目的 筛选、鉴定绵羊卵巢差异MicroRNA(miRNA)并研究其调控机制,为绵羊非繁殖季节发情的研究奠定基础。方法 使用Solexa测序技术进行筛选和分析,对发情和乏情绵羊卵巢,差异miRNA的数量和特征进行鉴定,通过实时荧光定量PCR进行检测。还进行了对miR-200c靶基因检测、GO注释和KEGG信号通路富集等的研究工作。并通过TargetScan技术、RNAhybrid技术等,成功找到了MAPK8-3′端非翻译区(3′UTR)与miR-200c的潜在互补结合位点,PCR扩增MAPK8(丝裂原活化蛋白激酶8)基因的3′UTR序列,并成功实现了在psiCHECK2的克隆,成功建立了MAPK8野生型/突变型重组的双荧光素酶报告质粒。接着把野生型psiheck2-MAPK8-3′UTR/突变型psiheck2-MAPK8-mut3′UTR的质粒,与miR-200c mimics/mimics-NC,转染至HEK 293T细胞中,从而通过双荧光素酶的系统,测定双荧光素酶的生物活性。结果 建立了OAN(乏情)和OEN(发情)非繁殖季节绵羊卵巢文库。鉴定出113个差异miRNA,9个为已知miRNA,104个为未知miRNA。在已知miRNA中有6个miRNA显著(P<0.05)上调、3个下调,其中上调倍数最高的为miR-200c。在104个未知的miRNA中,有52个miRNA显著(P<0.05)上调,52个显著(P<0.05)下调。获得候选新miRNA的长度分布主要集中在21~23 nt之间。荧光定量证实,miRNA表达的趋势与测序结果一致。用软件预测到miR-200c的27个靶基因。GO注释发现miR-200c在卵巢组织中介导细胞增殖、迁移、凋亡等过程。KEGG分析表明,miR-200c的靶基因涉及发情相关途径(MAPK信号途径、胰岛素信号通路和GnRH信号途径)以及与卵泡/黄体发育相关的途径,MAPK通路是富集基因最多的通路,共有25个基因被富集。干扰miR-200cmimics,会引起MAPK8基因的Wt型质粒的荧光表达明显减少(P<0.05),而Mut型则没有明显改变。结论 筛选出非繁殖季节发情差异显著(P<0.05)的miRNA有6个。试验研究初步证实了miR-200c与MAPK8的靶向关系,开展了miRNA家族调节绵羊非繁殖季节发情的试验,为深入研究其调控绵羊繁殖及卵泡发育机制提供了试验基础。
Abstract
Objective Screening and identification of microRNAs (miRNAs) in sheep ovaries and studying their regulatory mechanisms laid the foundation for the study of sheep estrus in the non-breeding season. Methods Screening and analysis using Solexa sequencing technology to identify the number and characterisation of differential miRNAs, by real-time fluorescence quantitative PCR, in oestrus and estrus sheep ovaries. Work was also carried out on miR-200c target gene detection, GO annotation and enrichment of the KEGG signalling pathway. The potential complementary binding site of MAPK8-3′-end untranslated region (3′UTR) and miR-200c was successfully found by TargetScan technology and RNAhybrid technology, and the 3′UTR sequence of MAPK8 (mitogen-activated protein kinase 8) gene was PCR amplified and cloning in psiCHECK2 was successfully achieved, and a dual luciferase reporter plasmid for MAPK8 wild-type/mutant recombination was successfully established. The plasmid of wild-type psiheck2-MAPK8-3′UTR/mutant psiheck2-MAPK8-mut3′UTR, with miR-200c mimics/mimics-NC, was then transfected into HEK 293T cells, thereby measuring the biological activity of the dual luciferase by a dual luciferase system. Results A library of OAN (absence) and OEN (estrus) non-breeding season sheep ovaries was established. 113 differential miRNAs were identified, 9 were known miRNAs and 104 were unknown miRNAs. Six of the known miRNAs were significantly (P<0.05) up-regulated and three were down-regulated, with the highest up-regulation fold being miR-200c. Of the 104 unknown miRNAs, 52 were significantly (P<0.05) up-regulated and 52 were significantly (P<0.05) down-regulated. The length distribution of the obtained candidate new miRNAs was mainly concentrated in the range of 21~23 nt. Fluorescence quantification confirmed that the trend of miRNA expression was consistent with the sequencing results. The software was used to predict the 27 target genes of miR-200c. GO annotation revealed that miR-200c mediates cell proliferation, migration, and apoptosis in ovarian tissue. KEGG analysis showed that the target genes of miR-200c were involved in estrus-related pathways (MAPK signalling pathway, insulin signalling pathway and GnRH signalling pathway) and pathways associated with follicular/luteal development, with the MAPK pathway being the most enriched pathway with a total of 25 genes enriched. Interfering with miR-200cmimics caused a significant decrease in fluorescent expression of the Wt-type plasmid of the MAPK8 gene (P<0.05), while the Mut-type was not significantly altered. Conclusion Six miRNAs were screened for significant (P<0.05) differences in non-breeding season estrus. The experimental study initially confirmed the targeting relationship between miR-200c and MAPK8, and carried out experiments on the miRNA family regulating estrus in sheep during the non-breeding season, providing an experimental basis for an in-depth study on its mechanism of regulating reproduction and follicle development in sheep.
关键词
绵羊 /
卵巢 /
非繁殖季节发情 /
miR-200c /
MAPK8
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Key words
sheep /
ovary /
non-breeding season estrus /
miR-200c /
MAPK8
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脚注
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基金
国家自然科学基金项目(32160770)
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