利用RNA干扰抑制具有多种剪接形式的RNA结合蛋白基因的表达

目的:构建具有多种剪接形式的RNA结合蛋白(RBPMS)基因siRNA的真核表达载体,观察其对RBPMS表达的影响。方法:利用RNA干扰(RNAi)技术,设计并合成了2条针对RBPMS基因的siRNA,将其克隆到siRNA表达载体pSliencer2.1-U6neo上。将重组质粒和带FLAG标签的RBPMS共转染293T人胚肾细胞,通过Western印迹检验RNAi效应。结果:测序证明成功构建了RBPMSsiRNA真核表达载体;Western印迹表明构建的siRNA能有效地抑制RBPMS基因的表达。结论:构建了RBPMSsiRNA的真核表达载体,该siRNA能有效地抑制RBPMS基因的表达。     

利用载体介导小干扰RNA诱导RSRC1基因沉默

目的：设计并构建人RSRC1基因小干扰RNA（siRNA）的真核表达载体,并观察其沉默效果。方法：以人RSRC1基因的cDNA序列为靶标,设计含有小发卡结构的2条寡核苷酸序列,并将其克隆到siRNA表达载体pSliencer2.1-U6neo上,转化大肠杆菌DH5α菌株,抽提质粒,测序正确后将重组质粒转染人胚肾293T细胞,通过Western blot和荧光分析检测其抑制效果。结果：重组体测序成功后,Western blot分析证明构建的siRNA能有效抑制外源性及内源性RSRC1表达;将siRNA重组质粒和带GFP标签的RSRC1共转染293T细胞,荧光显微镜下GFP的亮度明显减弱。结论：获得了2条人RSRC1siRNA真核表达载体,均能有效地抑制RSRC1基因表达。     

碳纳米管作为siRNA转运载体的研究进展

随着人们对RNA干扰分子机理的研究愈加深入,siRNA作为一种新的基因治疗药物极有可能为人类攻克癌症等难以治愈的疾病带来希望。然而,目前在RNA干扰应用中遇到的最大挑战就是如何有效地将siRNA导入靶细胞且不致引起严重的细胞毒性。碳纳米管在药物传递和基因传递等生物医学领域的潜在应用受到广泛关注;但要实现碳纳米管在基因治疗领域的应用,碳纳米管的功能化是关键,也是近几年来研究的重点。综述近年来碳纳米管作为siRNA转运载体在基因治疗领域的研究进展。     

利用小干扰RNA抑制小管间质性肾炎抗原相关蛋白的表达

目的：设计并构建TIN-ag-RP基因的小干扰RNA（siRNA）,检测其对小管间质性肾炎抗原相关蛋白（TIN—ag-RP）表达的干扰效果。方法：设计2条针对TIN-ag-RP基因的siRNA,并克隆到siRNA表达载体pSliencer2．1-U6 neo上;经酶切和测序证明构建成功后,将重组质粒和带FLAG标签的TIN-ag-RP基因共转染293T人胚肾细胞,通过Westernblot检验siRNA的干扰效果。结果：获得了2个TIN-og-RP基因siRNA真核表达载体,均能有效抑制TIN-ag-RP的表达,其中一条的抑制效率诂90％以上。结论：构肆的TIN-ag-RP某因的siRNA能有效抑制TIN-ag-RP的表达。     

RNA干涉技术

RNA干涉(RNAi)技术是利用一些小的双链RNA来高效、特异地阻断体内特定基因的表达,并促使mRNA降解,从而诱使细胞表现出特定基因缺失的表型。本从RNAi技术的历史、作用机制、研究策略、研究现状及应用前景等几个方面进行了综述,预测RNAi将会给基因治疗的发展带来新的希望。     

病毒siRNA介导的RNA干扰途径与机制

RNA干扰(RNAi)是真核生物体内重要的基因表达调控方式之一.RNAi的一种原始的作用是帮助生物体抵抗病毒,早期的研究表明无脊椎动物可以利用RNAi抵抗病毒,但是哺乳动物是否存在这一机制一直存在争议.最新的研究发现了哺乳动物RNAi抗病毒的强有力的证据,并且研究人员认为,这是一种之前被忽视的、全新的免疫途径.值得注意...     

mRNA translation is not a prerequisite for small interfering RNA-mediated mRNA cleavage

RNA interference constitutes a major means of eliminating mRNAs, yet how the small interfering RNAs (siRNA) within the RNA-induced silencing complex (RISC) finds its homologous target in the cell remains unknown. An attractive hypothesis is that RNA interference is linked to translation which allows RISC ready access to every translated mRNA. To test whether translation could direct siRNAs to mRNAs, chemical and biological inhibitors of translation and their effects on mRNA cleavage were tested. Our results show that mRNA degradation by siRNAs is not dependent on mRNA translation.     

RNA interference as a tool for exploring HIV-1 robustness

Short interfering RNAs (siRNAs) that target viral genes can efficiently inhibit human immunodeficiency virus type 1 (HIV-1) replication. Nevertheless, there is the potential for viral escape, particularly with a highly mutable target such as HIV-1. We present a novel strategy for anticipating and preventing viral escape using second-generation siRNAs. The evolutionary capacity of HIV-1 was tested by exerting strong selective pressure on a highly conserved sequence in the HIV-1 genome. We assayed the antiviral efficacy of five overlapping siRNAs directed against an essential region of the HIV-1 protease. Serial viral transfers in U87-CD4-CXCR4 cells were performed using four of the siRNAs. This procedure was repeated until virus breakthrough was detected. After several serial culture passages, resistant virus with a single point mutation in the targeted region was detected in the culture supernatants. The emergence of resistant virus was confirmed by molecular cloning and DNA sequencing of viral RNA. The most common escape route was the D30N mutation. Importantly, the addition of a second-generation siRNA that matched the D30N mutation restored viral inhibition and delayed development of escape variants. Passages performed with both siRNAs prevented the emergence of the D30N escape mutant and forced the virus to develop new escape routes. Thus, second-generation siRNAs can be used to block escape from RNA interference (RNAi) and to search for new RNAi escape routes. The protocol described here may be useful for exploring the sequence space available for HIV-1 evolution and for producing attenuated or deleterious viruses.     

靶向HIV-1pol的高效人工miRNA的构建与体外抗病毒能力评价

RNAi技术在抗HIV-1治疗研究中已显示出巨大潜力,获得可高效特异抑制HIV-1的RNAi元件是进行相关研究的重要基础。miRNA在抑制和表达方式上相比siRNA具有更多的优势。本研究即探讨构建可高效特异靶向HIV-1的人工miRNA元件。选择以保守性较好的HIV-1pol基因为靶区筛选高效保守的RNAi序列,设计了16个可靶向pol区高保守区段的RNAi靶点,构建表达载体与HIV-1感染性克隆进行共转染抑制实验,筛选显示pol1026序列兼具高保守性及高抑制效率特点。以天然miR-30a为基础骨架构建了靶向pol1026靶点的人工miRNA元件,通过与HIV-1感染性克隆质粒的共转染抑制实验验证获得了可有效抑制HIV-1表达的人工miRNA元件(miR-1026E)。通过与携带靶序列的报告质粒的共转染实验证明miR-1026E具有良好的靶点特异性。本研究进一步构建了携带miR-1026E表达元件的重组慢病毒,转导MT-4细胞并对转导后细胞进行克隆化筛选,获得稳定整合miR-1026E表达元件的MT-4-miR1026E细胞克隆,该细胞在体外攻毒实验中可高效抑制HIV-1的复制,具有显著的抑制HIV-1的能力。同时应用实时RT-PCR方法检测显示,miR-1026E在细胞中不会影响内源性代表miRNA(miR-181与miR-16)的表达水平和干扰素效应相关基因stat1的表达水平,具有良好的特异性。所获得的可特异高效抑制HIV-1复制的人工miRNA元件可为抗HIV-1研究提供重要参考。     

Nodamura virus B2 amino terminal domain sensitivity to small interfering RNA

Nodamura virus (NoV) B2, a suppressor of RNA interference, binds double stranded RNAs (dsRNAs) and small interfering RNAs (siRNAs) corresponding to Dicer substrates and products. Here, we report that the amino terminal domain of NoV B2 (NoV B2 79) specifically binds siRNAs but not dsRNAs. NoV B2 79 oligomerizes on binding to 27 nucleotide siRNA. Mutation of the residues phenylalanine49 and alanine60 to cysteine and methionine, respectively enhances the RNA binding affinity of NoV B2 79. Circular dichroism spectra demonstrated that the wild type and mutant NoV B2 79 have similar secondary structure conformations.     

小RNA分子与精子发生调控

近来研究发现小RNA(small RNAs)可作为转录后及翻译水平上基因表达调节的重要调节因子,利用小RNA来阐明调节精子发生的分子机制取得了显著进展。这些小RNA主要分为3类,即小干扰RNA(siRNA)、微小RNA(miRNA)以及与piwi蛋白相互作用的RNA(piRNA)。在减数分裂和精子发生过程中,小RNA具有多种生物学功能,如利用siRNA体外转染或体内注射来敲低特定基因从而研究该基因在精子发生过程中的作用;miRNA可能参与精子发生中有丝、减数及后减数分裂阶段的基因表达调节;piRNA主要参与调节雄性生殖细胞减数及后减数分裂的过程,在精子发生中起抑制反转录转座子(retrotransposons)的作用。文章对小RNAs合成、作用机制、功能及展望等最新进展进行了综述。     

RNA干扰机制的研究进展

RNA干扰(RNAi)广泛存在于各种生物体中,是参与细胞防御与分化调控的重要机制之一。RNAi的作用由双链RNA启动,通过在转录、转录后和翻译等多个水平上对同源基因表达的特异阻断和抑制来实现,清晰地阐明其作用机制将为功能基因组学、发育生物学,以及抗肿瘤、抗病毒的新策略研究提供重要的理论依据。本文综述了近年来有关RNAi机制的研究进展。     

Experimental Study on the Suppression of Human Nuclear Receptor hLRH-1 via a Vector-based RNA Interference

To explore the inhibitions of human nuclear receptor hLRH-1 via RNA interference, siRNAs expressing vectors pShLRH-1.1 and pShLRH-1.2, and targeting hLRH-1 were designed and constructed. The recombinants were introduced into hepatocellular carcinoma cells, BEL-7402, mediated by lipofectamin^TM. RT-PCR was carried out to examine the inhibition ratio of hLRH-1 expression. The same method was also applied to analyze the expression of farnesyl pyrophosphate synthetase (FPPS) gene. Our results demonstrated that after transient transfection, both pShLRH-1.1 and pShLRH-1.2 could trigger the efficient inhibition of hLRH-1 in cultured cells, BEL-7402. The inhibition ratios were up to 80%. By comparing with non-transfection and vector-transfection control, the expression of FPPS in cells with inhibition of hLRH-1 was up-regulated significantly. Thus, the inhibition of expression of hLRH-1 in cultured cells was achieved via RNA interference in this study. Our results also suggested that hLRH-1 acts as a negative regulator in FPPS expression.