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

doi:10.3864/j.issn.0578-1752.2024.05.014

摘要/Abstract

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。

关键词: 猪GCs凋亡, miR-34a, saRNA, NORHA

Abstract:

【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 increased the enrichment of AGO2, a core member of RNA induced transcriptional activation (RITA) complex, and H3K4me3, a transcription activated histone modifier, while or decreased the enrichment of H3K9me3, a transcription repressed histone modifier at MRE motif of miR-34a in the NORHA promoter. Co-transfection assay showed that knockdown of NORHA significantly reversed the upregulation of FOXO1 protein level and early apoptosis rate caused by miR-34a overexpression, as well as the downregulation of the BCL-2/BAX ratio. 【Conclusion】 Nuclear miR-34a was an endogenous small activating RNA (saRNA) in porcine GCs, and might be involved in the initiation of porcine follicular atresia by inducing early apoptosis in porcine GCs through target activating the transcription of the pro-apoptotic lncRNA NORHA.

Key words: porcine GC apoptosis, miR-34a, saRNA, NORHA

王思琪, 周春雪, 李玉琦, 杜星, 潘增祥, 李齐发. miR-34a通过激活lncRNA NORHA转录诱导猪卵巢颗粒细胞早期凋亡[J]. 中国农业科学, 2024, 57(5): 1000-1009.

WANG SiQi, ZHOU ChunXue, LI YuQi, DU Xing, PAN ZengXiang, LI QiFa. miR-34a Induces Early Apoptosis of Porcine Ovarian Granulosa Cells by Activating lncRNA NORHA Transcription[J]. Scientia Agricultura Sinica, 2024, 57(5): 1000-1009.

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https://www.chinaagrisci.com/CN/Y2024/V57/I5/1000

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寡核苷酸 Oligonucleotides	寡核苷酸序列 Oligonucleotide sequence (5′ to 3′)
mimics NC	UUGUACUACACAAAAGUACUG
miR-34a mimics	UGGCAGUGUCUUAGCUGGUUGU
NC-siRNA	UUCUCCGAACGUGUCACGUTT ACGUGACACGUUCGGAGAATT
NORHA-siRNA	GGAAAUAGCGAGUUGGGUUTT AACCCAACUCGCUAUUUCCTT

名称 Name	序列 Primer sequence (5′ to 3′)	退火温度 Annealing temp (℃)	产物长度 Product size (bp)
miR-34a	F: CGGGCTGGCAGTGTCTTAGC	60	56
miR-34a	R: CAGCCACAAAAGAGCACAAT	60	56
U6	F: GCTTCGGCAGCACATATACT	60	86
U6	R: TTCACGAATTTGCGTGTCAT	60	86
NORHA	F: GCTGAACCTGGGGAGTGTCTA	60	176
NORHA	R: TCTCTGTCCTTGTGGTGTCTGC	60	176
GAPDH	F: GGACTCATGACCACGGTCCAT	60	163
GAPDH	R: TCAGATCCACAACCGACACGT	60	163
BAX	F: CCGAAATGTTTGCTGACG	60	122
BAX	R: AGCCGATCTCGAAGGAAGT
BCL-2	F: TTCTTTGAGTTCGGTGGGG	60	195
BCL-2	R: CCAGGAGAAATCAAATAGAGGC	60	195
MRE-ChIP	F: CTTCCTGCTCCTGCTGTC	54	130
MRE-ChIP	R: GCCGTCGTCTGAAAGGAC	54	130
miR-34a茎环 miR-34a stem-loop	CCTGTTGTCTCCAGCCACAAAAGAGCAC AATATTTCAGGAGACAACAGGACAACCA