Improvement,identification, and target prediction for miRNAs in the porcine genome by using massive,public high-throughput sequencing data

Despite the broad variety of available microRNA (miRNA) research tools and methods, their application to the identification, annotation, and target prediction of miRNAs in nonmodel organisms is still limited. In this study, we collected nearly all public sRNA-seq data to improve the annotation for known miRNAs and identify novel miRNAs that have not been annotated in pigs (Sus scrofa). We newly annotated 210 mature sequences in known miRNAs and found that 43 of the known miRNA precursors were problematic due to redundant/missing annotations or incorrect sequences. We also predicted 811 novel miRNAs with high confidence, which was twice the current number of known miRNAs for pigs in miRBase. In addition, we proposed a correlation-based strategy to predict target genes for miRNAs by using a large amount of sRNA-seq and RNA-seq data. We found that the correlation-based strategy provided additional evidence of expression compared with traditional target prediction methods. The correlation-based strategy also identified the regulatory pairs that were controlled by nonbinding sites with a particular pattern, which provided abundant complementarity for studying the mechanism of miRNAs that regulate gene expression. In summary, our study improved the annotation of known miRNAs, identified a large number of novel miRNAs, and predicted target genes for all pig miRNAs by using massive public data. This large data-based strategy is also applicable for other nonmodel organisms with incomplete annotation information.     

花鲈cox6a基因与相关miRNA的鉴定及其在盐度适应中的表达调控分析

细胞色素c氧化酶（COX）是线粒体电子传递链的末端酶，COX6A是细胞色素c氧化酶的核编码亚基之一。为丰富花鲈（基因及相关miRNAs在花鲈渗透调节机制中的潜在作用，本研究开展了花鲈cox6a基因的miRNAs，并对其与）。系统进化树分析进一步确认了两个花鲈在垂体、脑、肝脏和肾脏中的表达量较高，基因的miRNA：miR-30e-5p、miR-223、miR-200a-3p和miR-155-5p，但没有发现可能靶定基因以及miR-30e-5p、miR-223、miR-200a-3p都呈现显著差异表达，而miR-155-5p仅在适应高盐环境时出现了显著的差异表达。这表明，基因和4个miRNAs在花鲈的渗透调节过程中发挥潜在作用。其中miR-30e-5p、miR-223和miR-200a-3p的表达趋势与cox6a2基因的表达调控。本研究为探讨花鲈适应不同盐度环境的渗透调节机制提供了基础数据。     

Affecting Lipid Metabolism Salivary MicroRNAs Expressions in Arabian Racehorses Before and After the Race

The active roles of microribonucleic acids (miRNAs) in gene regulation have made miRNAs a key point for the scientific world in the study of physiological processes. Although saliva includes the largest number of miRNAs, there is no miRNA study in saliva on horses has been found. Our study is the first study on miRNAs isolation from saliva in horses. In the present study, saliva was studied in Arabian racehorses to better understand the molecular mechanisms of expression levels that are effective in lipid metabolism of miRNAs and their target genes during the race. Identification of lipid metabolism of miRNAs and their target genes is an opportunity to provide information about biomarkers in Arabian racehorses on energy supply for race performance. Arabian racehorses have low glycogen content and high triglyceride storage capability, thanks to the high amount of oxidative type I fiber in their muscle tissue. Therefore, Arabian racehorses can provide higher levels of energy using more fat. The aim of this study is to determine the prerace and postrace expression levels of eight miRNAs in saliva that are known to affect lipid metabolism in Arabian racehorses. The expression level of eca-miR-33a was found to be statistically significant (P < .05). Target genes of eca-miR-33a have been copredicted as ABCA1, CROT, ABHD2, and SATB2, with three validated databases and other analysis tools. In conclusion, these findings revealed that both eca-miR-33a and its target genes could be potential core genes that play important roles in lipid metabolism in Arabian racehorses to provide energy during the race.     

miRNA在哺乳动物配子发生中的作用研究进展

哺乳动物配子发生的基因表达调控包括编码基因的阶段特异性表达调控及非编码基因的转录和转录后水平调控。微小RNA(microRNA, miRNA)作为一类小的非编码RNA, 通过识别靶基因非翻译区的结合位点, 导致mRNA降解或者蛋白质翻译抑制, 从而在转录后水平发挥调控作用。近年来, miRNA在哺乳动物生殖活动中的作用逐渐被揭示, 越来越多的研究表明, miRNA在哺乳动物精子发生、精子成熟、卵母细胞成熟、卵泡发育及早期胚胎发育等过程中都发挥着重要的调节作用, 其可通过调节支持细胞的增殖和凋亡或精原细胞、精母细胞及精细胞的细胞周期进程, 在精子发生的不同阶段发挥间接或直接调控作用, 也可通过调节卵母细胞、卵丘细胞以及颗粒细胞的增殖、凋亡、激素合成和细胞间作用, 对卵母细胞的发育和成熟过程进行调控。作者主要介绍了在哺乳动物配子发生过程中, miRNA的细胞和阶段特异性表达及其对靶基因的调节作用, 以期为深入研究哺乳动物配子发生的调控机制提供参考。     

利用生物信息学方法预测家蚕核型多角体病毒基因组编码的miRNA

microRNAs(miRNAs)是长度为18～25 nt的非蛋白质编码小RNA分子,可以通过抑制有关基因mRNA的翻译从而调控机体的生长发育、疾病发生等过程。在很多病毒中发现有miRNA的存在,这些miRNA可能在病毒基因的表达以及与宿主的相互作用中发挥作用。利用vMir软件预测家蚕核型多角体病毒(BmNPV)基因组可能编码的miRNA前体序列,对BmNPV基因组扫描分析后,得到7条能形成发夹结构的可能是miRNA的候选前体序列。以BmNPV添食感染5龄起蚕,72 h后取幼虫血淋巴提取总RNA,对这7条序列进行反转录PCR验证,其中扩增出的5条序列在BmNPV基因组中有相同的序列,推测这5条序列为BmNPV基因组编码的miRNA前体序列。进一步分析5条miRNA前体序列的发夹结构,并用反转录PCR的方法验证其成熟体序列,结果在5条miRNA前体序列共扩增出6条在BmNPV基因组中有相同序列的成熟体序列,它们分别位于不同的ORF之间,是BmNPV基因组编码的miRNA。利用在线靶基因预测程序RNAhybrid和RNA22预测到6条miRNA在BmNPV中的13个可能的靶基因,其中有6个靶基因是病毒的结构蛋白基因,2个为晚期表达因子基因,5个为未知功能基因。推测这些BmNPV基因组编码的miRNA可能与病毒感染宿主有关。     

鸡Lpin1基因3′-UTR遗传变异及其对miRNA结合位点的潜在效应

【目的】研究鸡Lpin1基因3′-UTR遗传变异/单倍型及其在品种间的分布，并预测这些变异对miRNA结合位点的潜在效应。【方法】根据鸡Lpin1基因组序列（GenBank登录号：NC_006090）设计一对特异性引物，采用PCR直接测序结合克隆测序的方法，进行鸡Lpin1基因3′-UTR及其邻近外显子在品种间的遗传变异/单倍型分析。【结果】①从6个品种中发现了8个变异位点，包含两个插入/缺失变异。这些变异均位于鸡Lpin1基因的3′-UTR，3′-UTR的变异频率为17SNPs/kb；②在鸡Lpin1基因3′-UTR区域，从固始鸡、卢氏绿壳蛋鸡中分别检测到7个变异位点，未检测到河南斗鸡品种内的变异；③用次要等位基因频率≥5%的6个变异位点（g.11A＞T，g.77C＞G，g.108_109delinsG，g.110_111delinsG，g.270A＞G和g.348G＞T）进行单倍型的构建和分析，从6个品种鸡中共检测到6种单倍型，其中P1和P4单倍型是群体的优势单倍型，频率均大于30%，河南斗鸡仅包含一种单倍型；④g.77C＞G变异可引起多个miRNA结合位点的增加/丢失。【结论】鸡Lpin1基因3′-UTR具有丰富的变异，3′-UTR变异位点/单倍型在品种间的分布存在明显的差异。     

Characterization of MicroRNA* Species in Peking Duck Skin

A substantial fraction of miRNA* species are conserved in animals and can repress activities of target genes. This study aims to investigate the miRNA* species in duck skin by using Solexa sequencing. We obtained a total of 96 miRNA* species in two skin small RNA libraries and identified 56 miRNA/miRNA* (miR/miR*) pairs. Nucleotide bias of miRNA* indicated that the priority was C>A>U>G for the first nucleotide and U>C>A>G for the last nucleotide. Comparison analyses showed that 3′-U accounted for a higher proportion in the 56 miR/miR* pairs. Among the top 20 expressed miRNA* species, 17 were shared by two libraries and most of the miRNA* species were highly conservative, especially in the “seed region”. miR-199a* were expressed highly in our samples, which was also previously shown abundant in mouse hair follicle. Furthermore, four miRNA* species were predicted to target their genes in signal pathways of feather follicle development and feather morphogenesis despite very low levels.     

microRNAs在成年羊驼皮肤组织中的表达

本研究旨在分析成年羊驼皮肤中miRNAs的表达,探讨miRNAs在皮肤和毛囊中的调控作用.利用多物种miRNA芯片与成年羊驼皮肤组织miRNAs杂交,来分析成年羊驼皮肤组织中miRNAs的表达.结果显示有20个miRNAs信号在4个样本中表达量高,microRNAs在羊驼皮肤表达,提示它们可能参与了皮肤和毛囊的更新、生长和发育.     

绵羊miRNA-411a与其靶基因FLT3的相互作用

miRNAs是一类长约22 nt的非编码单链小RNA分子,主要与靶基因的3′UTR结合参与生物体基因的表达和调控过程。已经证实许多miRNA通过对靶标的调控,在支原体肺炎感染过程中起重要作用。为验证miRNA-411a与预测的靶基因 FLT3间存在相互作用,采用生物信息学方法分析预测其二级结构,利用荧光定量PCR技术分析miRNA-411a的正常表达和过量表达后靶基因 FLT3的相对表达量,双荧光素酶报告基因试验验证两者的相互作用。结果表明,miRNA-411a与其靶基因 FLT3能形成典型的二级茎环结构;在过量表达miRNA-411a后,其靶基因 FLT3的表达量显著降低。综上所述,miRNA-411a是通过与 FLT3的3′UTR结合而发挥作用,确定 FLT3是miRNA-411a的靶基因。