MicroRNA-24-3p通过靶向P27促进猪颗粒细胞增殖并抑制细胞凋亡

doi:10.1016/j.jia.2023.04.008

摘要/Abstract

摘要：

卵巢卵泡发育状态直接影响母猪的繁殖性能，颗粒细胞作为卵泡中最大的细胞群，其增殖直接促进卵泡的生长，其凋亡直接导致卵泡的闭锁，因此，探究关键因子对颗粒细胞增殖、凋亡的作用及机制是至关重要的。miR-24-3p在高产大白×长白二元母猪卵巢组织中的表达水平显著高于低产母猪，并且miR-24-3p在各个物种间是高度保守的，KEGG和GO分析表明miR-24-3p可能与细胞增殖和凋亡有关，其靶基因也富集在Wnt和mTOR等与颗粒细胞生理功能相关的信号通路上。然而，miR-24-3p在颗粒细胞中的作用尚不清楚。在本研究中，我们通过流式细胞术、5-乙基-2′-脱氧尿苷(EdU)染色和细胞计数等试验，发现miR-24-3p促进了颗粒细胞增殖，增加S期细胞比例，上调细胞周期基因（CyclinB、CyclinD、CyclinE、CDK4）的mRNA和蛋白水平表达。此外，通过流式细胞术、RT-qPCR、Western Blot等方法，表明miR-24-3p能够抑制颗粒细胞凋亡并影响凋亡相关基因（BCL-2、BAX）的表达。在机制上，通过miRBase 、RNAhybrid等在线预测网站对miR-24-3p潜在的靶基因进行生物信息学分析，在5个靶基因预测网站中取交集，筛选出17个潜在基因。通过RT-qPCR、Western Blot、双荧光素酶报告试验并结合基因的生物学功能进行分析，结果显示，周期蛋白依赖性激酶抑制剂1B (P27/CDKN1B) 是miR-24-3p在细胞增殖和凋亡方面的靶基因。此外，恢复试验证明，过表达P27减弱了miR-24-3p对颗粒细胞增殖的促进作用，同时也减弱了miR-24-3p对颗粒细胞凋亡的抑制作用。这些结果表明miR-24-3p可通过靶向P27抑制其转录水平进而促进颗粒细胞增殖并抑制颗粒细胞凋亡。本研究从产仔数极端表型差异个体中筛选并鉴定影响繁殖性状的关键miRNA，并结合猪原代细胞培养模型、生物信息学分析、分子生物学试验阐明miR-24-3p影响颗粒细胞增殖凋亡的具体作用及机制。

Abstract:

Ovarian follicle development is associated with the physiological functions of granulosa cells (GCs), including proliferation and apoptosis. The level of miR-24-3p in ovarian tissue of high-yielding Yorkshire×Landrace sows was significantly higher than that of low-yielding sows. However, the functions of miR-24-3p on GCs are unclear. In this study, using flow cytometry, 5-ethynyl-2´-de-oxyuridine (EdU) staining, and cell count, we showed that miR-24-3p promoted the proliferation of GCs increasing the proportion of cells in the S phase and upregulating the expression of cell cycle genes, moreover, miR-24-3p inhibited GC apoptosis. Mechanistically, on-line prediction, bioinformatics analysis, a luciferase reporter assay, RT-qPCR, and Western blot results showed that the target gene of miR-24-3p in proliferation and apoptosis is cyclin-dependent kinase inhibitor 1B (P27/CDKN1B). Furthermore, the effect of miR-24-3p on GC proliferation and apoptosis was attenuated by P27 overexpression. These findings suggest that miR-24-3p regulates the physiological functions of GCs.

Key words: miR-24-3p , granulosa cells , proliferation , apoptosis

. MicroRNA-24-3p通过靶向P27促进猪颗粒细胞增殖并抑制细胞凋亡[J]. Journal of Integrative Agriculture, 2024, 23(4): 1315-1328.

Shengjie Shi, Lutong Zhang, Liguang Wang, Huan Yuan, Haowei Sun, Mielie Madaniyati, Chuanjiang Cai, Weijun Pang, Lei Gao, Guiyan Chu.

miR-24-3p promotes proliferation and inhibits apoptosis of porcine granulosa cells by targeting P27 [J]. Journal of Integrative Agriculture, 2024, 23(4): 1315-1328.

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