应用RNA干扰沉默猪内源性反转录病毒

目的:尝试应用RNA干扰(RNAi)沉默猪源PK-15细胞中的猪内源性反转录病毒(PERV),并通过反转录酶活性及pol基因相对荧光定量PCR检测沉默效果。方法:依据GenBank公布的PERV pol基因序列,采用Invitro-gen公司的BLOCK-iT RNAi Designer软件设计Stealth小干扰RNA(siRNA)序列;将合成的siRNA转染PK-15细胞,72 h后检测细胞上清PERV反转录酶活性及细胞内pol基因拷贝数并评价沉默效果。结果:反转录酶活性及pol基因拷贝数检测结果表明,设计的3条Stealth siRNA序列中,位于pol基因3272～3296 bp的序列能有效沉默PERV。结论:RNAi方法可有效使猪源PK-15细胞中的PERV沉默,为进一步研究天然抗病毒分子与PERV的相互作用提供了实验基础,同时也为猪源异种移植研究中去除PERV提供了一种可供尝试的方法。     

短双链RNA在病毒性心肌炎小鼠模型中的抗病毒研究

目的:通过在小鼠病毒性心肌炎动物模型研究短双链小干扰RNA(small interfering RNA,siRNA)对病毒感染和复制的抑制作用,研究RNAi在治疗病毒性疾病的可行性.方法:利用质粒载体将siRNA转染至HeLa细胞和Balb-c小鼠后感染病毒,荧光显微镜现察GFP表达量观察细胞内质粒转染效率和持续时间,通过病毒致细胞病变作用(CPE)保护实验病毒空斑形成实验检测病毒受抑制程度,动物模型中观察动物死亡率和易感组织病理变化评价siRNA的保护作用.结果:在HeLa细胞中针对CVB3 2B区的siRNA能显著抑制柯萨奇病毒B3的感染和复制,抑制率可达90%.动物模型中sigNA质粒可改善动物存活率(30%),并降低易感脏器中病毒含量,减轻病理反应.结论:针对CVB3基因组2B区的siRNA在病毒性心肌炎动物模型中具有保护作用.     

针对2B区域的短双链RNA抑制柯萨奇B3病毒感染HeLa细胞的特性研究

为了研究短双链RNA(Small interfering RNA,siRNA)对柯萨奇B组3型病毒(CVB3)复制的影响及其作用特性,合成针对CVB3基因组2B区的siRNA-2B,脂质体法转染HeLa细胞后感染CVB3病毒,观测转染效率及存留时间、毒性作用、病毒致细胞病变效应、病毒滴度、病毒RNA含量、siRNA-2B对重组基因的特异性降解及培养上清有限稀释后再感染情况.结果发现siRNA-2B能高效转染入HeLa细胞并存留长达48h,高剂量的siRNA-2B对培养细胞无明显毒性,siRNA-2B能特异性针对2B区有效地降解病毒RNA,能明显抑制病毒RNA的复制.随着转染浓度的增加,siRNA-2B的抗病毒作用逐渐增强.siRNA-2B还能明显降低CVB3的再感染能力.这些结果提示,针对基因组2B区的siRNA-2B可以明显抑制CVB3基因复制,有效控制病毒再感染,并具有高效性、特异性和量效关系等特点.为siRNA可能成为预防和治疗CVB3感染的新途径奠定基础.     

肿瘤睾丸抗原CT45-5基因小干扰RNA表达载体的构建及干扰效果鉴定

目的:构建肿瘤睾丸抗原CT45家族中5号成员基因(CT45-5)的小干扰RNA(siRNA),并检测其对HeLa及HeLa/Fhit细胞内源性CT45-5基因及外源脆性组氨酸三联体(Fhit)基因表达的干扰效果。方法:根据CT45-5基因序列,通过生物信息学方法对靶向CT45-5基因的siRNA进行预测,从中筛选出2对合理的CT45-5-siRNA,将其克隆到siRNA表达载体pSilencer 2.1-U6 Hygro中,转化大肠杆菌DH5α,挑取阳性克隆进行测序鉴定;将构建的CT45-5-siRNA重组载体转染人宫颈癌细胞HeLa及HeLa/Fhit细胞,Western印迹检测siRNA对HeLa及HeLa/Fhit细胞内源性CT45-5及外源Fhit基因表达的干扰效果。结果:构建了2个CT45-5-siRNA重组质粒,其中一条对CT45-5基因具有明显的干扰作用,并且Fhit基因表达也被抑制。结论:构建的CT45-5-siRNA为与CT45-5功能相关的基因研究奠定了基础。     

SiRNA抑制柯萨奇B3病毒的复制和表达

目的 研究观察体外合成siRNA对培养HELA细胞中柯萨奇B3病毒（Coxsackievirus B3,CVB3）的影响。方法根据siRNA靶序列设计原则,针对编码CVB3病毒聚合酶、VP1蛋白和5’非编码区基因组,特异性地体外合成三对siRNA,同时合成一对与CVB基因组序列无关的阴性对照siRNA。利用脂质体转染进入Hela细胞,用CVB3感染培养HELA细胞,观察转染后HELA细胞病变;采用RT-PCR技术检测感染CVB3各组的病毒RNA;用免疫荧光技术检测各组CVB3蛋白的表达;并用培养细胞上清液再感染HELA细胞观察病毒滴度。结果针对CVB3病毒聚合酶的siR-NA能有效的抑制病毒的复制和CVB3蛋白的表达,并能抑制病毒的再感染;而针对VP1蛋白和5’非编码区的siRNA能部分抑制病毒的复制和CVB3蛋白的表达。结论我们设计合成针对编码CVB3病毒聚合酶基因组的siRNA能有效抑制CVB3病毒复制和表达。     

细胞自噬促进柯萨奇病毒B3复制的研究

本研究探索柯萨奇病毒B3(Coxsackievirus B3,CVB3)感染引起的自噬与病毒复制之间的关系。CVB3感染HeLa细胞,并在病毒感染后6 h、8 h和10 h时检测LC3-Ⅰ蛋白、LC3-Ⅱ蛋白和p62蛋白的表达水平。结果显示CVB3病毒感染促使LC3-Ⅱ/LC3-Ⅰ比值升高,同时降低p62蛋白的表达。分别将自噬诱导剂雷帕霉素(Rapamy-cin)、自噬抑制剂3-甲基腺嘌呤(3-Methyladenine,3MA)或溶酶体抑制剂阿洛司他丁(Aloxistatin,E46D)预处理HeLa细胞2 h,CVB3感染药物处理细胞并在病毒感染6 h后收集细胞、检测CVB3病毒VP1蛋白的表达。结果显示雷帕霉素和E64D促使CVB3病毒VP1蛋白表达增加,而3MA降低CVB3病毒VP1蛋白的表达。本研究得出结论 CVB3病毒感染诱导自噬进而促进病毒复制。     

表达红荧光蛋白的重组柯萨奇病毒B组3型基因组稳定性分析

本文旨在分析含红荧光蛋白mCherry基因的重组柯萨奇病毒B组3型（coxsackievirus B3,CVB3）基因组的稳定性。用重组质粒pCVB3-mCherry转染HeLa细胞,观察细胞病变和mCherry的表达。收获病毒后,用噬斑实验纯化病毒并测定病毒毒力。将重组病毒CVB3-mCherry在HeLa细胞中连续传代,提取第2~6代重组病毒总RNA,经反转录-聚合酶链反应（RT-PCR）扩增出报告基因mCherry及CVB3部分序列,进行测序分析。结果表明, CVB3-mCherry转染的HeLa细胞出现细胞病变并表达红荧光蛋白mCherry;从第2代开始, CVB3-mCherry出现报告基因mCherry及部分CVB VP4基因序列丢失,基因序列丢失导致病毒开放读码框架移位。本研究表明,mCherry基因序列的插入导致CVB3基因组不稳定,随着病毒的传代逐渐丢失插入的报告基因mCherry及CVB3基因组的部分序列,病毒读码框移位,产生致死性突变株。因此,应用CVB3-mCherry时,病毒的传代次数应不超过2代,否则应重新从重组质粒中收获病毒,并对每代重组病毒进行纯化和毒力测定。     

泛素C端水解酶L1的RNA干扰载体的构建及效果评价

目的:获得抑制效果好的泛素 C 端水解酶 L1(UCH-L1)基小干扰 RNA(siRNA)干扰载体.方法:根据 Gen?Bank 中大鼠 UCH-L1基序列设计并合成4对 siRNA 寡核苷酸序列,将4对寡核苷酸序列退火成双链后分别插入siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR 中构建4个 siRNA 表达质粒,测序鉴定后将4个 siRNA 表达质粒分别转染 HEK293细胞,利用 Western 印迹和 qPCR 方法检测干扰效果;将干扰效果最好的质粒包装成腺病毒,感染大鼠血管平滑肌细胞(VSMC),并采用 TNFα干预诱导 UCH-L1表达升高,Western 印迹验证干扰效果.结果:测序分析证实4对 siRNA 寡核苷酸序列分别插入 siRNA 表达载体 pcDNA6.2-GW/EmGFP-miR;qPCR 检测与 Western 印迹均表明第3号 siRNA 表达载体对 UCH-L1表达的抑制程度最高,将其包装成腺病毒并转染 VSMC 能显著抑制 TNFα诱导的UCH-L1表达升高.结论:构建并筛选出干扰效果好的 UCH-L1 siRNA 干扰载体.     

柯萨奇病毒B组3型基因组剪切片段的序列分析

柯萨奇病毒B组(Coxsackievirus B,CVB)感染细胞时其基因组RNA存在不稳定现象,但产生机制尚不清楚。本研究将柯萨奇病毒B组3型(CVB3)感染细胞后,利用5′ cDNA末端快速扩增技术(5′ rapid amplification of cDNA ends,5′ RACE)扩增并克隆细胞内CVB3基因组片段,并对每条序列及其5′端的二级结构进行分析。结果获得的20条CVB3基因组片段,长度为 2 067～5 547 bp,片段断端主要分布于2Apro和2C编码区。RNAfold分析显示,这些片段多数在5′断点端形成二级茎-环结构。本研究显示,CVB在宿主细胞感染时可形成大量不完整基因组RNA片段,这些片段可在5′断点端形成局部双链结构,提示片段不是随机产生,可能是RNA酶剪切产物。此发现有助于理解CVB基因组不稳定的机制。     

A small interfering RNA targeting coxsackievirus B3 protects permissive HeLa cells from viral challenge

We examined the ability of small interfering RNAs (siRNAs) to disrupt infection by coxsackievirus B3 (CVB3). The incorporation of siRNAs dramatically decreased cell death in permissive HeLa cells in parallel with a reduction in viral replication. Three of four siRNAs had potent anti-CVB3 activity. The present study thus demonstrates that the antiviral effect is due to the downregulation of viral replication. In addition, an effective CVB3-specific siRNA had similar antiviral effects in other related enteroviruses possessing sequence homology in the targeted region. Because the CVB3-specific siRNA is effective against other enteroviruses, siRNAs have potential for a universal antienterovirus strategy.     

Inhibition of coxsackievirus B3 replication by small interfering RNAs requires perfect sequence match in the central region of the viral positive strand

Coxsackievirus B3 (CVB3) is the most common causal agent of viral myocarditis, but existing drug therapies are of limited value. Application of small interfering RNA (siRNA) in knockdown of gene expression is an emerging technology in antiviral gene therapy. To investigate whether RNA interference (RNAi) can protect against CVB3 infection, we evaluated the effects of RNAi on viral replication in HeLa cells and murine cardiomyocytes by using five CVB3-specific siRNAs targeting distinct regions of the viral genome. The most effective one is siRNA-4, targeting the viral protease 2A, achieving a 92% inhibition of CVB3 replication. The specific RNAi effects could last at least 48 h, and cell viability assay revealed that 90% of siRNA-4-pretreated cells were still alive and lacked detectable viral protein expression 48 h postinfection. Moreover, administration of siRNAs after viral infection could also effectively inhibit viral replication, indicating its therapeutic potential. Further evaluation by combination found that no enhanced inhibitory effects were observed when siRNA-4 was cotransfected with each of the other four candidates. In mutational analysis of the mechanisms of siRNA action, we found that siRNA functions by targeting the positive strand of virus and requires a perfect sequence match in the central region of the target, but mismatches were more tolerated near the 3' end than the 5' end of the antisense strand. These findings reveal an effective target for CVB3 silencing and provide a new possibility for antiviral intervention.     

Overexpression of 4EBP1, p70S6K,Akt1 or Akt2 differentially promotes Coxsackievirus B3-induced apoptosis in HeLa cells

Our previous studies have shown that the inhibition of phosphatidylinositol 3-kinase (PI3K) or mTOR complex 1 can obviously promote the Coxsackievirus B3 (CVB3)-induced apoptosis of HeLa cells by regulating the expression of proapoptotic factors. To further illustrate it, Homo sapiens eIF4E-binding protein 1 (4EBP1), p70S6 kinase (p70S6K), Akt1 and Akt2 were transfected to HeLa cells, respectively. And then, we established the stable transfected cell lines. Next, after CVB3 infection, apoptosis in different groups was determined by flow cytometry; the expressions of Bim, Bax, caspase-9 and caspase-3 were examined by real-time fluorescence quantitative PCR and western blot analysis; the expression of CVB3 mRNA and viral capsid protein VP1 were also analyzed by real-time fluorescence quantitative PCR, western blot analysis and immunofluorescence, respectively. At the meantime, CVB3 replication was observed by transmission electron microscope. We found that CVB3-induced cytopathic effect and apoptosis in transfected groups were more obvious than that in controls. Unexpectedly, apoptosis rate in Akt1 group was higher than others at the early stage after viral infection and decreased with the viral-infected time increasing, which was opposite to other groups. Compared with controls, the expression of CVB3 mRNA was increased at 3, 6, 12 and 24 h postinfection (p. i.) in all groups. At the meantime, VP1 expression in 4EBP1 group was higher than control during the process of infection, while the expressions in the other groups were change dynamically. Moreover, overexpression of 4EBP1 did not affect the mRNA expressions of Bim, Bax, caspase-9 and caspase-3; while protein expressions of Bim and Bax were decreased, the self-cleavages of caspase-9 and caspase-3 were stimulated. Meanwhile, overexpression of p70S6K blocked the CVB3-induced Bim, Bax and caspase-9 expressions but promoted the self-cleavage of caspase-9. In the Akt1 group, it is noteworthy that the expressions of Bim protein were higher than controls at 3 and 6 h p. i. but lower at 24 h p. i., and the expression of Bax protein were higher at 6 and 24 h p. i., while their mRNA expressions were all decreased. Furthermore, overexpression of Akt1 stimulated the procaspase-9 and procaspase-3 expression but blocked their self-cleavages. Overexpression of Akt2, however, had little effect on Bim, Bax and caspase-3, while prevented caspase-9 from self-cleavage at the late stage of CVB3 infection. As stated above, our results demonstrated that overexpression of 4EBP1, p70S6K, Akt1 or Akt2 could promote the CVB3-induced apoptosis in diverse degree via different mediating ways in viral replication and proapoptotic factors in BcL-2 and caspase families. As 4EBP1, p70S6K and Akt are the important substrates of PI3K and mammalian target of rapamycin (mTOR), we further illustrated the role of PI3K/Akt/mTOR signaling pathway in the process of CVB3-induced apoptosis.     

Sequence,chemical, and structural variation of small interfering RNAs and short hairpin RNAs and the effect on mammalian gene silencing

Small interfering RNAs (siRNAs) induce sequence-specific gene silencing in mammalian cells and guide mRNA degradation in the process of RNA interference (RNAi). By targeting endogenous lamin A/C mRNA in human HeLa or mouse SW3T3 cells, we investigated the positional variation of siRNA-mediated gene silencing. We find cell-type-dependent global effects and cell-type-independent positional effects. HeLa cells were about 2-fold more responsive to siRNAs than SW3T3 cells but displayed a very similar pattern of positional variation of lamin A/C silencing. In HeLa cells, 26 of 44 tested standard 21-nucleotide (nt) siRNA duplexes reduced the protein expression by at least 90%, and only 2 duplexes reduced the lamin A/C proteins to <50%. Fluorescent chromophores did not perturb gene silencing when conjugated to the 5'-end or 3'-end of the sense siRNA strand and the 5'-end of the antisense siRNA strand, but conjugation to the 3'-end of the antisense siRNA abolished gene silencing. RNase-protecting phosphorothioate and 2'-fluoropyrimidine RNA backbone modifications of siRNAs did not significantly affect silencing efficiency, although cytotoxic effects were observed when every second phosphate of an siRNA duplex was replaced by phosphorothioate. Synthetic RNA hairpin loops were subsequently evaluated for lamin A/C silencing as a function of stem length and loop composition. As long as the 5'-end of the guide strand coincided with the 5'-end of the hairpin RNA, 19-29 base pair (bp) hairpins effectively silenced lamin A/C, but when the hairpin started with the 5'-end of the sense strand, only 21-29 bp hairpins were highly active.     

Subtype- and species-specific knockdown of PKC using short interfering RNA

RNA interference (RNAi), the targeted mRNA degradation induced by double-stranded RNA (dsRNA), is a powerful tool for analyzing gene function in many organisms. Recently, it has been shown that RNAi is also applicable to cultured mammalian cells by using short interfering RNA (siRNA) [Nature 411 (2001) 494]. To examine whether this siRNA method is useful for analyzing the subtype-specific functions of protein kinase C (PKC), we first prepared siRNAs which target human alphaPKC and human deltaPKC and applied them into mammalian cells to suppress the expression of endogenous alphaPKC and deltaPKC, respectively. Each siRNA for alpha or deltaPKC specifically suppressed the endogenous expression of corresponding PKC subtype in human-derived cell lines such as HEK-293 and HeLa cells, but not in cells derived from rat species. The suppression level of deltaPKC reached maximum 48-72h after the transfection of siRNA. In addition, the siRNA targeting rat deltaPKC suppressed endogenous and exogenous rat deltaPKCs but not human deltaPKC, suggesting that siRNAs targeting PKCs effectively knocked down endogenous/exogenous PKCs in mammalian cells, in subtype- and species-specific manner. Furthermore, we also developed the method to discriminate the siRNA-transfected cells using the antibody recognizing thymine dimer. Our present results strongly suggest that siRNA method enable us to examine the subtype-specific function of PKC, not only by knockdown of the endogenous target PKC subtype, but also by subsequent compensation with the exogenous corresponding wild/mutant PKC derived from other species.     

Coxsackievirus B3 infection induces cyr61 activation via JNK to mediate cell death

Coxsackievirus B3 (CVB3), an enterovirus in the Picornavirus family, is the most common human pathogen associated with myocarditis and idiopathic dilated cardiomyopathy. We found upregulation of the cysteine-rich protein gene (cyr61) after CVB3 infection in HeLa cells with a cDNA microarray approach, which is confirmed by Northern blot analysis. It is also revealed that the extracellular amount of Cyr61 protein was increased after CVB3 infection in HeLa cells. cyr61 is an early-transcribed gene, and the Cyr61 protein is secreted into the extracellular matrix. Its function is related to cell adhesion, migration, and neuronal cell death. Here, we show that activation of the cyr61 promoter by CVB3 infection is dependent on JNK activation induced by CVB3 replication and viral protein expression in infected cells. To explore the role of Cyr61 protein in infected HeLa cells, we transiently overexpressed cyr61 and infected HeLa cells with CVB3. This increased CVB3 growth in the cells and promoted host cell death by viral infection, whereas down-expression of cyr61 with short interfering RNA reduced CVB3 growth and showed resistance to cell death by CVB3 infection. In conclusion, we have demonstrated a new role for cyr61 in HeLa cells infected with CVB3, which is associated with the cell death induced by virus infection. These data thus expand our understanding of the physiological functions of cyr61 in virus-induced cell death and provide new insights into the cellular factors involved.     

Heat shock protein 70 promotes coxsackievirus B3 translation initiation and elongation via Akt‐mTORC1 pathway depending on activation of p70S6K and Cdc2

We previously demonstrated that coxsackievirus B3 (CVB3) infection upregulated heat shock protein 70 (Hsp70) and promoted CVB3 multiplication. Here, we report the underlying mechanism by which Hsp70 enhances viral RNA translation. By using an Hsp70‐overexpressing cell line infected with CVB3, we found that Hsp70 enhanced CVB3 VP1 translation at two stages. First, Hsp70 induced upregulation of VP1 translation at the initiation stage via upregulation of internal ribosome entry site trans‐acting factor lupus autoantigen protein and activation of eIF4E binding protein 1, a cap‐dependent translation suppressor. Second, we found that Hsp70 increased CVB3 VP1 translation by enhancing translation elongation. This was mediated by the Akt‐mammalian target of rapamycin complex 1 signal cascade, which led to the activation of eukaryotic elongation factor 2 via p70S6K‐ and cell division cycle protein 2 homolog (Cdc2)‐mediated phosphorylation and inactivation of eukaryotic elongation factor 2 kinase. We also determined the position of Cdc2 in this signal pathway, indicating that Cdc2 is regulated by mammalian target of rapamycin complex 1. This signal transduction pathway was validated using a number of specific pharmacological inhibitors, short interfering RNAs (siRNAs) and a dominant negative Akt plasmid. Because Hsp70 is a central component of the cellular network of molecular chaperones enhancing viral replication, these data may provide new strategies to limit this viral infection.     

The effects of thiophosphate substitutions on native siRNA gene silencing

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.