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 effect on ovulation and lambing numbers in goats.
作者
刘杰
许香萍
邓铭
邹娴
江声伟
刘德武
柳广斌
孙宝丽
郭勇庆
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)
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
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)。
