Gene silencing by systemic delivery of synthetic siRNAs in adult mice

In mammalian cells, RNA duplexes of 21-23 nucleotides, known as small interfering RNAs (siRNAs) specifically inhibit gene expression in vitro. Here, we show that systemic delivery of siRNAs can inhibited exogenous and endogenous gene expression in adult mice. Cationic liposome-based intravenous injection in mice of plasmid encoding the green fluorescent protein (GFP) with its cognate siRNA, inhibited GFP gene expression in various organs. Furthermore, intraperitoneal injection of anti-TNF-alpha siRNA inhibited lipopolysaccharide-induced TNF-alpha gene expression, whereas secretion of IL1-alpha was not inhibited. Importantly, the development of sepsis in mice following a lethal dose of lipopolysaccharide injection, was significantly inhibited by pre-treatment of the animals with anti-TNF-alpha siRNAs. Collectively, these results demonstrate that synthetic siRNAs can function in vivo as pharmaceutical drugs.     

Identification of sequence features that predict competition potency of siRNAs

Small interfering RNAs (siRNAs) specifically knock-down target mRNAs via RNA interference (RNAi) mechanism. During this process, introduction of excess amount of exogenous siRNAs could lead to the saturation of cellular RNAi machinery. One consequence of RNAi machinery saturation is the competition between two simultaneously introduced siRNAs, during which one siRNA loses gene silencing activity. Although competition phenomena have been well characterized, the molecular and sequence features of siRNAs that specify the competition potency remain poorly understood. Here, for the first time, we performed a large-scale siRNA competition potency analysis by measuring the competition potency of 56 different siRNAs and ranking them based on their competition potency. We have also established an algorithm to predict the competition potency of siRNAs based upon the conserved sequence features of strong and weak competitor siRNAs. The present study supports our hypothesis that the competition potency of siRNAs is specified by the 5′-half antisense sequence and provides a useful guideline to design siRNAs with minimal RNAi machinery saturation.     

RNA interference induced by siRNAs modified with 4'-thioribonucleosides in cultured mammalian cells

Short interfering RNAs (siRNAs) variously modified with 4'-thioribonucleosides against the Photinus luciferase gene were tested for their induction of the RNA interference (RNAi) activity in cultured NIH/3T3 cells. Results indicated that modifications at the sense-strand were well tolerated for RNAi activity except for full modification with 4'-thioribonucleosides. However, the activity of siRNAs modified at the antisense-strand was dependent on the position and the number of modifications with 4'-thioribonucleosides. Since modifications of siRNAs with 4'-thioribonucleosides were well tolerated in RNAi activity compared with that of 2'-O-methyl nucleosides, 4'-thioribonucleosides might be potentially useful in the development of novel and effective chemically modified siRNAs.     

Transposon-based RNAi delivery system for generating knockdown cell lines

RNA interference is rapidly becoming a powerful tool for genetic analyses in mammalian systems. A potential drawback to transient small inhibitory RNA silencing is the short duration of downregulation it confers, usually only 24-72h. Viral-based vector systems for the long-term delivery of RNA hairpins have been developed, yet they require expertise in viral production and transduction. Here we describe a simple plasmid-based system for the generation of long-term gene knockdown utilizing RNA interference combined with the gene delivery capabilities of the mammalian Tc1-like transposon Sleeping Beauty. Designated Maleficent, this system is shown to downregulate exogenous expression of GFP in a constitutively positive cell line. In addition, targeting of the endogenously expressed lamin A gene results in long-term silencing with significant reduction in protein levels (> 95%). Maleficent therefore provides a relatively easy, efficient, and stable means of delivering RNAi hairpins to generate long-term gene-specific knockdown cell lines.     

哺乳动物中RNA干扰沉默基因表达的研究进展

RNA干扰及相关基因沉默导致的代谢通路变化已经彻底改变了人们对基因调控的理解。基因沉默技术已经被用来作为一种研究工具来控制某些细胞基因的表达,特异的si RNA导入不同的细胞需要不同的转染载体才能达到最大的转染效率,并且不会产生较大的细胞毒性。近年来尤其是碳纳米管等新型纳米材料载体的应用拓展了人们对传统脂质体和病毒载体的认识。首先介绍RNA干扰技术及其发展历程,随后对利用几种不同的载体来设计和进行RNA干扰试验进行了比较,最后对最初的脂质体到生物病毒再到高分子纳米材料介导的RNA干扰在疾病的治疗和临床诊断进行了展望。     

半乳糖凝集素-3(galectin-3)基因沉默对Eca109人类食管癌细胞的影响

半乳糖凝集素-3(galectin-3)是一种多功能的β-半乳糖苷结合凝集素,涉及包括细胞生长、粘附、增殖、进展、转移以及凋亡等多种生物学功能,在恶性肿瘤中高表达。以前的研究已经证实了galecin-3过表达在Eca109人食管癌细胞的生物学作用。本研究试图通过进行小干扰RNA(siRNA)介导的galectin-3沉默,以分析galectin-3沉默对食管癌细胞生物学行为的影响。我们采用Western blotting和RT-qPCR被用来证实在蛋白质和mRNA水平上的galectin-3低表达,使用细胞计数试剂盒-8评估细胞增殖,用Annexin V-PE/7-AAD细胞凋亡检测试剂盒和流式细胞术检测Eca109细胞的凋亡。研究结果表明,转染后72 h,si Gal-3组Eca109细胞增殖明显低于siRNA对照组和未处理组(p<0.001)。Transwell实验结果显示,与其他组相比,galecin-3的抑制作用显著降低Eca109细胞的迁移和侵袭能力(p<0.05)。与siRNA-对照组和未处理组相比,galectin-3敲低显著增加Eca109细胞的凋亡率(p<0.05)。敲低Eca109细胞中galecin-3的表达后,细胞增殖、迁移和侵袭能力下降,而细胞凋亡增强,说明galectin沉默可作为食管癌治疗的新策略。     

Functional comparison of single- and double-stranded siRNAs in mammalian cells

The concept of small interfering RNA (siRNA) has been extended to include not only short double-stranded RNA of 19-25bp, but also single-stranded antisense RNA of the same length, since such single-stranded antisense siRNAs were recently found to be able to inhibit gene expression as well. We made comprehensive comparison of double- and single-stranded siRNA functions in RNA interference (RNAi), targeting multiple sites and different mRNAs, measuring RNAi effects at different time-points and in different cell lines, and examining response curves. Duplex siRNAs were found to be more potent than single-stranded antisense siRNAs. This was verified by the observation that single-stranded antisense siRNAs, which were inefficient in some cases when used alone, could be rescued from inefficiency by sequentially transfecting with the sense siRNAs. This result suggests that the structural character of siRNA molecules might be a more important determinant of siRNA efficiency than the cellular persistence of them.     

RNA干扰(RNAi)文库研究进展

RNAi是由双链RNA(dsRNA)引发的转录后基因沉默现象,由dsRNA产生的小分子siRNA会导致生物体内同源转录产物特异性降解,是基因表达调控的重要方式之一。目前RNAi技术已发展成为遗传分析强有力的工具,在基因功能分析鉴定方面发挥越来越大的作用。构建大规模的RNAi文库进而转变成RNAi突变体库是功能基因组学研究的重要手段,因此如何利用简单经济的方法构建特定物种的高效RNAi文库就成为关键问题。综述了目前构建RNAi文库的不同方法以及每种构建方法的优点和存在的不足,为不同研究目的的RNAi文库的构建提供参考。     

RNA interference and its application in bone-related diseases

RNA interference (RNAi) is the most exciting insight in biology in past decades, which provided new perspectives into the genome-wide surveys of gene function by targeted degradation of mRNA with the introduction of small interfering RNAs (siRNAs) or small hairpin RNAs (shRNAs) in a large variety of organisms, and turned out to be a more efficient and convenient method compared with the traditional knockout pathway. What's more, as the enhancement of its stability and improvement of its delivery vehicles, RNAi is bound to be a practical tool in determine gene function first in vitro and then in vivo. In this paper, we will focus on the recent achievements of RNAi and also depict the development of RNAi as a potentially powerful tool in studying bone-related diseases.     

Analysis of RNA Silencing in Agroinfiltrated Leaves of <Emphasis Type="Italic">Nicotiana Benthamiana</Emphasis> and <Emphasis Type="Italic">Nicotiana Tabacum</Emphasis>

In this study we analyse several aspects of cytoplasmic RNA silencing by agroinfiltration of DNA constructs encoding single- and double-stranded RNAs derived from a GFP transgene and from the endogenous Virp1 gene. Both types of inductors resulted after 2–4 days in much higher concentration of siRNAs in the agroinfiltrated zone than normally seen during systemic silencing. More specifically, infiltration of two transgene hairpin constructs resulted in elevated levels of siRNAs. However, differences between the two constructs were observed: the antisense–sense arrangement was more effective than the sense–antisense order. For both double-stranded forms, we observed a relative increase of the 24-mer size class of siRNAs. When a comparable hairpin construct of the endogenous Virp1 gene was assayed, the portion of the 24-mer siRNA class remained low as observed for all kinds of single-stranded inducers. The lack of increase of Virp1-derived 24-mers was independent of the expression level, as demonstrated by agroinfiltration into a transgenic plant that overexpressed Virp1 and showed the same pattern. Using transducer constructs, we could detect within a week transitive silencing from GFP to GUS sequences in the infiltrated zone and in either direction 5′–3′ and 3′–5′. Conversely, for the endogenous Virp1 gene neither transitive silencing nor the induction of systemic silencing could be observed. These results are discussed in view of the current models of RNA silencing.     

Effect of siRNAs on HSV-1 plaque formation and relative expression levels of RR mRNA

RNA interference (RNAi) is a process by which introduced small interfering RNA (siRNA) can cause the specific degradation of mRNA with identical sequences. The human herpes simplex virus type 1 (HSV-1) RR is composed of two distinct homodimeric subunits encoded by UL39 and UL40, respectively. In this study, we applied siRNAs targeting the UL39 and UL40 genes of HSV-1. We showed that synthetic siRNA silenced effectively and specifically UL39 and UL40 mRNA expression and inhibited HSV-1 replication. Our work offers new possibilities for RNAi as a genetic tool for inhibition of HSV-1 replication.     

RNA interference: the story of gene silencing in plants and humans

RNA interference is an exciting field of functional genomics that can silence viral genes. This property of interfering RNA can be used to combat viral diseases of plants as well as animals and humans. It is a short sequence of nucleic acid that can bind to the mRNA of the gene and interferes the process of its expression. It is diverse in occurrence as well as in applications. It occurs from nematodes to fungi and can cause gene silencing in plants, animals and human beings. Small interfering RNAs are used to silence plant viral genes and in production of therapeutic drugs against Hepatitis or Immuno-deficiency viruses in human. In this review, we will discuss the history, mechanism and applications of RNA interference in plant, animal and human research.     

Small interference RNA-mediated knockdown of sperm associated antigen 9 having structural homology with c-Jun N-terminal kinase-interacting protein

Recently, we reported a novel testis-specific sperm associated antigen 9 (SPAG9) protein, a new member of the JNK-interacting protein family, having a functional role in sperm-egg fusion [N. Jagadish, R. Rana, R. Selvi, D. Mishra, M. Garg, S. Yadav, J.C. Herr, K. Okumura, A. Hasegawa, K. Koyama, A. Suri, Biochem. J. 389 (2005) 73-82]. NCBI Blast searches revealed SPAG9 nucleotide sequence similarities with ESTs of various cancerous tissues. In the present study, we compared the efficiency of two independent SPAG9 specific small interfering RNA (siRNA) constructs, BS/U6/spag9 and BS/U6/spag9-I, to ablate the SPAG9 expression in mammalian cells. A positive correlation between the ratio of target gene versus siRNA and the suppression of SPAG9 expression was observed. Further, the cotransfection of BS/U6/spag9 with pcDNA-SPAG9 and pFlag-CMV2-JNK-3 resulted in specific suppression of SPAG9 without affecting JNK-3 expression. The present investigation will eventually extend the application of SPAG9 siRNA in in vivo targeting experiments that aim to define the SPAG9 functional genomics in tumor and reproductive biology.     

siRNA介导多药耐药相关蛋白(MRP1)的表达沉默

利用pSIREN-RetroQ载体构建了3个沉默多药耐药相关蛋白（MRP1）基因表达质粒pSI REN-siRNAs.并通过限制性内切酶酶切鉴定和DNA测序鉴定,将截断MRP和全长MRP1 cDNA分别克隆到真核表达载体pEGFP-N2和pcDNA3.1中,产生了pEGFP-MRP1T和pcDNA-MRP1表达质粒．质粒pEGFP-MRP1T分别与3个pSIREN-siRNAs共转染HEK293细胞沉默MRP1T-GFP靶基因,pSIREN-siRNA1作为阴性对照.荧光显微镜下显示结果表明,与pSIREN-siRNA1相比,pSIREN-siRNA2和pSIREN-siRNA3产生的siRNA能够有效沉默MRP1T-GFP融合蛋白的表达．为了沉默全长MRP1基因的表达,pcDNA-MRP1分别与3个pSIREN-siRNAs共转染HEK293细胞．Western印迹和MTT分析表明,pSIREN-siRNA2和pSIREN- siRNA3能有效抑制190 kD MRP1在HEK293细胞中的表达,而pSIREN-siRNA1则不能．pSIREN-siRNA2和pSIREN-siRNA3能逆转MRP1转染HEK293细胞产生的多药耐药性．RNA二级结构预测结果分析表明,siRNA1靶序列mRNA局部自由能热动力参数ΔG低于siRNA2和siRNA3靶序列mRNA局部自由能热动力参数,siRNA1的GC含量和Tm值高于siRNA2和siRNA3．这些数据提示,siRNA和局部靶结构可能影响siRNA对MRP1 mRNA表达的沉默作用．     

Design of LNA-modified siRNAs against the highly structured 5' UTR of coxsackievirus B3

This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5' UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays.     

植物RNA沉默的分子机制研究进展

RNA沉默（RNA silencing）是真核生物中的一种抵抗外源遗传因子（病毒、转座子或转基因）及调控基凶表达的防御机制。参与植物RNA沉默的酶及蛋白质主要包括6种RNA依赖的RNA聚合酶、4种Dicer-like（DCL）核酸内切酶和10种Argonautes蛋白。植物中4条RNA沉默途径分别由微小RNA（miRNAs）和3种小干扰RNA（siRNAs）介导,包括反式作用siRNAs（ta－siRNAs）、天然反义siRNAs（natsiRNAs）和异染色质siRNAs（hc-siRNAs）。在植物RNA沉默的系统性传播中,由DCL4或DCL2将dsRNAs裁剪为次级SiRNAS,以放大RNA沉默信号和增强沉默效应。