动物病毒microRNA的研究进展

MicroRNAs(miRNAs)是一种含有约22～25个核苷酸的非编码单链RNA分子,广泛存在于人类及其他各种生物中。它通过与靶mRNA特异性的碱基互补配对,引起靶mRNA降解或者抑制其翻译,从而调节基因的转录后表达水平。病毒microRNA是新发现的一类miRNA,综述了近年来病毒microRNA的产生、作用机制、生物学功能及其在动物上的研究进展。     

血清微小RNA在中药肝毒性临床前评价中的应用研究

通过对19种具有潜在肝脏毒性的中草药进行临床前安全性评价,在观测体重、摄食量、血清生化指标、脏器系数与肝脏组织病理学改变的同时,探讨血清微小RNA(miRNA)作为新型候选标志物在中草药肝损伤诊断中的应用价值。结果表明:与溶剂对照组相比,血清miRNA-122水平显著升高的给药组有6组,而血清谷丙转氨酶(ALT)和谷草转氨酶(AST)水平显著升高的分别为2组和3组,其中五倍子组表现为急性药源性肝损伤。在血清miRNA-122的时间相关性和剂量相关性分析中,与血清ALT和AST相比,血清miRNA-122呈现出更高的敏感性,表现为更早期和更大幅度的显著升高。说明血清miRNA-122可作为中草药肝损伤的新型候选诊断标志物,与肝脏组织病理学改变具有良好的相关性,且较传统血清生化学指标更为敏感。     

猪血管内皮细胞中与猪瘟病毒相关microRNA的初步筛选

本试验旨在筛选猪脐静脉血管内皮细胞(SUVECs)内作用于猪瘟病毒(CSFV)的microRNA。通过构建CSFV3′和5′非翻译区(Untranslated region,UTR)的双荧光素酶报告基因载体,经生物信息学预测可能与CSFV相互作用的microRNA,然后瞬时共转染重组载体与microRNA的抑制物,最后通过发光检测仪测定荧光素酶活性。结果表明,成功构建了CSFV5′非翻译区(373 bp)和3′非翻译区(252 bp)的报告基因载体,并合成了4条预测出的microRNA(ssc-mi R-let7c、ssc-mi R-106a、ssc-mi R-18、ssc-mi R-139)的抑制物,共转染后发光检测仪检测到荧光素酶的活性被不同程度抑制。本研究发现microRNA作用位点主要在CSFV的3′-UTR,而且以上4种microRNA都对CSFV3′-UTR有不同程度的抑制作用,其中ssc-mi R-18的抑制作用比较明显。     

microRNAs作为结核分枝杆菌感染标志物的研究进展

微小RNA(microRN.As)是一类长度约为22个核苷酸的非编码单链RNA分子,它参与基因转录后的表达调控,在免疫系统的调控和运行中具有重要作用。结核分枝杆菌是典型的胞内寄生菌,其感染能够引起人畜共患结核病。从microRNAs与结核免疫机制的相关性以及在结核分枝杆菌感染过程中巨噬细胞、外周血单核细胞及血清中microRNAs表达谱变化等方面,进行综述microRNAs作为结核分枝杆菌感染标志物的研究现状,以期为动物结核病检测提供理论参考。     

Comprehensive Characteristics of MicroRNA Expression Profile Conferring to Rhizoctonia solani in Rice

MicroRNAs(miRNAs) are about 22 nucleotides regulatory non-coding RNAs that play versatile roles in reprogramming plant responses to biotic and abiotic stresses. However, it remains unknown whether miRNAs confer the resistance to necrotrophic fungus Rhizoctonia solani in rice. To investigate whether miRNAs regulate the resistance to R. solani, we constructed 12 small RNA libraries from susceptible and resistant rice cultivars treated with water/pathogen at 5 h post inoculation(hpi), 10 hpi and 20 hpi, respectively. By taking the advantage of next-generation sequencing, we totally collected 400–450 known mi RNAs and 450–620 novel miRNAs from the libraries. Expression analysis of mi RNAs demonstrated different patterns for known and novel miRNAs upon R. solani challenge. Thirty-four mi RNA families were identified to be expressed specifically in rice, and most of them were involved in plant disease resistance. A particular Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway analysis result revealed that a great majority of target genes of regulated miRNAs belonged to the pathway of plant-pathogen interaction. Moreover, miR444 b.2, miR531 a, mir1861 i, novel_miR1956 and novel_miR135 conferred response to R. solani infection confirmed by Northern blot. Our global understanding of miRNA profiling revealed that the regulation of mi RNAs may be implicated in the control of rice immunity to R. solani. Analysis of the expression of miRNAs will offer the community with a direction to generate appropriate strategies for controlling rice sheath blight disease.     

Profiling microRNA expression in bovine alveolar macrophages using RNA-seq

MicroRNAs (miRNAs) are important regulators of gene expression and are known to play a key role in regulating both adaptive and innate immunity. Bovine alveolar macrophages (BAMs) help maintain lung homeostasis and constitute the front line of host defense against several infectious respiratory diseases, such as bovine tuberculosis. Little is known, however, about the role miRNAs play in these cells. In this study, we used a high-throughput sequencing approach, RNA-seq, to determine the expression levels of known and novel miRNAs in unchallenged BAMs isolated from lung lavages of eight different healthy Holstein–Friesian male calves. Approximately 80 million sequence reads were generated from eight BAM miRNA Illumina sequencing libraries, and 80 miRNAs were identified as being expressed in BAMs at a threshold of at least 100 reads per million (RPM). The expression levels of miRNAs varied over a large dynamic range, with a few miRNAs expressed at very high levels (up to 800,000 RPM), and the majority lowly expressed. Notably, many of the most highly expressed miRNAs in BAMs have known roles in regulating immunity in other species (e.g. bta-let-7i, bta-miR-21, bta-miR-27, bta-miR-99b, bta-miR-146, bta-miR-147, bta-miR-155 and bta-miR-223). The most highly expressed miRNA in BAMs was miR-21, which has been shown to regulate the expression of antimicrobial peptides in Mycobacterium leprae-infected human monocytes. Furthermore, the predicted target genes of BAM-expressed miRNAs were found to be statistically enriched for roles in innate immunity. In addition to profiling the expression of known miRNAs, the RNA-seq data was also analysed to identify potentially novel bovine miRNAs. One putatively novel bovine miRNA was identified. To the best of our knowledge, this is the first RNA-seq study to profile miRNA expression in BAMs and provides an important reference dataset for investigating the regulatory roles miRNAs play in this important immune cell type.     

猪背最长肌与腰大肌microRNA表达谱分析

【目的】为研究microRNA在猪肌肉生长发育中的作用。【方法】采用Illumina高通量测序技术对猪背最长肌和腰大肌的microRNA转录组进行测定,并鉴定差异表达的microRNA。【结果】在猪背最长肌和腰大肌文库中分别产生了15.22M和17.52M的序列;两个文库共检测到的695个microRNA,其中有363个共表达、193个差异极显著(P<0.01)。【结论】说明microRNA在不同骨骼肌类型中存在差异表达。     

循环microRNA在冠心病及其中医证候诊断中的作用

microRNA是一类长度约为22个核苷酸的内源性非编码RNA,并能以特定的方式与靶基因结合,从而抑制靶基因的翻译或使其降解。microRNA只有在受到致病因素（如环境因素）的影响以及病理状态下,其表达量才会发生改变,因此microRNA具有成为各种疾病的诊断生物标志物的可能。目前有大量研究表明microRNA参与冠心病发生发展,但其作用机制尚未完全阐明,本文从microRNA的特性、microRNA与冠心病、microRNA与中医证候等方面对近年来国内外的研究做一综述。     

小鼠miR367逆转录病毒载体的构建与检测

为了探明小鼠体细胞重编程过程中miR367的作用机制,试验构建小鼠miR367逆转录病毒载体。根据NCBI上小鼠miR367的相关序列,从小鼠基因组中扩增miR367并将其连接到pMD18-T载体,然后对重组质粒进行双酶切并回收目的片段。将目的片段与pMXs逆转录病毒载体连接,然后依次经过酶切、PCR筛选阳性克隆和测序,最终构建逆转录病毒载体miR367-pMXs;采用磷酸钙法将miR367-pMXs转染plat-E细胞,以包装逆转录病毒颗粒;用逆转录病毒颗粒侵染小鼠胚胎成纤维细胞,在侵染后的第5天,提取被侵染细胞的总RNA,逆转录后获得cDNA,并采用Q-PCR方法检测侵染细胞中的miR367表达量。结果表明,被逆转录病毒侵染后,小鼠胚胎成纤维细胞中miR367的相对表达量比侵染前约提高了170倍。上述结果表明,成功构建小鼠miR367的逆转录病毒载体,为开展体细胞重编程机制的相关研究提供依据。     

水稻Os-miR390前体基因的克隆、载体构建及转化

【目的】microRNA是一类长度为21~23 nt的非编码小分子RNA,对动植物的正常发育具有非常重要的调节作用。水稻Os-miR390的功能可能与生长素应答相关,拟采用转基因方法研究其生物学功能。【方法】根据水稻Os-miR390的前体RNA结构特点合理设计引物,从水稻基因组中克隆出其前体DNA片段,将其构建在玉米UbiI启动子驱动的表达载体中,并转入水稻。【结果】利用PCR成功地从水稻基因组中扩增出了Os-miR390前体基因,并将其构建在UbiI水稻组成型启动子下。经农杆菌介导,将其成功转入水稻愈伤,获得抗性幼苗,经过PCR检测,得到阳性转基因植株。【结论】T0代转基因植株表现叶片黄化的特征,待T1至T2代性状稳定后结合研究靶基因可深入研究其功能。