RNA干扰的机制及其应用

RNA干扰(RNA interference,RNAi)是真核生物中高度保守的,由小分子干扰RNA(small interfering RNA,si RNA)介导的转录后基因沉默现象,在基因功能的研究中得到了非常广泛的应用,并有望成为小核酸药物在疾病治疗中发挥重要作用。现对近年来RNAi的作用机制、si RNA的产生途径、引起RNAi副作用的原因以及RNAi表达载体的设计这四个方面的国内外研究进展进行了总结,对RNAi与CRISPR技术在应用中的关系进行了探讨,展望了RNAi技术未来的发展。     

RNA干涉的临床前实验研究进展

RNA干涉（RNA interference,RNAi）是一种非常保守的细胞现象,在基础研究和疾病治疗方面具有重大的应用前景。尤其引人注目的是,新的RNAi方法学与已建立的转基因策略相结合,有效地将组织特异性RNAi导入到患者体内,有望治疗人类疾病。本文综述了RNAi的机制与在其临床前的实验研究,简要介绍了RNAi的新用途,讨论了RNAi基因治疗存在的问题,展望了RNAi基因治疗的应用前景。     

RNAi生物技术作物环境风险评估研究进展

RNA干涉(RNAi)在昆虫遗传和功能基因研究方面广泛应用。近年来,RNAi被认为是具有应用潜力的害虫防治新方法。具有良好抗虫性状的RNAi生物技术作物已研究成功,预示其商业化应用成为可能。因此,有关RNAi作物的生态风险是商业化应用前人们所关心的问题。要建立RNAi生物技术作物环境安全评价准则,监管者、受益各方及风险评估者必须要了解RNAi理论及其在生物技术领域的应用。科学分析并准确提出RNAi作物的生态风险问题,如非期望的基因沉默、靶外结合或脱靶效应、靶标害虫的抗性、小干涉RNA(si RNA)的环境持久性和不确定性等,并通过研究获得科学数据,将为政府依法监管提供依据。RNAi生物技术作物的环境风险评估主要包括功能基因及其表达特征(如ds RNA序列、长度、表达浓度及沉默效果的持续性等)、杀虫谱及对非靶标生物的影响、环境中的残留问题、功能性状的持续稳定性等。现行的生物技术作物环境风险评估方法和内容需要进一步修改完善,以适应今后RNAi生物技术作物的发展和应用。     

RNAi技术的医疗应用潜力

RNA干扰是指双链RNA在细胞内特异性地诱导同源互补的mRNA降解,从而阻断相应基因表达的现象。RNAi发展成为一种新型的基因治疗方式的进程取决于哺乳动物砌RNAi的研究进展。在大多数哺乳动物细胞中,直接导入长dsRNA引发的非特异性基因沉默掩盖了RNAi效应,而多种有效的双链RNA导入方式在一定程度上解决了这一问题。初步的实验结果表明,用RNAi治疗癌症、病毒感染等疾病的设想有可能变成现实。     

RNAi及其抑制口蹄疫病毒复制的研究进展

RNA干扰(RNA interference,RNAi)是指由双链RNA(double strand RNA,dsRNA)介导的序列特异的RNA降解过程。已经证明,在植物和昆虫细胞中RNAi是其主要的抗病毒免疫机制,至今为止几乎没有发现在病毒感染哺乳动物细胞过程中诱发有效的抗病毒RNAi反应。因此,人们希望能够利用人工方法在哺乳动物细胞中建立有效的抗病毒RNAi防御策略。迄今为止,对多种哺乳动物病毒的研究结果令人振奋。主要围绕RNAi的分子基础、基本策略及其在抑制口蹄疫病毒复制中的研究现状作了综述。     

c-FLIP的siRNA促进TRAIL诱导的癌细胞凋亡的研究

肿瘤凋亡因子TRAIL(TNF-related apoptosis-inducing ligand)是TNF家族的成员之一,其不论在体内还是体外均可选择性诱导癌细胞凋亡。初期临床试验已经证实TRAIL或死亡受体激动剂抗体在癌症治疗中的安全性。另外,也有研究表明,许多癌细胞对TRAIL有耐受性,究其原因是凋亡通路中抗凋亡蛋白c-FLIP和IAP等的阻遏。更多研究发现,si RNA靶向抑制c-FLIP的同时结合广谱IAP拮抗剂AT406,进一步提高了TRAIL诱导的癌细胞凋亡,因此联合使用c-FLIP抑制剂或拮抗剂、IAP拮抗剂以及TRAIL或死亡受体激动剂抗体这三类药物可能是理想的抗癌新方法。综述了c-FLIP的发现、结构、功能和其参与死亡受体介导的信号通路,以及多种si RNA应用于c-FLIP以提高癌细胞对TRAIL敏感性和si RNA条件优化中的一些进展,并讨论了目前癌症临床治疗中si RNA的应用前景及存在的问题,最后提出较安全有效的基于TRAIL介导的癌症治疗新方法。     

利用RNAi逆转录病毒载体系统对人朊病毒蛋白表达的稳定抑制

朊病毒是引起可传染的致死性海绵状脑病的致病因子,细胞中正常的朊病毒蛋白(PrPC)在该疾病病程发展中起着必不可少的作用。同时,PrPC已被证明在胃癌、乳腺癌等癌症中发挥着保护癌细胞的作用。根据人源PrPC(HuPrPC)cDNA序列,本研究设计了4种19nt的siRNA,将其构建成RNAi逆转录病毒载体系统,进行了其对HuPrPC表达的抑制效应的分析,从中获得了能高效稳定抑制HuPrPC表达的3种靶向序列,其中si626(5′-GGTTGAGCAGATGTGTATC-3′)的抑制效果最为明显,其抑制效率可达85%以上。随后,利用筛选出的si292和si626的稳定干扰细胞系进行了细胞浸润性实验,结果发现,PrPC干扰细胞系细胞浸润能力显著下降。这为进一步研究朊病毒疾病的基因治疗、以PrPC为靶标进行PrPC相关癌症的辅助治疗研究奠定了一定的基础。     

以两亲性细胞膜穿透肽为基础的siRNA递送策略

小干扰RNA(si RNA)的发展给针对病理障碍特异性基因的靶向治疗带来了巨大的希望。然而,细胞膜对带负电荷分子的低渗透性,细胞对si RNA的摄取能力差,成为了si RNA临床应用的主要障碍。虽然学者们提出了一系列si RNA递送的方法,但是截止到目前,仍没有递送si RNA的通用方法。细胞膜穿透肽(cell-penetrating peptides,CPPs)的发现为si RNA非侵袭性的进入细胞提供了一种非常有前景的运载工具,已被成功地应用于治疗性si RNA分子的体内和体外实验的递送。最近,一种新的以两亲性CPPs为基础的si RNA递送系统-CADY(a secondary amphipathic peptide,Ac-GLWRALWRLLRSLWRLLWRA-cysteamide)受到了高度关注,它能与si RNA形成稳定的非共价复合物,并在原代和悬浮细胞系中高效地递送si RNA,具有极高的应用前景。     

RNAi技术研究新进展

RNA干扰(RNAi)是指双链RNA在细胞内特异性地诱导同源互补的mRNA降解,从而阻断相应基因表达的现象。RNAi在生物界中广泛存在,其发生过程主要分为3个阶段:起始阶段、扩增阶段和效应阶段。它在维持基因组稳定、基因表达调控等方面发挥重要生物学作用。随着人们对RNAi研究的不断深入,目前RNA干扰技术作为基因沉默的一个工具,已被广泛用于基因功能研究、疾病的靶点治疗和寻找新的药物靶标等方面的研究。     

长链非编码RNA在胃癌中的研究进展

长链非编码RNA(1ong non-coding RNA,lnc RNA)是一组长度超过200 bp、缺少特异开放阅读框、不具备完整蛋白编码功能的RNA,其在表观遗传学调控以及转录和转录后调控等方面发挥重要作用。目前,在乳腺癌和肝癌中对Lnc RNA的研究颇多,而对胃癌中Lnc RNA的报道却刚刚兴起。近几年越来越多的研究发现,在胃癌中有很多特异表达的Lnc RNA与胃癌的发生、发展、侵袭、转移密切相关。本文结合国内外最新研究就lnc RNA在胃癌中的研究进展作一简要综述,主要介绍了Lnc RNA在肿瘤研究中的最新发现,尤其是其与胃癌发生发展的密切联系,旨在为胃癌的诊断和治疗提供新思路。     

miRNA与siRNA胃癌相关研究的现状及进展

RNA干扰是表观遗传学的研究热点,它参与基因复制后表达调控,并与肿瘤发生密切相关。近年对RNA干扰研究较多的是微小RNA和小干扰RNA。文章概述了微小RNA和小干扰RNA的基本理论,并综述它们在胃癌研究中的现状及进展。认为RNA干扰分析和应用是研究胃癌相关基因功能及作用机制的有效方法,并将对胃癌的诊治产生巨大影响。     

RNA interference against polo-like kinase-1 in advanced non-small cell lung cancers

Worldwide, approximately one and a half million new cases of lung cancer are diagnosed each year, and about 85% of lung cancer are non-small cell lung cancer (NSCLC). As the molecular pathogenesis underlying NSCLC is understood, new molecular targeting agents can be developed. However, current therapies are not sufficient to cure or manage the patients with distant metastasis, and novel strategies are necessary to be developed to cure the patients with advanced NSCLC.RNA interference (RNAi) is a phenomenon of sequence-specific gene silencing in mammalian cells and its discovery has lead to its wide application as a powerful tool in post-genomic research. Recently, short interfering RNA (siRNA), which induces RNAi, has been experimentally introduced as a cancer therapy and is expected to be developed as a nucleic acid-based medicine. Recently, several clinical trials of RNAi therapies against cancers are ongoing. In this article, we discuss the most recent findings concerning the administration of siRNA against polo-like kinase-1 (PLK-1) to liver metastatic NSCLC. PLK-1 regulates the mitotic process in mammalian cells. These promising results demonstrate that PLK-1 is a suitable target for advanced NSCLC therapy.     

Photosensitizing carrier proteins for photoinducible RNA interference

RNA interference (RNAi) is being widely explored as a tool in functional genomics and tissue engineering, and in the therapy of intractable diseases, including cancer and neurodegenerative diseases. Recently, we developed a photoinducible RNAi method using photosensitizing carrier proteins, named CLIP-RNAi (CPP-linked RBP-mediated RNA internalization and photoinduced RNAi). Novel carrier proteins were designed for this study to establish a highly efficient delivery system for small interfering RNA (siRNA) or short hairpin RNA (shRNA) and to demonstrate light-dependent gene silencing. In addition, the results suggested that the dissociation of the siRNA (or shRNA) from carrier proteins in the cytoplasm is a critical event in CLIP-RNAi-mediated gene silencing.     

A siRNA system based on HSP70 promoter results in controllable and powerful gene silencing by heat‐induction

RNAi is a powerful tool for gene‐specific knockdown and gene therapy. However, the imprecise expression of siRNA limits the extensive application of RNAi in gene therapy. Here we report the development of a novel controllable siRNA expression vector pMHSP70psil that is initiated by HSP70 promoter. We determined the efficiency of the controllable siRNA system by targeting the gama‐synuclein (SNCG) gene in breast cancer cells MCF‐7. The results show that the controllable siRNA system can be induced to initiate siRNA expression by heat‐induction. The silencing effect of SNCG occurs at a relatively low level (10.1%) at 37°C, while it is significantly increased to 69.4% after heat induction at 43°C. The results also show that the controllable siRNA system inhibits proliferation of cancer cells by heat‐shock. Therefore, this RNAi strategy holds the promise of the high efficiency in gene knockdown at targeted times and locations, avoiding systemic side effects. It provides, for the first time, an approach to control siRNA expression by heat‐shock. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1289–1297, 2013     

Selection and optimization of asymmetric siRNA targeting the human c-MET gene

The silencing of specific oncogenes via RNA interference (RNAi) holds great promise for the future of cancer therapy. RNAi is commonly carried out using small interfering RNA (siRNA) composed of a 19 bp duplex region with a 2-nucleotide overhang at each 3′ end. This classical siRNA structure, however, can trigger non-specific effects, which has hampered the development of specific and safe RNAi therapeutics. Previously, we developed a novel siRNA structure, called asymmetric shorter-duplex siRNA (asiRNA), which did not cause the non-specific effects triggered by conventional siRNA, such as off-target gene silencing mediated by the sense strand. In this study, we first screened potent asiRNA molecules targeting the human c-MET gene, a promising anticancer target. Next, the activity of a selected asiRNA was further optimized by introducing a locked nucleic acid (LNA) to maximize the gene silencing potency. The optimized asiRNA targeted to c-MET may have potential as a specific and safe anticancer RNAi therapeutic.     

Modified high-density lipoprotein modulates aldosterone release through scavenger receptors via extra cellular signal-regulated kinase and Janus kinase-dependent pathways

A proliferation-inducing ligand (APRIL) is overexpressed in most tumor cells and tissues, especially in tumors of the alimentary system, such as colorectal cancer (CRC), gastric cancer, and liver cancer. RNA interference (RNAi) has been proved to be a powerful tool for gene knockdown and holds great promise for the treatment of cancer. In this study, the efficacy of RNAi targeting APRIL was analyzed via relevant experiments on human CRC xenografted in BALB/c nude mice. Both the mRNA and protein levels of APRIL were examined after intratumoral injection of APRIL small interfering RNA (siRNA). Meanwhile, pathological tools were utilized to observe the alterations on the aspects of proliferation, metastasis, apoptosis and cellular necrosis by means of detecting proliferating cell nuclear antigen, Ki-67, MMP-2, MMP-9, TIMP-3, TIMP-4, Bcl-2, Bax and Bcl-xL of CRC. In addition, terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) and hematoxylin and eosin staining were also conducted to examine cell apoptosis and necrosis. It was found that grafted human colorectal tumor growth and metastasis were obviously inhibited while tumor cell apoptosis and necrosis were induced after in vivo APRIL siRNA injection into nude mice. The data indicated that silencing of the APRIL gene using RNAi may serve as a novel therapeutic strategy for treatment of CRC.     

小干扰RNA的研究进展

RNA干扰（RNA interference,RNAi）是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mR_NA产生特异性降解,进而引起不同水平的基因沉默。其效应分子主要是小干扰RNA（siRNA）。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。     

小干扰RNA的研究进展

RNA干扰(RNA interference,RNAi)是近年发展起来的一种新技术。RNAi是指通过外源性或内源性的双链RNA在体内诱导靶基因mRNA产生特异性降解,进而引起不同水平的基因沉默,其效应分子主要是小干扰RNA(siRNA)。siRNA是生物界普遍存在的一种抵御外来基因和病毒感染的基因调控方式,也是一种重要的研究工具。大量的研究工作致力于设计合理的siRNA片段用于基因功能研究,并将其作为一种治疗方法用于肿瘤、病毒性疾病等基因治疗以及药物靶向研究。因此本文对siRNA的作用机制、设计原则及其在临床应用中的缺点和解决方法进行综述。