miR-542-3p生物信息学分析及对未成熟猪睾丸支持细胞的影响  

Bioinformatics analysis of miR-542-3p and its effects on immature pig testis Sertolicells

作　　者：尹艳飞 蒋芊芊 冉茂良[2] 翁波 YIN Yanfei;JIANG Qianqian;RAN Maoliang;WENG Bo(Xiangxi National Vocational and Technical College,Hunan Xiangxi 416007,China;Hunan Provincial Key Laboratory of Genetic Improvement of Animals,College of Animal Science and Technology,Hunan Agricultural University,Changsha 410128,China)

机构地区：[1]湘西民族职业技术学院,湖南湘西416007 [2]湖南农业大学动物科学技术学院畜禽遗传改良湖南省重点实验室,长沙410128

出　　处：《黑龙江畜牧兽医》2024年第11期47-56,119,共11页Heilongjiang Animal Science And veterinary Medicine

基　　金：湖南省自然科学基金面上项目(2023JJ60247)。

摘　　要：为了探讨miR-542-3p的生物信息学信息及其对未成熟猪睾丸支持细胞的影响,试验通过AliBaba 2.1和AnimalTFDB 3.0软件预测miR-542-3p转录因子结合位点(TFBS);并分析miR-542-3p在不同物种间的保守性;采用CCK-8试剂、ATP试剂盒、流式周期检测试剂盒和实时荧光定量PCR方法探究过表达miR-542-3p对未成熟猪睾丸支持细胞的影响;利用TargetScan、miRDB和miWalk 3种在线软件预测miR-542-3p靶基因后取交集,并对猪源靶基因进行GO功能注释和KEGG信号通路富集分析;最后利用Cytoscape 3.2.0软件构建TF-miR-542-3p-靶基因相互作用网络。结果表明:经预测miR-542-3p受GATA1、TBP、Sp1、Ap1、SRF等多个转录因子调控,且在多物种间具有高度的保守性;miR-542-3p可显著或极显著抑制未成熟猪睾丸支持细胞增殖活性(P<0.05或P<0.01),极显著降低细胞内ATP含量(P<0.01),显著或极显著抑制细胞增殖基因和周期基因的相对表达量(P<0.05或P<0.01),进而抑制细胞周期和细胞增殖;转染miR-542-3p mimic后G1期猪睾丸支持细胞比例显著升高(P<0.05),S期细胞比例极显著降低(P<0.01);共发现7个猪源miR-542-3p潜在靶基因,miR-542-3p调控的UBE2E1基因相对表达量极显著下降(P<0.01),KCMF1、ILK基因相对表达量显著下降(P<0.05),HIPK3基因相对表达量极显著升高(P<0.01),AP3D1、PLEKHM3基因相对表达量显著升高(P<0.05),各靶基因主要在激酶活性和结合上发挥作用;miR-542-3p靶基因主要参与MAPK、cAMP、TGF-β和cGMP-PKG等信号通路;构建的TF-miR-542-3p-靶基因相互作用网络包含5个转录因子和50个mRNA。说明miR-542-3p受多种转录因子调控,在多物种间高度保守,能够抑制未成熟猪睾丸支持细胞增殖,并通过调控下游靶基因的表达参与MAPK、cAMP和TGF-β等信号通路,从而调控未成熟猪睾丸支持细胞生长。In order to investigate the bioinformatics information of miR-542-3p and its effects on immature pig testis Sertoli cells,in this experiment,AliBaba 2.1 and AnimalTFDB 3.0 software were used to predict the transcription factor binding site(TFBS)of miR-542-3p,and the conservatism of miR-542-3p among different species was analyzed.The effects of overexpression of miR-542-3p on immature pig testis Sertoli cells were investigated by CCK-8 kit,ATP kit,flow cycle detection kit and real-time PCR.Three online software TargetScan,miRDB and miWalk were used to predict the miR-542-3p target gene and then intersected,and the pig-derived target genes was annotated with CO function and the KEGG signaling pathway enrichment analysis was performed.Finally,Cytoscape 3.2.O software was used to construct a TF-miR-542-3p-target genes interaction network.The results showed that miR-542-3p was predicted to be regulated by multiple transcription factors such as GATA1,TBP,Spl,Apl,and SRF,and had high conservatism among multiple species.miR-542-3p significantly or very significantly inhibited the proliferative activity of immature pig testis Sertoli cells(P<0.05 or P<0.01);significantly reduced intracellular ATP content(P<0.O1);significantly or very significantly inhibited the relative expression of cell proliferation and cyclerelated genes(P<0.05 or P<0.01),and thereby inhibited cell cycle and proliferation.After transfection with miR-542-3p mimic,the proportion of Sertoli cells in Gl phase of pig testis was significantly increased(P<0.05),and the proportion of cells in S phase was extremely significantly decreased(P<0.01).A total of 7 potential target genes of pig-derived miR-542-3p were discovered;the relative expression of miR-542-3p-regulated UBE2E1 gene decreased significantly(P<0.01);the relative expression of KCMF1 and ILK genes decreased significantly(P<0.05);the relative expression of HIPK3 gene was significantly increased(P<0.01);the relative expression of AP3D1 and PLEKHM3 genes was significantly increased(P<0.05).Each target

关 键 词：miR-542-3p 生物信息学 未成熟公猪 睾丸支持细胞 靶基因 

分 类 号：S828.2[农业科学—畜牧学]