miR-26a-5p通过靶向PTEN基因调控山羊卵巢颗粒细胞的增殖

doi:10.16431/j.cnki.1671-7236.2025.08.020

中国畜牧兽医 ›› 2025, Vol. 52 ›› Issue (8): 3715-3725.doi: 10.16431/j.cnki.1671-7236.2025.08.020

• 遗传繁育 • 上一篇

miR-26a-5p通过靶向PTEN基因调控山羊卵巢颗粒细胞的增殖

王尧悦¹, 史倩倩², 罗琪³, 高林娜², 吴浩⁴, 张建丽², 丁强²

1. 江苏农林职业技术学院畜牧兽医学院, 镇江 212400;
2. 江苏省农业科学院畜牧研究所, 江苏省畜禽精准育种工程研究中心, 农业农村部种养结合重点实验室, 南京 210014;
3. 西北农林科技大学动物科技学院, 杨凌 712100;
4. 海安市大公镇畜牧兽医站, 南通 226600

收稿日期:2024-11-25 发布日期:2025-08-02
通讯作者: 丁强 E-mail:dingqiang198907@163.com
作者简介:王尧悦，E-mail：18792629437@163.com。
基金资助:
江苏农林职业技术学院高层次人才引进项目(2024rc17)；国家自然科学青年基金项目(32102550)

miR-26a-5p Targets PTEN Gene to Regulate the Proliferation of Goat Ovarian Granulosa Cells

WANG Yaoyue¹, SHI Qianqian², LUO Qi³, GAO Linna², WU Hao⁴, ZHANG Jiangli², DING Qiang²

1. School of Animal Husbandry and Veterinary Medicine, Jiangsu Vocational College of Agricultural and Forestry, Zhenjiang 212400, China;
2. Key Laboratory of Crop and Animal Integrated Farming, Ministry of Agriculture and Rural Affairs, Jiangsu Province Engineering Research Center for Precision Animal Breeding, Institute of Animal Science, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;
3. College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China;
4. Dagong Town Animal Husbandry Veterinary Station in Hainan Province, Nantong 226600, China

Received:2024-11-25 Published:2025-08-02

摘要/Abstract

摘要： 【目的】颗粒细胞的增殖受miRNA等多种因素的调控，其中，miR-26a-5p在山羊生长卵泡中显著富集，或与卵泡生长发育相关。试验旨在研究miR-26a-5p在山羊颗粒细胞增殖作用机制，以期丰富卵泡发育中分子调控网络。【方法】采用实时荧光定量PCR分析miR-26a-5p在不同大小山羊卵泡中的表达情况；体外培养鉴定卵巢颗粒细胞，并在颗粒细胞中分别过表达和抑制miR-26a-5p。利用EdU增殖和MTT试验检测颗粒细胞转染后的增殖情况；利用双荧光素酶报告系统和Western blotting验证miR-26a-5p与靶基因PTEN间的互作关系。通过Western blotting分析颗粒细胞过表达或干扰miR-26a-5p表达后PI3K/Akt信号通路中相关蛋白的表达情况。【结果】在不同大小的山羊卵泡中均检测到miR-26a-5p的表达，miR-26a-5p在卵母细胞中也有表达，其中，与其他直径卵泡(＜2、4～5和＞5 mm)的颗粒细胞相比，miR-26a-5p在中小卵泡(直径2～3 mm)颗粒细胞中显著富集(P＜0.05)。在体外培养的颗粒细胞中，过表达miR-26a-5p组增殖细胞比例显著高于NC mimics组(P＜0.05)；转染miR-26a-5p inhibitor的颗粒细胞与转染NC inhibitor相比增殖比例显著减少(P＜0.05)。双荧光素酶报告试验结果显示，与突变组相比，miR-26a-5p靶向PTEN基因3'-UTR的种子序列显著降低了荧光素酶活性(P＜0.05)。Western blotting结果显示，与转染NC mimics组相比，颗粒细胞中过表达miR-26a-5p可显著降低PTEN蛋白的表达(P＜0.05)，过表达miR-26a-5p 96 h仍可检测到磷酸化蛋白p-AKT的表达上调及p-AKT/AKT比例增加(P＜0.05)。【结论】卵巢颗粒细胞中miR-26a-5p通过直接靶向抑制PTEN基因的表达来活化AKT蛋白，激活PI3K/Akt信号通路，促进颗粒细胞增殖。

关键词: miR-26a-5p; PTEN基因; 颗粒细胞增殖; PI3K/Akt信号通路

Abstract: 【Objective】 Granulosa cells (GCs) proliferation is regulated by many factors,such as miRNAs,miR-26a-5p was significantly enriched in the growing follicles of goats,which might have a regulated role during the folliclar development.This study was aimed to reveal the functions of miR-26a-5p in goat GCs,in order to enrich the molecular regulatory network in follicular development. 【Method】 Real-time quantitative PCR was used to analyze the expression of miR-26a-5p in goat follicles with different sizes.The ovarian GCs were cultured and identified in vitro.The proliferation of GCs was detected using EdU and MTT assays after with overexpressed and inhibited miR-26a-5p expression.The interaction between miR-26a-5p and the target gene PTEN was verified using a dual luciferase reporter system and Western blotting,respectively.Western blotting was used to analyze the expression of proteins related to PI3K/Akt signaling pathway in GCs after overexpression or interference with miR-26a-5p expression. 【Result】 The expression of miR-26a-5p was detected in goat follicles of varying sizes,including in oocytes.miR-26a-5p was significantly enriched in GCs of small-to-medium follicles (2-3 mm in diameter) compared with GCs from follicles of other diameters (＜2,4-5,and ＞5 mm) (P＜0.05).In in vitro cultured GCs,the proportion of proliferating cells was significantly higher in miR-26a-5p overexpression group than that in control group (transfected with NC mimics) (P＜0.05).Conversely,transfection with miR-26a-5p inhibitor significantly reduced the proliferation rate compared with NC inhibitor group (P＜0.05).Dual-luciferase reporter assays revealed that compared with mutant group,miR-26a-5p overexpression significantly reduced luciferase activity by targeting the seed sequence in the 3'-UTR of PTEN gene (P＜0.05).Western blotting results showed that compared with NC mimics group, miR-26a-5p overexpression significantly downregulated the expression of PTEN protein in GCs (P＜0.05).Furthermore,elevated phosphorylated the expression of p-AKT protein and an increased p-AKT/AKT ratio were still detectable 96 h after miR-26a-5p overexpression (P＜0.05). 【Conclusion】 miR-26a-5p directly targeted and suppressed the expression of PTEN gene in ovarian GCs,thereby activated the AKT protein and PI3K/Akt signaling pathway,which promoted GCs proliferation.

Key words: miR-26a-5p; PTEN gene; granulosa cell proliferation; PI3K/Akt signaling pathway

中图分类号:

S814.8

王尧悦, 史倩倩, 罗琪, 高林娜, 吴浩, 张建丽, 丁强. miR-26a-5p通过靶向PTEN基因调控山羊卵巢颗粒细胞的增殖[J]. 中国畜牧兽医, 2025, 52(8): 3715-3725.

WANG Yaoyue, SHI Qianqian, LUO Qi, GAO Linna, WU Hao, ZHANG Jiangli, DING Qiang. miR-26a-5p Targets PTEN Gene to Regulate the Proliferation of Goat Ovarian Granulosa Cells[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3715-3725.

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miR-26a-5p通过靶向PTEN基因调控山羊卵巢颗粒细胞的增殖

miR-26a-5p Targets PTEN Gene to Regulate the Proliferation of Goat Ovarian Granulosa Cells

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