单双羔绵羊卵巢组织差异表达microRNAs筛选

doi:10.3864/j.issn.0578-1752.2015.10.016

摘要/Abstract

摘要： 【目的】全面了解绵羊卵巢组织miRNAs表达情况，分析产单羔和产双羔绵羊卵巢miRNAs表达差异，从而为探讨miRNAs在繁殖力调控中的作用提供依据。【方法】应用多物种miRNA芯片对绵羊卵巢组织miRNA进行表达分析。首先选择经产单羔羊和双羔羊，发情后采集双侧卵巢提取总RNA，分离小片段 RNA与miRNA芯片杂交，然后进行数据分析获得绵羊卵巢组织miRNAs表达谱；利用生物信息学软件，以q-value ≤ 5%，且Fold Change ≥2 或≤0.5的标准筛选单羔羊和双羔羊卵巢差异表达miRNAs，并利用q-PCR技术验证芯片结果；分别采用microT和miRDB两种方法预测差异表达miRNAs的靶基因，然后合并两种方法结果数据取二者的交集，用在线软件对靶基因进行GO功能注释。【结果】在检测的来自所有物种的miRNAs中，有5 448个miRNAs在单羔和双羔羊卵巢组织中共同表达，22个在单羔羊卵巢中特异性表达，15个在双羔羊卵巢中特异性表达；对绵羊中已报道的103个miRNAs，比较其在两组母羊卵巢组织中的表达变化，共获得11个差异表达miRNAs，其中4个上调表达，7个下调表达；随机选择一个上调和一个下调表达的miRNAs进行q-PCR验证，定量结果与芯片结果一致，说明芯片结果准确、可信；预测获得7个miRNAs的靶基因，分别是：oar-miR-370-5p、oar-miR-376b-5p、oar-miR-381-5p、oar-miR-412-5p、oar-miR-541-3p、oar-miR-544-5p和oar-miR-1185-5p，每个miRNA获得的靶基因个数分别为：115、71、1、5、8、135和23个。GO注释结果显示，miR-376b-5p和miR-1185-5p的靶基因主要参与形成细胞内组分和细胞器，在分子功能分类中，绝大部分基因为连接分子类和催化活性分子类，在生物学过程分类中，主要参与细胞增殖、分化、凋亡过程和代谢过程；同时miR-376b-5p的靶基因IGF-1、DAZL、MTOR、MET、NEDD4和miR-376b-5p的靶基因AHR参与生殖过程的调控。【结论】成功构建了绵羊卵巢miRNAs表达谱，获得产单羔母羊和产双羔母羊卵巢组织差异表达miRNAs，这些miRNAs可能与绵羊卵泡发育和产羔数多少有关。

关键词: 绵羊, 卵巢, miRNAs, 芯片

Abstract: 【Objective】This study was conducted to get a comprehensive understanding of miRNAs expression in sheep ovary, and to analyze their expression differences between sheep producing single lamb and twins, thus providing a basis for exploring the role of miRNAs in the regulation of fertility. 【Method】 Multi-species miRNA microarray was used to profile ovarian miRNAs expression in sheep. Firstly, single-lamb-producing and twin-producing ewes were selected. Ovaries were collected after estrus for total RNA extraction, and small RNA fragments were isolated and hybridized with miRNA microarray. Then data analysis was performed to obtain sheep ovarian miRNAs profile. Differentially expressed microRNAs were selected by bioinformatics software with the standard of q-value ≤ 5% and Fold Change ≥2 or ≤0.5, and q-PCR technique was employed to testify the microarray results. Two different methods, microT and miRDB, were used, respectively, and then data were merged and to predict target genes. Finally, online software was used to perform GO function annotation for target genes. 【Result】 Among all the detected miRNAs, a total of 5448 miRNAs were detected in sheep ovaries, of which 22 were specifically expressed in single-lamb-producing group, while 15 were in twin-producing group; For the miRNAs reported in sheep, expression differences were compared between two groups, and obtained 11 differentially expressed miRNAs, with 7 down-regulated and 4 up-regulated. Tow miRNAs were randomly selected to perform q-PCR validation, and the results were consistent with the microarray results, illustrating that the microarray results were accurate and credible. Target genes were obtained for 7 miRNAs: oar-miR-370-5p, oar-miR-376b-5p, oar-miR-381-5p, oar-miR-412-5p, oar-miR-541-3p, oar-miR-544-5p and oar-miR-1185-5p，and the number of target genes for each miRNAs was: 115, 71, 1, 5, 8, 135 and 23. The GO annotation results showed that target genes of miR-376b-5p and miR-1185-5p are mainly involved in forming intracellular components and organelles; in the molecular function category, the vast majorities of genes are the connection and catalytic activity molecules; in biological process, mainly involved in cell proliferation, differentiation, apoptosis and metabolism. Meanwhile, the target genes of miR-376b-5p ( IGF-1, DAZL, MTOR, MET and NEDD4 ) and miR-376b-5p ( AHR ) take part in reproduction regulation.【Conclusion】Sheep ovarian miRNAs expression profiling was successfully constructed, and differentially expressed miRNAs between single-lamb-producing and twin-producing sheep were obtained. These miRNAs may be involved in sheep follicular development and litter size regulation.

Key words: sheep, ovary, miRNAs, microarray

祁云霞，刘晓芳，张萍，何小龙，邢玉梅，达赖，特日格勒，刘永斌，荣威恒. 单双羔绵羊卵巢组织差异表达microRNAs筛选[J]. 中国农业科学, 2015, 48(10): 2039-2048.

QI Yun-xia, LIU Xiao-fang, ZHANG Ping, HE Xiao-long, XING Yu-mei, Dalai, Terigele, LIU Yong-bin, RONG Wei-heng. Differentially Expressed microRNAs Screening Between Ovaries of Sheep Producing Single Lamb and Twins[J]. Scientia Agricultura Sinica, 2015, 48(10): 2039-2048.

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