RNAi的研究及应用

RNA干扰（RNA interference,RNAi）是由双链RNA（double-stranded RNA,dsRNA）引发的转录后基因沉默机制．RNAi可以调节和关闭基因的表达,进而调控细胞的各种高级生命活动,是真核生物中普遍存在的抵抗病毒入侵、抑制转座子活动、调控基因表达的监控机制．目前RNAi的研究取得了很大进展,有可能为肿瘤基因治疗提供新策略．     

RNAi与RNAi技术

RNA干扰(RNA interference, RNAi)是近年来发展起来的研究生物体基因表达、调控与功能的一项崭新技术,它利用了由小干扰RNA(small interfering RNA, siRNA)引起的生物细胞内同源基因的特异性沉默(silencing)现象,其本质是siRNA与对应的mRNA特异结合、降解,从而阻止mRNA的翻译.RNAi是生物进化的结果,是生物体对病毒基因外源核酸侵入的一种保护性反应.它普遍存在于各种生物中,具有抗病毒、稳定转座子及监控异常表达mRNA的生物学功能.RNA干扰现象不仅能提供一种经济、快捷、高效的抑制基因表达的技术手段,而且有可能在基因功能测定、基因治疗等方面开辟一条新思路.     

RNAi鉴定药物靶点及siRNAs治疗疾病

RNAi是与靶基因序列同源的双链RNA（dsRNA）所诱导的一种特异性基因沉默现象。RNAi是生物体进化过程中抵御外来基因和病毒感染的进化保守机制,是真核生物中存在的一种抗病毒入侵、抑制转座子活动、调控基因表达的监控机制,具有重大生物学意义。RNA干扰技术的发现及其作为基因敲除工具在模式生物、哺乳动物细胞和体内的成功应用的例子确保了它在药学研究中的作用。越来越多的研究显示RNAi在药物研发中显示出极大潜力,目前已有部分RNAi药物进入临床试验阶段。本文将从两个方面来论述RNAi在药学的应用：一是RNAi在药物靶点鉴定中的运用;二是siRNAs作为药物在疾病治疗中的作用。     

RNA干扰技术的研究概述

RNA干扰是真核生物中由双链RNA诱发同源mRNA降解,使靶基因沉默的现象。本文概述了RNAi技术作为基因调控的有效工具,在生物学基础研究、农业领域和医药领域的相关应用。     

RNAi的研究进展

RNAi是通过双链RNA的介导特异性降解相应序列的mRNA,从而导致转录后水平的基因沉默．广泛存在于真菌、植物和动物等真核生物中．RNAi是自然界存在的生物抵抗病毒入侵、抑制转座子活动的一种防御机制,现已发展成为功能基因组学研究的重要手段之一．     

RNAi的研究进展

RNAi是通过双链RNA的介导特异性降解相应序列的mRNA,从而导致转录后水平的基因沉默．广泛存在于真菌、植物和动物等真核生物中．RNAi是自然界存在的生物抵抗病毒入侵、抑制转座子活动的一种防御机制,现已发展成为功能基因组学研究的重要手段之一．     

RNA 干扰作用及其技术

RNA干扰作用(RNAi)是通过双链RNA介导的特异性转录后基因沉默，这一过程已在拟南芥、线虫和真菌等多种模式生物中得到揭示．RNAi主要通过双链RNA被核酸酶切割成的小干扰RNA(siRNA)，由siRNA介导识别并靶向切割同源靶mRNA分子而实现的．RNAi具有高效性和高特异性，已成为关闭基因的一项新技术，在基因功能研究和疾病的基因治疗中具有广阔的应用前景．     

RNAi的革命

通过实验手段向细胞内导入长的双链RNA或者转基因可以产生一些短片段的双链RNA,这些短片段的双链RNA可以通过促使特定基因的mRNA降解来高效、特异地阻断体内特定基因的表达,使细胞出现特定基因缺失的表型,称为RNA干扰(RNA interference,RNAi)。SiRNA(small interference RNA)就是这种短片段双链RNA分子,能够以序列同源互补的mRNA为靶目标,降解特定的mRNA。基本上所有的生物体内也都存在一种内源的小分子RNA,为单链RNA,由基因组转录生成,但是不编码蛋白质,它们的功能在于调节mRNA的翻译,称为miRNA,是由具有发夹结构的前体剪切产生的。RNAi的发现具有划时代的意义,它不仅揭示了细胞内基因沉默的机制,而且有望成为后基因组时代基因功能分析的有力工具,极大地促进了人类揭示生命奥秘的进程。     

RNAi在药物研究中的研究进展

RNA干扰是双链RNA分子在mRNA水平上诱发的序列特异性的转录后基因表达沉默。随着RNAi分子机制研究的深入和应用的发展,RNAi已经应用在药物研究的各个领域,特别在药物开发上能够解决一些如药物靶点的鉴定、优化药靶等瓶颈问题,在节约研发时间的同时加速药物的临床研究。RNAi是药物研究的领域具有很大潜力的新型技术工具。     

RNAi的技术研究进展

RNA干扰是一些小的双链RNA可以高效、特异的阻断体内特定基因表达,促使mRNA降解,诱使细胞表现出特定基因缺失的表型,也是体内抵御外在感染的一种重要保护机制。本文对RNA i的发现过程及作用机理进行了综述,本技术已经成为最近生物医学领域的焦点之一,将应用于基因功能和信号传导系统,上下游分子相互关系的研究可能为肿瘤基因治疗提供新的策略,在病毒性、寄生病原性、突变基因、癌基因和癌相关基因表达引起的疾病的基因治疗之中也有了新的突破。     

RNA干涉技术

RNAi是双链RNA介导的、序列特异性的转录后基因沉默，自1998年发现至今已有很大进展。本文综述了RNA干涉的分子机理及其应用研究进展。     

基于支持向量机的siRNA降解效率预测

为了辅助siRNA的设计,从已发表文献中共收集到573个siRNA的实验数据,使用基于统计学习理论的支持向量机(SVM)方法,提取了siRNA序列的碱基对关联性(BBC)特征,然后使用十倍交叉验证方法,对siRNA沉默目标基因的效率进行了预测.结果表明,基于支持向量机,选用多项式核作为核函数的算法具有最高的AUC值(0.73,ROC曲线图)和最高的r值(0.43,Pearson相关系数分析),优于以前基于打分的算法.结果说明,在以后的siRNA的设计中应该更多关注碱基之间的关联信息.     

Inhibition of chemotherapy-related breast tumor EMT by application of redox-sensitive siRNA delivery system CSO-ss-SA/siRNA along with doxorubicin treatment

Metastasis is one of the main reasons causing death in cancer patients. It was reported that chemotherapy might induce metastasis. In order to uncover the mechanism of chemotherapy-induced metastasis and find solutions to inhibit treatment-induced metastasis, the relationship between epithelial-mesenchymal transition (EMT) and doxorubicin (DOX) treatment was investigated and a redox-sensitive small interfering RNA (siRNA) delivery system was designed. DOX-related reactive oxygen species (ROS) were found to be responsible for the invasiveness of tumor cells in vitro, causing enhanced EMT and cytoskeleton reconstruction regulated by Ras-related C3 botulinum toxin substrate 1 (RAC1). In order to decrease RAC1, a redox-sensitive glycolipid drug delivery system (chitosan-ss-stearylamine conjugate (CSO-ss-SA)) was designed to carry siRNA, forming a gene delivery system (CSO-ss-SA/siRNA) down-regulating RAC1. CSO-ss-SA/siRNA exhibited an enhanced redox sensitivity compared to nonresponsive complexes in 10 mmol/L glutathione (GSH) and showed a significant safety. CSO-ss-SA/siRNA could effectively transmit siRNA into tumor cells, reducing the expression of RAC1 protein by 38.2% and decreasing the number of tumor-induced invasion cells by 42.5%. When combined with DOX, CSO-ss-SA/siRNA remarkably inhibited the chemotherapy-induced EMT in vivo and enhanced therapeutic efficiency. The present study indicates that RAC1 protein is a key regulator of chemotherapy-induced EMT and CSO-ss-SA/siRNA silencing RAC1 could efficiently decrease the tumor metastasis risk after chemotherapy.

     

Effect of interrupted endogenous BMP/Smad signaling on growth and steroidogenesis of porcine granulosa cells

Bone morphogenetic proteins (BMPs) play a critical role in the growth and steroidogenesis of granulosa cells (GCs). BMP signals act through membrane-bound heteromeric serine/threonine kinase receptors. Upon ligand binding, BMPs activate intracellular Smad proteins and regulate growth and apoptosis in various cell types. The objective of this study was to demonstrate the effects of BMP/Smad signal on growth and steroidogenesis of porcine GCs. A strategy of RNA interference (RNAi)-mediated ‘gene silencing’ of Smad4, a core molecule mediating the intracellular BMP/Smad signal transduction pathways, was used to interrupt endogenous BMP/Smad signaling. Results indicate that Smad4-small interfering RNA (siRNA) caused specific inhibition of Smad4 mRNA and protein expression after transfection. Interrupted endogenous BMP/Smad signaling significantly inhibited growth, and induced apoptosis of porcine GCs, while decreasing estradiol production. In addition, interrupted BMP/Smad signaling significantly (P<0.05) changed the expression of Cyclin D2, CDK4, Bcl-2, and Cyp19a1. These findings provide new insights into how BMP/Smad signaling regulates the growth and steroidogenesis of porcine GCs.     

A Laboratory-intensive Course on RNA Interference and Model Organisms

RNA interference (RNAi) is a powerful method to silence gene expression in a variety of organisms and is generating interest not only as a useful tool for research scientists but also as a novel class of therapeutics in clinical trials. Here, we report that undergraduate and graduate students with a basic molecular biology background were able to demonstrate conceptual knowledge and technical skills for using RNAi as a research tool upon completion of an intensive 8-wk RNAi course with a 2-h lecture and 5-h laboratory per week. Students were instructed on design of RNAi experiments in model organisms and perform multiweek laboratory sessions based on journal articles read and discussed in class. Using Nicotiana benthamiana, Caenorhabditis elegans, and mammalian cell culture, students analyzed the extent of silencing using both qualitative assessment of phenotypic variations and quantitative measurements of RNA levels or protein levels. We evaluated the course over two semesters, each with a separate instructor. In both semesters, we show students met expected learning outcomes as demonstrated by successful laboratory experiment results, as well as positive instructor assessments of exams and lab reports. Student self-assessments revealed increased confidence in conceptual knowledge and practical skills. Our data also suggest that the course is adaptable to different instructors with varying expertise.     

RNA interference against interleukin-5 attenuates airway inflammation and hyperresponsiveness in an asthma model

Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5×106 IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.     

How golden is silence? Teaching undergraduates the power and limits of RNA interference

It is hard and getting harder to strike a satisfying balance in teaching. Time dedicated to student-generated models or ideas is often sacrificed in an effort to “get through the syllabus.” I describe a series of RNA interference (RNAi) experiments for undergraduate students that simultaneously explores fundamental concepts in gene regulation, develops cutting-edge laboratory skills, and embraces student-directed learning. Students design a small interfering RNA (siRNA) against luciferase, add it to cells expressing this gene, and then quantitatively assess the siRNA's effect on both intended and unintended targets, using a luciferase assay and a DNA microarray. Because both RNAi and microarray technologies are relatively new, with no clear consensus on their analysis or limitations, students are encouraged to explore different approaches to the design of their reagents and interpretations of their data. The ability to creatively formulate a hypothesis-driven experimental approach to a scientific question and to critically evaluate collected data is stressed. Equally important, this experiment emphasizes how modern scientific ideas emerge, are debated, tested, and decided.