miR-495-3p对山羊卵巢颗粒细胞功能的影响

doi:10.11843/j.issn.0366-6964.2022.02.011

摘要/Abstract

摘要： 旨在探究miR-495-3p对山羊卵巢颗粒细胞功能的影响及作用机制。本研究选取健康的3~4月龄大足黑山羊母羊，收集卵巢颗粒细胞，利用miR-495-3p模拟物（mimics）和抑制物（inhibitor）构建过表达和抑制模型，通过流式细胞术检测细胞凋亡和周期，ELISA分析颗粒细胞的雌二醇（E2）和孕酮（P4）分泌，采用qRT-RCR、Western blot检测细胞凋亡、增殖、周期及类固醇激素合成相关基因的mRNA和蛋白的表达水平。结果显示，miR-495-3p显著促进卵巢颗粒细胞凋亡（P<0.01），并且促进BAX表达（P<0.05），抑制BCL2表达（P<0.01）；过表达miR-495-3p呈时间依赖性抑制细胞增殖活力，显著降低PCNA的表达（P<0.05）；过表达miR-495-3p显著增加G2/M期细胞百分比（P<0.05），并显著降低细胞周期相关基因CDK4（P<0.001）和CCND1（P<0.05）的mRNA水平，抑制miR-495-3p极显著降低CCNE1的mRNA水平（P<0.01）；miR-495-3p促进颗粒细胞E2和P4分泌；过表达miR-495-3p显著提高3β-HSD（P<0.001）、CYP11A1（P<0.01）、CYP19A1（P<0.05）的mRNA和蛋白表达水平，干扰miR-495-3p极显著抑制3β-HSD（P<0.01）、促进CYP11A1的表达（P<0.01）。上述结果表明，miR-495-3p促进颗粒细胞凋亡，抑制增殖，将细胞周期阻滞在G2/M期，刺激类固醇激素分泌，从而影响山羊卵泡的发育。

关键词: miR-495-3p, 山羊卵巢颗粒细胞, 凋亡, 类固醇激素

Abstract: The objective of this study was to explore the effect of miR-495-3p on the function of goat ovarian granulosa cells and its regulation mechanism. In this study, healthy 3-4 months female Dazu black goats were selected to collect ovarian granulosa cells, miR-495-3p mimics and inhibitor were used to construct overexpression and inhibition cell models. The phenotype of apoptosis and cell cycle were detected by flow cytometry. The secretion of estradiol (E2) and progesterone (P4) in granulosa cells were tested by ELISA kits. The qRT-PCR and Western blot were used to detect mRNA and protein expression levels of genes involved in cell apoptosis, proliferation, cycle and steroid hormone synthesis, respectively. The results showed that miR-495-3p significantly promoted ovarian granulosa cell apoptosis (P<0.01), enhanced BAX expression (P<0.05) and inhibited BCL2 expression (P<0.01) in ovarian granulosa cell. Moreover, overexpression of miR-495-3p inhibited cell proliferation in a time-dependent manner and significantly reduced the expression of PCNA (P<0.05). Meanwhile, overexpression of miR-495-3p significantly increased the percentage of cells at G2/M phase (P<0.05), and significantly reduced the mRNA level of cell cycle-related genes CDK4 (P<0.001) and CCND1 (P<0.05). Others, inhibition of miR-495-3p significantly reduced the mRNA level of CCNE1 gene (P<0.01). Meanwhile, miR-495-3p could promote the secretion of E2 and P4 in granulosa cells. Overexpression of miR-495-3p significantly increased the mRNA and protein expression levels of 3β-HSD (P<0.001), CYP11A1 (P<0.01), CYP19A1 (P<0.05). Interference with miR-495-3p significantly inhibited 3β-HSD(P<0.01), promoted CYP11A1 expression (P<0.01). Overall, the above results indicated that miR-495-3p can promote granulosa cell apoptosis, inhibit proliferation, arrest in the G2/M phase of cell cycle, stimulate the secretion of steroid hormones, thereby affect the follicular development of goat.

Key words: miR-495-3p, goat ovarian granulosa cells, apoptosis, steroid hormones

中图分类号:

S827.3

王磊, 何莉娜, 唐雪, 李碧筠, 黄思艺, 王钰锟, 徐德军, 赵中权. miR-495-3p对山羊卵巢颗粒细胞功能的影响[J]. 畜牧兽医学报, 2022, 53(2): 436-446.

WANG Lei, HE Lina, TANG Xue, LI Bijun, HUANG Siyi, WANG Yukun, XU Dejun, ZHAO Zhongquan. Effects of miR-495-3p on Ovarian Granulosa Cell Functions in Goat[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(2): 436-446.

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