miR-34a通过激活lncRNA NORHA转录诱导猪卵巢颗粒细胞早期凋亡  

miR-34a Induces Early Apoptosis of Porcine Ovarian Granulosa Cells by Activating lncRNA NORHA Transcription

作　　者：王思琪 周春雪 李玉琦 杜星[1] 潘增祥[1] 李齐发[1] WANG SiQi;ZHOU ChunXue;LI YuQi;DU Xing;PAN ZengXiang;LI QiFa(College of Animal Science and Technology,Nanjing Agricultural University,Nanjing 210095)

机构地区：[1]南京农业大学动物科技学院,南京210095

出　　处：《中国农业科学》2024年第5期1000-1009,共10页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2022YFD1600903);国家自然科学基金(32072693)。

摘　　要：【目的】探索miR-34a诱导GCs凋亡的RNA激活机制,为揭示猪卵泡闭锁的分子机理、筛选调控母猪繁殖的小核酸调节物提供依据。【方法】利用生物信息学方法分析猪miR-34a特征及其与潜在靶基因lncRNA NORHA启动子的结合情况。利用核质分离技术分析猪GCs中miR-34a的亚细胞定位。qPCR检测miR-34a对猪GCs中NORHA、凋亡标志基因BCL-2和BAX表达的影响。利用荧光素酶活性分析验证miR-34a对NORHA启动子转录活性的靶向调控关系。利用染色质免疫沉淀(ChIP)检测miR-34a对猪GCs中NORHA启动子miRNA反应元件(MRE)处AGO2和组蛋白修饰因子富集情况。利用western blot和流式细胞术分析miR-34a通过NORHA对下游因子FOXO1的表达和细胞凋亡的调控作用。【结果】猪miR-34a为基因间miRNA,其序列在哺乳动物中具有高度保守性。亚细胞定位分析显示,miR-34a在猪GCs中细胞核与细胞质均有分布。生物信息学分析显示miR-34a的MRE位于NORHA启动子-194 nt至-173 nt处,且两者结合自由能为-23.9 kcal·mol^(-1)。qPCR结果表明miR-34a显著上调猪GCs中NORHA的表达。荧光素酶活性分析显示,miR-34a极显著上调NORHA启动子报告载体活性,而对MRE突变型启动子活性没有显著影响。ChIP试验显示,miR-34a可增加NORHA启动子MRE处RNA诱导转录激活(RITA)复合物核心成员AGO2和转录激活型组蛋白H3K4me3的富集,而减少转录抑制型组蛋白H3K9me3的富集。共转试验显示,敲减NORHA可显著或极显著逆转由miR-34a过表达引起的FOXO1蛋白水平和早期凋亡率的上调,以及BCL-2/BAX比值的下调。【结论】细胞核miR-34a是猪GCs中的内源性小激活RNA(saRNA),通过靶向激活促凋亡lncRNA NORHA的转录,诱导猪GCs早期凋亡(细胞膜完整),可能参与猪卵泡闭锁的启动。【Objective】miR-34a has been shown to be a pro-apoptotic factor in porcine ovarian granulosa cells(GCs)in our previous study.The aim of this study was to explore the RNA activation mechanism by which miR-34a induced apoptosis in the nucleus,so as to provide a basis for uncovering the molecular mechanism of porcine follicular atresia,and to screen small nucleic acid regulator that regulated procine reproduction.【Method】Bioinformatics methods were used to analyze the characteristics of the porcine miR-34a,the binding sites of miR-34a to the promoter region of target lncRNA NORHA,and the binding capacity.The subcellular localization of miR-34a was analyzed in porcine GCs by using nucleocytoplasmic fractionation.The effects of miR-34a on the expression of NORHA,apoptosis marker genes BCL-2 and BAX in porcine GCs were determined by qPCR.Luciferase assay was used to verify the regulatory relationship of miR-34a on the transcriptional activity of the NORHA promoter.Chromatin immunoprecipitation(ChIP)was utilized to detect the enrichment of AGO2 and histone modifiers at the miRNA response element(MRE)in the NORHA promoter in porcine GCs by miR-34a.The regulatory effect of miR-34a on downstream factor FOXO1 expression and GC apoptosis via NORHA were analyzed by western blot and flow cytometry.【Result】The porcine miR-34a was an intergenic miRNA,and its sequence was highly conserved among mammals.Subcellular localization analysis showed that miR-34a was distributed in both nucleus and cytoplasm in porcine GCs.Bioinformatic analysis revealed that the MRE of miR-34a was located at-194 nt--173 nt in the NORHA promoter,and the binding free energy of the two compounds was-23.9 kcal·mol^(-1).qPCR showed that miR-34a significantly upregulated NORHA expression in porcine GCs.Luciferase assay revealed that miR-34a significantly upregulated the activity of the reporter vector with NORHA promoter,whereas it had no significant effect on the activity of promoter with the mutated MRE of miR-34a.ChIP assay showed that miR-34a

关 键 词：猪GCs凋亡 MIR-34A saRNA NORHA 

分 类 号：S828[农业科学—畜牧学]