miR-144-5p靶向WNT5a对山羊卵巢颗粒细胞增殖、凋亡的影响  

Effect of miR-144-5p Targeting WNT 5a on the Proliferation and Apoptosis of Goat Ovarian Granulosa Cells

作　　者：刘杰[1,2] 许香萍 邓铭 邹娴[1,2] 江声伟[1,2] 刘德武 柳广斌 孙宝丽[1,3,4] 郭勇庆[1,3,4] 李耀坤 LIU Jie;XU Xiangping;DENG Ming;ZOU Xian;JIANG Shengwei;LIU Dewu;LIU Guangbin;SUN Baoli;GUO Yongqing;LI Yaokun(College of Animal Science,South China Agricultural University,Guangzhou 510642,China;Guangdong Key Laboratory of Animal Breeding and Nutrition,State Key Laboratory of Livestock and Poultry Breeding,Institute of Animal Science,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China;National Engineering Research Center for Livestock and Poultry Breeding,South China Agricultural University,Guangzhou 510642,China;Guangdong Key Laboratory of Agricultural Animal Genomics and Molecular Breeding,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学动物科学学院,广州510642 [2]广东省农业科学院动物科学研究所,畜禽育种国家重点实验室,广东省畜禽育种与营养研究重点实验室,广州510640 [3]华南农业大学畜禽育种国家地方联合工程研究中心,广州510642 [4]华南农业大学,广东省农业动物基因组学与分子育种重点实验室,广州510642

出　　处：《畜牧兽医学报》2023年第6期2421-2435,共15页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省现代农业产业技术体系项目(2021KJ127);广东省科技特派员项目(KTP20210344)。

摘　　要：旨在探究miR-144-5p靶向WNT5a对山羊颗粒细胞增殖和凋亡的影响。本研究选用2岁健康雷州母山羊的卵巢颗粒细胞来过表达和抑制表达miR-144-5p及WNT5a,通过荧光定量PCR(real-time qPCR, RT-qPCR)、Western blot、双荧光素酶报告基因、CCK-8等技术来探究miR-144-5p与WNT5a对颗粒细胞增殖和凋亡的影响。结果表明,miR-144-5p在大卵泡颗粒细胞中的表达量极显著高于小卵泡颗粒细胞(P<0.01),WNT5a在大卵泡颗粒细胞中的表达量极显著低于小卵泡颗粒细胞(P<0.01);CCK-8检测发现,miR-144-5p抑制了GCs增殖,过表达miR-144-5p后,抑凋亡基因BCL-2的mRNA和蛋白表达极显著下调(P<0.01),促凋亡基因BAX的mRNA和蛋白表达极显著上调(P<0.01);Wnt/β-Catenin通路CTNNB1 mRNA和β-Catenin蛋白水平极显著降低(P<0.01);另外,miR-144-5p能靶向WNT5a抑制其表达。同时,CCK-8检测结果表明,WNT5a可促进GCs增殖,过表达WNT5a后,抑凋亡基因BCL-2的mRNA和蛋白水平极显著上调(P<0.01),促凋亡基因BAX的mRNA显著下调(P<0.05)、蛋白水平极显著下调(P<0.01),Wnt/β-Catenin通路CTNNB1 mRNA和β-Catenin蛋白水平极显著提高(P<0.01)。综上所述,在GCs中,miR-144-5p靶向负调控WNT5a的表达,调控Wnt/β-Catenin通路,进而上调BAX/BCL-2的比值来抑制颗粒细胞增殖并促进其凋亡,这可能对山羊的排卵数和产羔数产生一定的影响。Aiming The study aimed to investigate the effect of miR-144-5p targeting WNT 5a on proliferation and apoptosis of goat granulosa cells.Ovarian granulosa cells from 2-year-old healthy Leizhou female goats were selected to overexpress and repress miR-144-5p and WNT 5a,and the effects of miR-144-5p and WNT 5a on granulosa cell proliferation and apoptosis were investigated by fluorescent quantitative PCR(real-time qPCR,RT-qPCR),Western blot,dual luciferase reporter gene,CCK-8 and other techniques.The results showed that the expression of miR-144-5p was highly significantly higher in large follicular granulosa cells than that in small follicular granulosa cells(P<0.01),and the expression of WNT 5a was highly significantly lower in large follicular granulosa cells than that in small follicular granulosa cells(P<0.01);CCK-8 assay revealed that miR-144-5p inhibited the proliferation of GCs,and overexpression of miR-144-5p resulted in highly significant downregulation of mRNA and protein expression of the apoptosis suppressor gene BCL-2(P<0.01)and highly significant upregulation of mRNA and protein expression of the pro-apoptotic gene BAX(P<0.01);Wnt/β-Catenin pathway gene CTNNB 1 mRNA andβ-Catenin protein levels were highly significantly reduced(P<0.01);In addition,miR-144-5p could target WNT 5a to inhibit its expression.Meanwhile,CCK-8 assay results showed that WNT 5a promoted the proliferation of GCs,and overexpression of WNT 5a significantly upregulated the mRNA and protein levels of the apoptosis suppressor gene BCL-2(P<0.01),significant downregulation of significantly downregulated the mRNA level(P<0.05)and protein level(P<0.01)of the pro-apoptotic gene BAX,and the Wnt/β-Catenin pathway gene CTNNB 1 mRNA andβ-Catenin protein levels were highly significantly increased(P<0.01).In summary,in GCs,miR-144-5p targets and negatively regulates WNT 5a expression,regulates the Wnt/β-Catenin pathway,and then upregulates the BAX/BCL-2 ratio to inhibit granulosa cell proliferation and promote apoptosis,which may have an eff

关 键 词：颗粒细胞 miR-144-5p WNT 5a 山羊 繁殖 

分 类 号：S827.3[农业科学—畜牧学]