A brief review of microRNA and its role in PRRSV infection and replication

Porcine reproductive and respiratory syndrome virus (PRRSV), a single-stranded RNA virus, mainly infects cells of monocyte/macrophage lineage. Recently, host microRNAs were shown to be capable of modulating PRRSV infection and replication by multiple ways such as targeting viral genomic RNA, targeting viral receptor and inducing antiviral response. MicroRNAs are small RNAs and have emerged as important regulators of virus-host cell interactions. In this review, we discuss the identified functions of host microRNAs in relation to PRRSV infection and propose that cellular microRNAs may have a substantial effect on cell or tissue tropism of PRRSV.     

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.     

木薯sRNA 测序分析及其开花相关 microRNA的挖掘

为研究木薯花序与叶片中 sRNA 表达特性,探索 microRNA 对其开花过程的调控。本研究以木薯品种“华南八号”花序和幼叶为材料,利用 RNA-seq 技术进行 sRNA 测序分析;并筛选开花相关 microRNAs 进行表达特性分析。从花序和幼叶测序结果中分别获得 12 092 109 条和 11 499 655 条 sRNA 序列。花序中筛选出 139 条已知 microRNAs 和253 条新预测 microRNA,分别预测得到 992 和 3 736 条靶基因;幼叶中筛选出 134 条已知 microRNA 和 191 条新预测microRNAs,分别预测得到 979 和 2 603 条靶基因。最终筛选出 8 种与开花调控相关和 3 种花色调控相关的 microRNAs。表达分析显示,miRNAs 在两样品间呈现出较大的整体性差异,并且在功能与代谢通路上也不尽相同;开花相关microRNAs 中表达差异较大的为 miR156、miR172 和 miR169,其中 miR156 表达量在花序中高于幼叶,而 miR172 表达量在花序中低于幼叶,推测其功能为抑制后续的开花过程,避免过度开花。本研究分析了木薯花序与幼叶中 sRNA的整体特性,通过表达差异分析初步明确了 microRNAs 对木薯开花的调控,为进一步深入探索奠定了基础。     

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.     

落叶松体胚发育中12个针叶树特异microRNAs表达分析

以落叶松体细胞胚胎发育8个阶段的材料构建小RNA文库,进行了Solexa测序、miRNA鉴定、靶基因预测、qRT-PCR定量分析等研究。结果表明:发现的87个miRNAs中,29个为针叶树特异的,来自12个家族,分别为miR946、miR947、miR950、miR951、miR1311、miR1312、miR1313、miR1314、miR1316、miR3701、miR3702和miR3704,其中25个长度为22nt,且在小RNA文库中的最低拷贝数为18(miR1311),最高拷贝数为33 009(miR950)。发现12个miRNA家族的34个靶基因可能参与生长发育、抗病、AGO反馈调节等多方面遗传调控;qRT-PCR分析表明:72%miRNAs表达次高峰在后期单胚或早期子叶胚,在中期子叶胚最低,最高峰在后期子叶胚,分别与子叶形成、胚胎后熟、胚胎休眠的生理活动相吻合,而miRNA前体的表达模式不同于miRNA,前体变化幅度小于成熟体。     

Differential microRNA expression profiling and target gene prediction in the muscle tissues of clenbuterol-fed Chinese miniature swine

MicroRNAs (miRNAs) regulate gene expression by inhibiting the translation of target RNA. Clenbuterol (CLB) is a β-adrenergic agonist that has the ability to modify muscle characteristics by promoting protein synthesis and inhibiting the growth of fat. To determine the expression profile of miRNAs in the muscle of CLB-treated Chinese miniature swine and to predict their target genes, 13 microRNAs were detected and analyzed. We found that miR-133, miR-206, and miR-1 exhibited the highest expression, although no difference in expression levels was observed between the control and the treatment groups, whereas miR-29a and miR-29b were differentially expressed in the CLB-treated muscles. Furthermore, we analyzed the relationship between miR-29a/b expression, muscle growth, and candidate target genes, and determined that the expression of the four target genes (HnRNPF, SWI/SNF, TPM1, and LPL) was regulated by miR-29a and miR-29b and were functionally related to muscle development.     

水稻microRNA单核苷酸多态性

microRNA(miRNA)是一类内源性非编码小RNA,长度约22个核苷酸.miRNA通过与靶基因rnRNA特定位点结合,调控植物生长、发育和胁迫耐受性.pre-miRNA的单核苷酸突变会影响miRNA的成熟过程和调控功能,因此,研究miRNA基因中的单核苷酸多态性(SNP)分布对研究miRNA功能分化和基因进化有重...     

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。