RNA干扰的研究进展

RNA干扰(RNA interference,RNAi)是一种由双链RNA诱发的转录后水平的基因沉默现象,是近几年发展起来的基因表达调节新机制。RNAi广泛存在于真菌、植物和动物中,这种调控可以由siRNA、shRNA及miRNA等小分子RNA参与。在此主要对RNAi的研究进展如背景、分子调控机制、存在的问题和应用前景等进行了综述。     

病毒miRNA的研究进展

MicmRNA（miRNA）是指一组由多细胞生物产生的小片段非编码RNA,是真核生物基因调控重要的组成成分。近些年来有研究者在病毒中也同样发现编码产生的miRNA,其可通过RNA干扰途径参与调节感染过程以及促进癌症的发生。因此阻止病毒小RNA生成有望成为治疗病毒相关疾病的新方法。     

小分子RNA表达载体构建的新方法——MicroRNA前体PCR置换法

MicroRNA(miRNA)基因的终产物是进化上高度保守的、具有基因表达调控功能的非编码小分子RNA。miRNA的特征性发卡环前体(pre-miRNAs)在体内经数步加工后,与AGO蛋白构成沉默复合体(RISC)以行使其功能。如将已知pre-miRNAs上的miRNA成熟链和互补链(miRNA*)分别置换为有待研究的小分子RNA,并构建表达载体转化植株,利用转化株内源的miRNA成熟加工体系,可以产生预设的小分子RNA。利用已知拟南芥miRNA前体为模板,使用替换PCR的方法,人工构建了gso8-ap-miRNA-pBI121表达载体。利用农杆菌介导法转化拟南芥,多数T1代植株表现出提早开花的突变表型。该方法是一种简便、高效的构建小分子RNA表达载体的方法。     

真核生物中的微小RNA及其功能研究进展

真核生物中存在两种主要的非编码RNA(non-coding RNA),在真核生物中发挥重要作用。一类为微小RNA(microRNA,miRNA),另一为小干扰RNA(siRNA)。miRNA大小为19～25nt,在体内与蛋白质形成核糖核蛋白复合体(miRNP),在真核基因的表达调控,生长发育中起重要作用。siRNA在RNA干扰(RNA地 interference,RNAi)途径中起定位特异mRNA的作用。miRNA与siRNA有联系也有区别。miRNA在真核生物中的调控机制具有保守性。     

siRNA与miRNA在生物基因调控中的沉默机制比较

siRNA和miRNA的沉默机制是生物基因调控的重要手段之一. 小干扰RNA（small interfering RNA,siRNA）是RNA干扰的引发物,激发与之互补的目标mRNA沉默. 非编码RNA中的微小RNA（microRNA,miRNA）,能够识别特定的目标mRNA,通过与mRNAs的3′ 非翻译区结合,影响该目标蛋白的翻译水平. siRNA和miRNA的基因调控机制对生物学研究及疾病的病因和治疗等有直接影响. 本文主要对siRNAs和miRNAs的生物起源及沉默机制进行比较性论述：提出Dicers酶蛋白、Ago蛋白以及20 nt~25 nt的双链RNAs的 3类大分子是RNA沉默的特征结构,并进行了说明性论述|总结性叙述了siRNA和miRNA的2类小分子经典沉默机制,并提出其异同点. 最后,本文根据近期研究进展,对siRNA和miRNA的生物起源及沉默机制提出了新的疑问.     

脊椎动物病毒与微RNA

抗病毒作用是RNA干扰(RNAi)在植物及低等动物中的一个重要功能。一方面,宿主细胞编码并表达短干扰RNA(siRNA),对入侵细胞的病毒产生抑制作用;另一方面,病毒编码并表达特定的RNA或蛋白质,以对抗宿主细胞的RNAi。在部分脊椎动物病毒中已经发现多种由病毒编码的微RNA(miRNA),它们对病毒及细胞基因的表达有重要的调节作用。同时,某些细胞miRNA也可影响脊椎动物病毒的复制。然而,RNAi在脊椎动物细胞中是否具有广谱抗病毒活性、脊椎动物病毒又是否普遍编码miRNA及普遍具备拮抗RNAi的机制?目前尚无定论,有待于进一步的研究加以阐明。     

人工 microRNA 技术研究进展

RNA干扰（RNAi）是由双链RNA触发的在mRNA水平进行的特异靶序列的基因沉默现象,广泛存在于动物、植物和病毒中,主要包括小干扰RNA（siRNA）及微小RNA（miRNA）两种作用途径。人工miRNA（amiRNA）是将天然miRNA的成熟序列替换成人工设计的靶向其他感兴趣基因的反义序列,通过天然miRNA的生成和作用途径达到RNAi的效果,具有干扰效果明显、作用迅速、毒性低等优点,拥有广阔的应用前景。我们对基于amiRNA的基因沉默技术进行了较为系统的介绍和总结,梳理了该技术的优缺点和适用范围,并展望了其进一步发展的方向和应用前景。     

一类新的基因沉默小RNA-piRNA（piwi-interacting RNA）

piRNA是单链非编码小分子RNA,长度约26-31nt,大部分集中在29-30nt,5’端具有尿嘧啶偏向性（约86％）,能够与Argonaute蛋白家族中的Piwi亚家族蛋白相互结合而产生作用。piRNA的功能主要是维持基因组中转座子的正常沉默状态,以防止基因组中转座子爆发而引起相应基因的改变。piRNA与siRNA及miRNA均是近些年发现的非编码小RNA,它们均可通过一套相应的机制进行RNA干扰,在转录、转录后甚至翻译水平对靶基因及蛋白进行调节,它们之间既有联系又有区别。piRNA数据库的建立将对这类小分子RNA的研究有很大的促进作用。     

RNA干扰技术的原理与应用

RNA干扰(RNA interference,RNAi)是由双链RNA(double-stranded RNA,dsRNA)所引起的序列特异性基因沉默,是真核生物中一种非常保守的机制,它与协同抑制(cosuppression)、转座子沉默(transposon silencing)以及发育等许多重要的生物学过程密切相关。RNA干扰依赖于小干扰RNA(Small interference RNA,siRNA)与靶序列之间严格的碱基配对,具有很强的特异性,涉及众多基因和蛋白复合物,构成了一个以小RNA为核心的真核基因表达调控系统,它可以在染色质水平、转录水平、转录后水平和翻译水平参与基因表达的调节。RNA干扰技术为人们迅速、准确的剖析基因的功能,分析基因之间错综复杂的联系和相互作用提供了极为有用的工具,同时也为人们预防和治疗癌症和病毒疾病提供了新的思路。     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

植物microRNA的保守性及其分布的分析

MicroRNA(miRNA)是真核生物中具有重要调控作用的小分子非编码RNA。本文对miRNA官网miRBase数据库Release 22.1中隶属于植物界的绿藻门、苔藓植物门、蕨类植物门、裸子植物门、被子植物门共计82个物种的miRNA进行了统计分析。miRBase共收录植物miRNA 前体8 615个,成熟miRNA 10 414条,隶属于2 892个miRNA家族。绿藻门miRNA与其他4个门miRNA无同源性;对其他4个门植物miRNA的保守性进行研究,发现存在于2个植物门的miRNA家族有26个,属于中度保守miRNA家族;14个miRNA家族存在于3个及3个以上植物门中,属于高度保守miRNA家族,其中7个miRNA家族系苔藓、蕨类、裸子和被子植物共有,是植物中最保守的miRNA。分析表明,超过30个miRNA家族的植物有35种。进一步对40个中度或者高度保守miRNA在35种植物中的分布进行研究,发现miRNA家族及其成员在物种间的分布存在较大的差异。这些分布上的差异一方面反映不同植物中miRNA的研究深度不同,另一方面也反映出miRNA在植物进化过程中的适应性调整。研究不同植物中miRNA家族的分布,可在miRNA水平为植物早期进化同源性的研究提供分子依据。     

miRNA–miRNA interaction implicates for potential mutual regulatory pattern

Background

Natural or endogenous sense/antisense miRNAs, located on sense and antisense strands in the same genomic region, respectively, are detected recently. However, little is known about these miRNA pairs, especially for their distributions in different animal species. We herein present systematic analysis of them in human, mouse and rat miRNAs, and their expression patterns based on deep sequencing datasets.

Methods and results

The phenomenon of miRNA–miRNA interaction could be detected in different animal species. The common miRNAs pairs were found across species. These miRNA pairs could form miRNA:miRNA duplex with complete complementary structure, and were prone to be located on specific chromosomes. They might be homologous miRNA genes (especially in human), or clustered in a gene cluster (especially in rat), or simultaneously detected in different genomic regions due to multicopy pre-miRNAs. Remarkably, some miRNA pairs, located in different genomic regions, also showed complementarity as well as endogenous sense/antisense miRNAs. Based on published deep sequencing datasets, one member of miRNA pairs always was abundantly expressed, whereas another was quite rare. Rare common target mRNAs of these miRNA pairs were predicted.

Conclusions

Interaction between miRNAs and significant expression divergence implied complex potential mutual regulatory pattern in the miRNA world. The study would enrich miRNA regulatory network.     

Identification of the highly accumulated microRNA*s in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa)

Plant microRNAs (miRNAs) are crucial for the regulation of gene expression, which is involved in almost all the important biological processes. In the cytoplasm, the miRNA strand is selectively incorporated into a specific Argonaute (AGO)-associated gene silencing complex, while the miRNA* is degraded rapidly. Thus, most miRNA*s were thought to be biologically meaningless. Interestingly, several recent reports in both plants and animals have shaken this notion. Many miRNA*s were demonstrated to possess regulatory roles in gene expression. However, the low accumulation levels of most miRNA*s raise the question whether the activities of this small RNA (sRNA) species are widespread in plants. Here, by using publicly available sRNA high-throughput sequencing data, we found that the accumulation levels of several miRNA*s could be much higher than those of their miRNA partners in certain organs, mutants and/or AGO-associated silencing complexes of both Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). Based on target prediction and degradome sequencing data-based validation, some of these highly accumulated miRNA*s were indicated to possess cleavage-based potential regulatory role on certain targets. Besides, some interesting biological interpretations were obtained based on the accumulation patterns of the miRNA*s, the annotations of the target genes, and literature mining. Taken together, the expanded list of the highly accumulated miRNA*s along with their potential target genes discovered in this study further strengthened the current notion that certain members of the miRNA* species are biologically relevant, which needs further inspection.     

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).     

A convenient system for highly specific and sensitive detection of miRNA expression

Since the first miRNA was discovered in 1993, miRNAs have become a hotspot for biological research. In order to feed this demand, a robust method is required to detect miRNA gene expression. Development of a detection method is more difficult for miRNAs than for long RNAs, such as mRNA, owing to their small size. Existing methods have limitations; thus, new methods are required. We describe a new system for detecting miRNA expression, which can distinguish miRNA from its precursor and has single-nucleotide resolution. It has single molecule and multiplex detection potential. It may be performed as a polymerase chain reaction (PCR) method, a blotting method, or a macroarray method according to the analyst''s preference. This personalized system provides a convenient tool for the detection of miRNA gene expression.     

环状RNA:潜在的新型miRNA活性调控分子

环状RNA(circular RNA,circRNA)是近年来RNA领域最新的研究热点.它是一类由特殊的选择性剪切产生且在真核细胞中广泛表达的环形内源性RNA分子.研究发现,circRNA富含microRNA(miRNA)结合位点,可以发挥竞争性内源RNA作用,作为miRNA"海绵"来解除对其靶基因的抑制效应.近年来,circRNA作为一种新型调控分子调控miRNA功能的发挥,受到众多研究者的青睐.本文综述circRNA的产生机制,及其调控miRNA的最新研究进展与研究方法等.     

MicroRNA靶向病毒携带的microRNA模拟靶序列对病毒的抑制作用

Micro RNA模拟靶序列(target mimic,TM)通过竞争性结合miRNA,从而干扰miRNA对靶标m RNA的调控.我们前期工作发现:以黄瓜花叶病毒(Cucumber mosaic virus,CMV)作为载体在植物体内表达TM序列有效地抑制了miRNA的活性或稳定性,从而消减了miRNA对靶基因的调控.但是,miRNA与CMV携带的TM序列的结合在一定程度上抑制了病毒的积累.研究分析了miRNA靶向病毒携带的TM序列对病毒抑制作用的内在原因.a.通过RNA印迹分析CMV携带不同miRNA TM序列对病毒积累的影响,进一步明确miRNA靶向病毒携带的TM序列对病毒的抑制作用;b.利用GFP作为报告基因,通过荧光显微镜、蛋白质印迹以及RNA印迹分析TM序列对重组病毒积累的影响;c.以GFP作为报告基因,利用荧光显微镜观察和免疫印迹方法分析模拟靶序列对GFP翻译的影响;d.利用CMV病毒的反式复制系统分析miRNA模拟靶序列对病毒负链RNA合成的影响.结果表明,多种植物内源的miRNA靶向CMV基因组携带的miRNA TM序列,在不同程度上抑制了病毒的积累,miRNA与其TM序列的结合抑制GFP蛋白的翻译和负链的合成.植物内源的miRNA通过与病毒基因组携带的miRNA模拟靶序列结合,通过抑制病毒蛋白的翻译以及病毒负链RNA的合成,从而降低了病毒的积累水平.基于该论文的研究结果有可能建立一种抗病毒的新方法.