAs, one utilizing an expression plasmid (pAVU6+27) and the other utilizing a recombinant lentivirus harboring the pAVU6+27-derived expression cassette. Although 5'UTR has been considered to be the most suitable region for therapeutic siRNA and/or shRNA because of its extremely high degree of sequence conservation, we observed only a faint suppression of an HCV subgenomic replicon by shRNA against 5'UTR. In both plasmid-and lentivirus-mediated expression systems, shRNAs against NS3-1 and NS5B suppressed most efficiently the replication of the HCV replicon without suppressing host cellular gene expression. Synthetic siRNA against NS3-1 also inhibited replication of the HCV replicon in a dose-dependent manner. Taken together, the present results imply the possibility that the recombinant lentivirus expressing shRNA against NS3-1 would be a useful tool to inhibit HCV-1b infection.     

Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and MicroRNA biogenesis

Although inhibition of RNA interference (RNAi) by plant virus proteins has been shown to enhance viral replication and pathogenesis in plants, no viral gene product has as yet been shown to inhibit RNAi in vertebrate cells. Here, we present evidence demonstrating that the highly structured approximately 160-nucleotide adenoviral VA1 noncoding RNA can inhibit RNAi at physiological levels of expression. VA1, which is expressed at very high levels in adenovirus-infected cells, potently inhibited RNAi induced by short hairpin RNAs (shRNAs) or human microRNA precursors but did not affect RNAi induced by artificial short interfering RNA duplexes. Inhibition appeared to be due both to inhibition of nuclear export of shRNA or premicro-RNA precursors, competition for the Exportin 5 nuclear export factor, and inhibition of Dicer function by direct binding of Dicer. Together, these data argue that adenovirus infection can result in inhibition of RNAi and identify VA1 RNA as the first viral gene product able to inhibit RNAi in human cells.     

PolyC-Binding Protein 1 Interacts with 5′-Untranslated Region of Enterovirus 71 RNA in Membrane-Associated Complex to Facilitate Viral Replication

Enterovirus 71 (EV71) is one causative agent of hand, foot, and mouth disease (HFMD), which may lead to severe neurological disorders and mortality in children. EV71 genome is a positive single-stranded RNA containing a single open reading frame (ORF) flanked by 5′-untranslated region (5′UTR) and 3′UTR. The 5′UTR is fundamentally important for virus replication by interacting with cellular proteins. Here, we revealed that poly(C)-binding protein 1 (PCBP1) specifically binds to the 5′UTR of EV71. Detailed studies indicated that the RNA-binding K-homologous 1 (KH1) domain of PCBP1 is responsible for its binding to the stem-loop I and IV of EV71 5′UTR. Interestingly, we revealed that PCBP1 is distributed in the nucleus and cytoplasm of uninfected cells, but mainly localized in the cytoplasm of EV71-infected cells due to interaction and co-localization with the viral RNA. Furthermore, sub-cellular distribution analysis showed that PCBP1 is located in ER-derived membrane, in where virus replication occurred in the cytoplasm of EV71-infected cells, suggesting PCBP1 is recruited in a membrane-associated replication complex. In addition, we found that the binding of PCBP1 to 5′UTR resulted in enhancing EV71 viral protein expression and virus production so as to facilitate viral replication. Thus, we revealed a novel mechanism in which PCBP1 as a positive regulator involved in regulation of EV71 replication in the host specialized membrane-associated replication complex, which provides an insight into cellular factors involved in EV71 replication.     

Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

Multiple shRNA expressing vector enhances efficiency of gene silencing

RNA interference (RNAi) is the process of sequence-specific gene silencing. However, RNAi efficiency still needs to be improved for effective inhibition of target genes. We have developed an effective strategy to express multiple shRNAs (small hairpin RNA) simultaneously using multiple RNA Polymerase III (Pol III) promoters in a single vector. Our data demonstrate that multiple shRNAs expressed from Pol III promoters have a synergistic effect in repressing the target gene. Silencing of endogenous cyclophilin A (CypA) or key HIV viral genes by multiple shRNAs results in significant inhibition of the target gene.     

非完全互补shRNA对RNA聚合酶II启动子调控的RNAi的影响

RNA干扰（RNAi）是一种转录后基因沉默技术,可有效诱导序列特异性基因沉默．由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA可有效介导RNAi效应,为组织特异性基因沉默提供了一条新的途径．但是,由RNA聚合酶Ⅱ启动子调控表达的小发卡RNA（shRNA）在序列上与靶基因非完全互补对RNAi效应的影响鲜有报道．本文初步探索RNA聚合酶Ⅱ启动子调控表达的shRNA碱基发生突变或缺失对RNAi效应的影响．研究表明,靶向hTERT mRNA的碱基突变shRNA显著降低RNAi效应,而靶向GFP mRNA的碱基缺失shRNA对RNAi效应没有显著影响．本研究为非完全互补shRNA对RNAi效应的进一步深入研究提供了理论与实验依据．     

Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery

RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.     

Interactome analysis of the EV71 5′ untranslated region in differentiated neuronal cells SH‐SY5Y and regulatory role of FBP3 in viral replication

Enterovirus 71 (EV71), a single‐stranded RNA virus, is one of the most serious neurotropic pathogens in the Asia‐Pacific region. Through interactions with host proteins, the 5′ untranslated region (5′UTR) of EV71 is important for viral replication. To gain a protein profile that interact with the EV71 5′UTR in neuronal cells, we performed a biotinylated RNA‐protein pull‐down assay in conjunction with LC–MS/MS analysis. A total of 109 proteins were detected and subjected to Database for Annotation, Visualization and Integrated Discovery (DAVID) analyses. These proteins were found to be highly correlated with biological processes including RNA processing/splicing, epidermal cell differentiation, and protein folding. A protein–protein interaction network was constructed using the STRING online database to illustrate the interactions of those proteins that are mainly involved in RNA processing/splicing or protein folding. Moreover, we confirmed that the far‐upstream element binding protein 3 (FBP3) was able to bind to the EV71 5′UTR. The redistribution of FBP3 in subcellular compartments was observed after EV71 infection, and the decreased expression of FBP3 in host neuronal cells markedly inhibited viral replication. Our results reveal various host proteins that potentially interact with the EV71 5′UTR in neuronal cells, and we found that FBP3 could serve as a positive regulator in host cells.     

Human immunodeficiency virus type 1 escape is restricted when conserved genome sequences are targeted by RNA interference

RNA interference (RNAi) is a cellular mechanism in which small interfering RNAs (siRNAs) mediate sequence-specific gene silencing by cleaving the targeted mRNA. RNAi can be used as an antiviral approach to silence the human immunodeficiency virus type 1 (HIV-1) through stable expression of short-hairpin RNAs (shRNAs). We previously reported efficient HIV-1 inhibition by an shRNA against the nonessential nef gene but also described viral escape by mutation or deletion of the nef target sequence. The objective of this study was to obtain insight in the viral escape routes when essential and highly conserved sequences are targeted in the Gag, protease, integrase, and Tat-Rev regions of HIV-1. Target sequences were analyzed of more than 500 escape viruses that were selected in T cells expressing individual shRNAs. Viruses acquired single point mutations, occasionally secondary mutations, but—in contrast to what is observed with nef—no deletions were detected. Mutations occurred predominantly at target positions 6, 8, 9, 14, and 15, whereas none were selected at positions 1, 2, 5, 18, and 19. We also analyzed the type of mismatch in the siRNA-target RNA duplex, and G-U base pairs were frequently selected. These results provide insight into the sequence requirements for optimal RNAi inhibition. This knowledge on RNAi escape may guide the design and selection of shRNAs for the development of an effective RNAi therapy for HIV-1 infections.     

Short interfering RNA-directed inhibition of hepatitis B virus replication

RNA interference (RNAi) is the process by which double-stranded RNA directs sequence-specific degradation of mRNA. In mammalian cells, RNAi can be triggered by 21-nucleotide duplexes of short interfering RNA (siRNA). We examined effects of siRNA on hepatitis B virus (HBV) replication. Human hepatoma cells were transfected with HBV DNA and siRNA against HBV-pregenome RNA. Transfection experiments demonstrated that the siRNA reduced the amount of HBV-pregenome RNA and resulted in reduction of the levels of replicative intermediates and viral protein. Our results indicate that siRNA-mediated gene silencing inhibits HBV replication through suppression of viral RNA, which may be useful as a potential therapeutic modality.     

Far upstream element binding protein 1 binds the internal ribosomal entry site of enterovirus 71 and enhances viral translation and viral growth

Enterovirus 71 (EV71) is associated with severe neurological disorders in children, and has been implicated as the infectious agent in several large-scale outbreaks with mortalities. Upon infection, the viral RNA is translated in a cap-independent manner to yield a large polyprotein precursor. This mechanism relies on the presence of an internal ribosome entry site (IRES) element within the 5'-untranslated region. Virus-host interactions in EV71-infected cells are crucial in assisting this process. We identified a novel positive IRES trans-acting factor, far upstream element binding protein 1 (FBP1). Using binding assays, we mapped the RNA determinants within the EV71 IRES responsible for FBP1 binding and mapped the protein domains involved in this interaction. We also demonstrated that during EV71 infection, the nuclear protein FBP1 is enriched in cytoplasm where viral replication occurs. Moreover, we showed that FBP1 acts as a positive regulator of EV71 replication by competing with negative ITAF for EV71 IRES binding. These new findings may provide a route to new anti-viral therapy.     

Inhibition of gammaherpesvirus replication by RNA interference

RNA interference (RNAi) is a conserved mechanism in which double-stranded, small interfering RNAs (siRNAs) trigger a sequence-specific gene-silencing process. Here we describe the inhibition of murine herpesvirus 68 replication by siRNAs targeted to sequences encoding Rta, an immediate-early protein known as an initiator of the lytic viral gene expression program, and open reading frame 45 (ORF 45), a conserved viral protein. Our results suggest that RNAi can block gammaherpesvirus replication and ORF 45 is required for efficient viral production.