miR-148a-3p靶向PPARγ基因抑制鹅颗粒细胞孕酮的合成  

miR-148a-3p Inhibits the Production of Progesterone by Targeting PPARγ in Granulosa Cells of Goose

作　　者：邓艳 解广娟 胡深强 李亮 王继文 DENG Yan;XIE Guangjuan;HU Shenqiang;LI Liang;WANG Jiwen(Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province,Sichuan Agricultural University,Chengdu 613111,China)

机构地区：[1]四川农业大学畜禽遗传资源发掘与创新利用四川省重点实验室,成都613111

出　　处：《畜牧兽医学报》2021年第6期1571-1581,共11页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家现代水禽产业技术体系(CARS-43-6)。

摘　　要：旨在探究miR-148a-3p是否通过靶基因PPARγ调节鹅卵泡颗粒细胞中类固醇激素的合成。本研究选用12只健康的处于产蛋高峰期的天府肉鹅母系母鹅分别用于组织样品采集和颗粒细胞培养。利用qPCR检测miR-148a-3p在鹅不同阶段卵泡颗粒层中的表达;通过MEGA 7软件分析miR-148a-3p在不同物种的保守性,预测miR-148a-3p的靶基因,采用双荧光素酶报告试验验证miR-148a-3p与靶基因的靶标关系;在鹅颗粒细胞中转染miR-148a-3p mimics和inhibitor,qPCR检测miR-148a-3p对靶基因及其下游基因表达水平的影响;同时利用ELISA技术检测激素含量的变化。结果表明,miR-148a-3p在不同物种中高度保守;在鹅卵泡发育过程中,miR-148a-3p的表达量随卵泡直径的增加呈先升高后下降的趋势。在鹅卵泡颗粒细胞体外培养过程中,miR-148a-3p mimics能显著下调3β-HSD的表达(P<0.05),抑制孕酮的合成(P<0.05);而miR-148a-3p inhibitor的作用则相反(P<0.05)。生物信息学分析和双荧光素酶报告试验证实,PPARγ为miR-148a-3p的靶基因。在颗粒细胞中,miR-148a-3p mimics能显著降低PPARγ的表达(P<0.05),而miR-148a-3p inhibitor能显著上调PPARγ的表达(P<0.05)。当干扰PPARγ后,3β-HSD的表达量显著降低(P<0.05),孕酮的含量也降低。这些结果表明,在鹅等级卵泡颗粒细胞中,miR-148a-3p可靶向结合PPARγ来抑制3β-HSD的表达,从而降低颗粒细胞孕酮的合成。The aim of this study was to explore whether miR-148a-3p could target PPARγto regulate the production of steroid hormone in granulosa cells of goose.In the present study,12 healthy and high-laying maternal Tianfu meat female geese were used for sample collection and culture of granulosa cells in vitro.Quantitative polymerase chain reaction(qPCR)was used to detect the expression profile of miR-148a-3p at different stages of granulosa layers in follicles of goose.MEGA 7 software was used to identify the conservation of miR-148a-3p in different species.Target genes of miR-148a-3p were predicted and validated via double luciferase report system.miR-148a-3p mimics and inhibitor were transfected into goose granulosa cells,and the expression levels of target gene and downstream genes were examined by qPCR.Meanwhile,ELISA was used to measure the changes of hormone levels.The results showed that miR-148a-3p was highly conserved in different species.The expression level of miR-148a-3p firstly increased and then decreased during the follicles development of goose.In the culture of goose granulosa cells in vitro,the expression level of 3β-HSD and the progesterone production significantly decreased after the transfection of miR-148a-3p mimics(P<0.05),while the above indicators increased after the transfection of miR-148a-3p inhibitor(P<0.05).Bioinformatic analysis and double luciferase report system demonstrated that PPARγwas the target gene of miR-148a-3p.The expression level of PPARγsignificantly decreased after transfection of miR-148a-3p mimics in granulosa cells(P<0.05),while that increased after transfection of miR-148a-3p inhibitor(P<0.05).After interfering PPARγ,the expression level of 3β-HSD significantly decreased(P<0.05),and the progesterone production also decreased.These results demonstrated that miR-148a-3p could target PPARγto inhibit the expression of 3β-HSD and decrease the progesterone production in granulosa cells of goose.

关 键 词：miR-148a-3p PPARΓ 鹅 颗粒细胞 孕酮 3Β-HSD 

分 类 号：S835.2[农业科学—畜牧学]