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。
【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.
作者
王尧悦
史倩倩
罗琪
高林娜
吴浩
张建丽
丁强
WANG Yaoyue;SHI Qianqian;LUO Qi;GAO Linna;WU Hao;ZHANG Jiangli;DING Qiang(School of Animal Husbandry and Veterinary Medicine,Jiangsu Vocational College of Agricultural and Forestry,Zhenjiang 212400,China;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;College of Animal Science and Technology,Northwest A&F University,Yangling 712100,China;Dagong Town Animal Husbandry Veterinary Station in Hainan Province,Nantong 226600,China)
出处
《中国畜牧兽医》
北大核心
2025年第8期3715-3725,共11页
China Animal Husbandry & Veterinary Medicine
基金
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)。
