microRNAs相关新型调控技术与疾病治疗

MicroRNA（miRNA）是一类在进化中高度保守的非编码小分子单链RNA(19～25 nt）,在转录后水平调控中发挥重要作用. 越来越多的证据表明,miRNA广泛参与了机体生长发育、细胞增殖与凋亡等多种重要的生理过程,并在病毒感染、恶性肿瘤、心血管与内分泌疾病等多种病理状态下呈异常表达,影响许多疾病的发生、发展及预后. 对miRNA的表达水平进行可控性干预可能成为一种有效的治疗手段. 本文综述了目前用于干预miRNA水平的调控技术,以及疾病治疗相关给药系统的研究进展,提示miRNA相关新型调控技术具有较为广阔的临床应用前景.     

microRNA调控的生物网络

生物网络是生物体内各种分子通过相互作用来完成各种复杂的生物功能的一个体系。网络水平的研究,有助于我们从整体上理解生物体内各种复杂事件发生的内在机制。microRNA（miRNA）是一类在转录后水平调控基因表达的小RNA分子。研究结果表明,miRNA调控的靶基因分布范围很广,因此必然与目前所研究的生物网络有着各种各样的联系。对这种关系的揭示,将对阐明miRNA的调控规律起到重要的作用。本文重点讨论了miRNA调控的基因调控网络、蛋白质相互作用网络以及细胞信号传导网络的特征。此外,还总结了miRNA调控的网络模体（motif）和miRNA协同作用网络的特征。     

微小RNA分析技术研究进展

微小RNA（miRNA）是一类起重要调控作用的非编码小分子RNA。准确分析组织或细胞中miRNA的表达水平是研究其生物学功能的基础。近年,研究者开发出多种方法检测不同生理、病理过程中miRNA的差异表达,发现miRNA的异常表达与癌症等多种疾病密切相关。目前,miRNA已逐渐成为重要的疾病诊断生物标志物乃至治疗靶点。miRNA的分析技术贯穿miRNA的研究和药物研发过程,并起到关键作用。我们针对miRNA的不同研究阶段所采用的定性和定量分析方法,着重阐述了用于初始miRNA研究的克隆测序类技术、分析miRNA表达谱的高通量芯片技术、研究具体目标miRNA及其前体表达的qPCR和改良Northern印迹技术,以及将修饰后miRNA作为药物的药代动力学评价技术。     

哺乳动物乳汁miRNA的研究进展

作为真核生物体内重要的调控分子,miRNA可存在于包括血清、血浆、唾液、尿液在内的多种动物体液之中。最近的研究表明哺乳动物的乳汁中也分泌miRNA,表明miRNA有可能通过哺乳传递给新生个体,从而调控新生个体发育过程中的某些关键生理、生化途径,协助其正常发育。这一发现很可能揭开了哺乳动物世代间miRNA进行功能基因表达调控的研究序幕。文章对哺乳动物乳汁中miRNA的发现过程,这些miRNA在乳汁中的存在形态及其分离技术,以及其中数个重要miRNA的已知功能作简要概述,并探讨了相关的后续研究任务及其面临的挑战。     

miRNA调控药用植物生长发育和次生代谢

microRNA(miRNA)作为一类内源性的短链非编码RNA,广泛存在于真核细胞中,主要通过对转录本剪切和抑制翻译等方式,参与转录后基因的表达调控。近年来研究表明,多种药用植物中鉴定出大量的miRNA。这些miRNA对药用植物的生长发育和次生代谢产物合成具有调控功能。次生代谢产物是药用植物的主要有效成分,研究miRNA对药用植物次生代谢过程的调控作用具有十分重要的意义。本文综述了miRNA在植物中的产生途径、作用方式和体内功能,在此基础上重点介绍了miRNA对药用植物生长发育和次生代谢产物生物合成的调控作用,并对药用植物miRNA的研究进行了展望,以期为提高药用植物产量,高效获得药用植物有效成分以及临床应用开拓新的思路。     

植物miRNA的进化

鉴于miRNA在植物基因表达调控中的重要作用, 人们已经开展对植物miRNA的预测、鉴定、功能和进化等方面的研究。随着许多模式植物基因组测序的完成, miRNA的基因组学和进化信息的整合为miRNA的起源和进化研究提供了越来越多的证据和假说, 然而尚未见关于植物miRNA进化方面的系统报道。文章从miRNA的起源以及相应的几种假说、miRNA的产生和消亡、miRNA的功能进化等几方面来分析和综述植物miRNA进化的研究进展。     

MicroRNA对多细胞动物复杂性进化的影响

MicroRNA(miRNA)是一种长度约为22个碱基的非编码单链小分子RNA。作为一类重要的转录后基因表达调控因子,miRNA参与了广泛的生物学过程,如发育时程调控、细胞分化、凋亡、肿瘤以及病毒抵抗等。然而,除了在个体发生过程中的重要功能外,越来越多的研究表明,miRNA在系统发生中也扮演着关键的角色。基因表达模式的不同被广泛地认为是物种内和物种间表型差异的根源,动物物种间miRNA的保守性和多样性研究提示miRNA对物种间表型差异以及动物进化起着重要的作用。文章介绍了miRNA产生过程和作用机制,重点探讨了miRNA在动物进化过程中的作用,从miRNA的进化速度、miRNA表达的时空特异性、miRNA作用靶位点变异以及miRNA基因的扩增与丢失4个方面论述miRNA介导的基因调控网络对多细胞动物发育复杂性进化的影响,推测miRNA在多细胞动物进化过程中驱动了复杂性的增加。     

落叶松体胚发育中5个miRNA前体与成熟体的表达

利用同源比对或RACE克隆了5个落叶松(Larix leptolepis) miRNA前体。结果显示, 在各物种miRNA前体间, 成熟序列高度相似, 但其它序列相似度差异大, 序列相似度与亲缘关系有关。采用qRT-PCR分析了5个miRNA、前体和靶基因在落叶松体胚8个发育阶段的表达变化。结果显示, miRNA表达最高峰出现在后期子叶胚, 暗示与促进胚胎休眠有关; 表达次高峰出现时期不同, 表现为miR397和miR408在PEMIII, miR398在早期单胚, miR156和miR166在早期子叶胚, 表明其与保持薄细胞壁、质子传递、顶端分生组织形成等调控有关。miRNA成熟体表达与前体含量不呈线性相关, 可能受多重调控。研究结果对于阐明MIR基因进化、表达调控及在体胚发育中的调控功能具有重要理论意义。     

昆虫microRNA的研究进展

MicroRNA (miRNA)是20世纪90年代发现的一类由内源基因编码的长度约21~24 nt的非编码单链RNA分子, 广泛存在于真核生物中, 对基因的转录后调控起着非常重要的作用。本文简要介绍了miRNA的产生与调控机制, 同时从昆虫miRNA的发现鉴定、 靶基因预测与功能验证, 昆虫miRNA的序列特征与进化, 果蝇和非果蝇类昆虫miRNA生物学功能以及供昆虫miRNA研究的网络平台等方面对昆虫miRNA的最新进展进行了综述, 旨在为进一步研究昆虫miRNA提供借鉴和参考。对昆虫miRNA的研究表明其参与调控细胞分化、 增殖及凋亡、 胚胎发育、 器官发生、 形态构建、 生理代谢、 环境协调、 行为认知、 免疫防御等几乎所有的生物过程。因此, 深入研究其生物功能、 调控网络和开发应用等可能成为今后一段时间昆虫miRNA研究的重要内容。     

miRNA在植物病原调控方面的研究进展

miRNA是一类在真核生物体内普遍存在的,长度在22 nt左右的单链小RNA分子。大量研究发现,miRNA可广泛参与植物的生长发育、新陈代谢、物质运输、逆境响应及病原防御等多种生理生化过程。目前已经从植物中鉴定到大量的miRNA,但其中参与植物病原调控相关miRNA的研究较少。miRNA作为一种重要的转录调控因子,可参与调控病原相关分子模式触发的免疫反应和效应因子触发的免疫反应。拟南芥中,miRNA393通过靶向生长素受体基因对植物生长素进行负调控,从而在抵御细菌侵染方面发挥作用;水稻中,miRNA528可响应水稻条纹病毒(RSV)的侵染,人为提高miRNA528水平有助于维持水稻对RSV的抗性。阐述了miRNA的作用机制,与植物细菌、真菌和病毒侵染相关的miRNA研究进展,总结了葡萄,苹果,梨和桃等具有重要经济价值果树中病原调控相关miRNA研究情况,旨在为今后miRNA在植物,特别是在果树抗病研究方面提供全面的理论依据。     

MiR133b-mediated inhibition of EGFR-PTK pathway promotes rAAV2 transduction by facilitating intracellular trafficking and augmenting second-strand synthesis

Recombinant adeno-associated virus (rAAV) is an extremely attractive vector in the in vivo delivery of gene therapy as it is safe and its genome is simple. However, challenges including low permissiveness to specific cells and restricted tissue specificity have hindered its clinical application. Based on the previous studies, epidermal growth factor receptor-protein tyrosine kinase (EGFR-PTK) negatively regulated rAAV transduction, and EGFR-positive cells were hardly permissive to rAAV transduction. We constructed a novel rAAV-miRNA133b vector, which co-expressed miRNA133b and transgene, and investigated its in vivo and in vitro transduction efficiency. Confocal microscopy, live-cell imaging, pharmacological reagents and labelled virion tracking were used to analyse the effect of miRNA133b on rAAV2 transduction and the underlying mechanisms. The results demonstrated that miRNA133b could promote rAAV2 transduction and the effects were limited to EGFR-positive cells. The increased transduction was found to be a direct result of decreased rAAV particles degradation in the cytoplasm and enhanced second-strand synthesis. ss-rAAV2-miRNA133b vector specifically increased rAAV2 transduction in EGFR-positive cells or tissues, while ss-rAAV2-Fluc-miRNA133b exerted an antitumor effect. rAAV-miRNA133b vector might emerge as a promising platform for delivering various transgene to treat EGFR-positive cell-related diseases, such as non-small-cell lung cancer.     

A novel therapeutic strategy for alleviating atrial remodeling by targeting exosomal miRNAs in atrial fibrillation

Atrial fibrillation (AF) is one of the most frequent cardiac arrhythmias, and atrial remodeling is related to the progression of AF. Although several therapeutic approaches have been presented in recent years, the continuously increasing mortality rate suggests that more advanced strategies for treatment are urgently needed. Exosomes regulate pathological processes through intercellular communication mediated by microribonucleic acid (miRNA) in various cardiovascular diseases (CVDs). Exosomal miRNAs associated with signaling pathways have added more complexity to an already complex direct cell-to-cell interaction. Exosome delivery of miRNAs is involved in cardiac regeneration and cardiac protection. Recent studies have found that exosomes play a critical role in the diagnosis and treatment of cardiac fibrosis. By improving exosome stability and modifying surface epitopes, specific pharmaceutical agents can be supplied to improve tropism and targeting to cells and tissues in vivo. Exosomes harboring miRNAs may have clinical utility in cell-free therapeutic approaches and may serve as prognostic and diagnostic biomarkers for AF. Currently, limitations challenge pharmaceutic design, therapeutic utility and in vivo targeted delivery to patients. The aim of this article is to review the developmental features of AF associated with exosomal miRNAs and relate them to underlying mechanisms.     

Viral delivery of miR-196a ameliorates the SBMA phenotype via the silencing of CELF2

Spinal and bulbar muscular atrophy (SBMA) is an inherited neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) tract of the androgen receptor (AR-polyQ). Characteristics of SBMA include proximal muscular atrophy, weakness, contraction fasciculation and bulbar involvement. MicroRNAs (miRNAs) are a diverse class of highly conserved small RNA molecules that function as crucial regulators of gene expression in animals and plants. Recent functional studies have shown the potent activity of specific miRNAs as disease modifiers both in vitro and in vivo. Thus, potential therapeutic approaches that target the miRNA processing pathway have recently attracted attention. Here we describe a novel therapeutic approach using the adeno-associated virus (AAV) vector–mediated delivery of a specific miRNA for SBMA. We found that miR-196a enhanced the decay of the AR mRNA by silencing CUGBP, Elav-like family member 2 (CELF2). CELF2 directly acted on AR mRNA and enhanced the stability of AR mRNA. Furthermore, we found that the early intervention of miR-196a delivered by an AAV vector ameliorated the SBMA phenotypes in a mouse model. Our results establish the proof of principle that disease-specific miRNA delivery could be useful in neurodegenerative diseases.     

Inhibition of microRNA with antisense oligonucleotides

Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.     

microRNAs: a new emerging class of players for disease diagnostics and gene therapy

microRNAs (miRNAs) are a new class of non-protein-coding small RNAs, which regulate the expression of more than 30% protein-coding genes. The unique expression profiles of different miRNAs in different types of cancers and at different stages in one cancer type suggest that miRNAs can function as novel biomarkers for disease diagnostics and may present a new strategy for miRNA gene therapy. Anti-miRNAs and antisense oligonucleotides (ASO) have been employed to inhibit specific miRNA expression in vitro and in vivo for investigational and clinical purposes. Although miRNA-based diagnostics and gene therapy are still in their infancy, their huge potentials will meet our need for future disease diagnostics and gene therapy. High efficient delivery of miRNAs into targeted sites, designing accurate anti-miRNA/ASOs, and related biosafety issues are three major challenges in this field.     

Development of a microRNA delivery system based on bacteriophage MS2 virus-like particles

Recently, microRNA (miRNA)-mediated RNA interference has been developed as a useful tool in gene function analysis and gene therapy. A major obstacle in miRNA-mediated RNAi is cellular delivery, which requires an efficient and flexible delivery system. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for RNA and drug delivery. However, MS2 VLP-mediated miRNA delivery has not yet been reported. We therefore used an Escherichia coli expression system to produce the pre-miR 146a contained MS2 VLPs, and then conjugated these particles with HIV-1 Tat(47-57) peptide. The conjugated MS2 VLPs effectively transferred the packaged pre-miR146a RNA into various cells and tissues, with 0.92-14.76-fold higher expression of miR-146a in vitro and about two-fold higher expression in vivo, and subsequently suppressed its targeting gene. These findings suggest that MS2 VLPs can be used as a novel vehicle in miRNA delivery systems, and may have applications in gene therapy.     

MicroRNA在靶向治疗缺血性心脏病中的研究进展

MicroRNA（MiRNA,miR）通过调节信使RNA（mRNA）的表达,广泛参与心血管系统细胞的增殖、迁移、分化、凋亡等病理生理过程,在心血管系统疾病的发生发展过程中起着重要的调控作用。越来越多的研究表明,针对缺血性心血管疾病的发病机制,通过特异性调节miRNA的活性,抑制相关蛋白的表达,对各种缺血性心脏病具有显著的治疗作用。但目前开发miRNA靶向治疗药物尚缺乏大规模的临床试验研究,其有效性和安全性需进一步证实。本文旨在综述MicroRNA在靶向治疗缺血性心脏病中的研究进展,以期为开发MiRNA靶向治疗药物治疗缺血性心脏病提供更多的理论依据。