miR-195-5p靶向CLDN6调控多囊卵巢综合征大鼠卵巢颗粒细胞的增殖、凋亡  

miR-195-5p targeting CLDN6 regulates the proliferation and apoptosis of ovarian granulosa cells in rats withpolycystic ovarysyndrome

作　　者：余慧 王静 杜丹[1] 杨帆[1] 王脉桃 YU Hui;WANG Jing;DU Dan;YANG Fan;WANG Maitao(The First People's Hospital of Chenzhou,Chenzhou,Hunan 423000,China)

机构地区：[1]郴州市第一人民医院,湖南郴州423000

出　　处：《中国优生与遗传杂志》2024年第7期1357-1363,共7页Chinese Journal of Birth Health & Heredity

基　　金：湖南省自然科学基金(2024JJ7053);郴州市第一人民院院内科研项目(N2019019)。

摘　　要：目的 探讨微小RNA-195-5p(miR-195-5p)/紧密连接蛋白6(CLDN6)对多囊卵巢综合征(PCOS)大鼠卵巢颗粒细胞增殖、凋亡的调控作用机制研究。方法 取10只SD大鼠,分为正常组(5只)和PCOS组(5只),构建PCOS模型。分离培养PCOS大鼠卵巢颗粒细胞,分为Con组、miR-NC组、miR-195-5p组、si-NC组、si-CLDN6组、miR-195-5p+pcDNA组、miR-195-5p+pcDNA-CLDN6组。采用实时定量PCR(qRT-PCR)检测miR-195-5p和CLDN6mRNA在正常大鼠及各组PCOS大鼠卵巢颗粒细胞中的表达水平。双荧光素酶报告实验验证miR-195-5p、CLDN6靶向关系;甲基噻唑基四唑法(MTT)检测PCOS大鼠卵巢颗粒细胞增殖活性、平板克隆实验检测PCOS大鼠卵巢颗粒细胞克隆形成能力、流式细胞术测定细胞凋亡率、Westernblot检测细胞CLDN6、活化型半胱氨酸蛋白酶9(cleavedcaspase 9)、活化型半胱氨酸蛋白酶3(cleaved-caspase 3)、B淋巴细胞瘤2(Bcl-2)相关X蛋白(Bax)表达水平。结果 miR-195-5p在PCOS大鼠中表达水平较正常大鼠低,CLDN6蛋白表达水平较正常大鼠高(P<0.05)。miR-195-5p靶向调控CLDN6蛋白表达水平。过表达miR-195-5p或干扰CLDN6蛋白表达抑制PCOS大鼠卵巢颗粒细胞的增殖,促进细胞凋亡(P<0.05)。上调CLDN6蛋白表达水平可部分逆转过表达miR-195-5p对PCOS大鼠卵巢颗粒细胞增殖、凋亡的作用(P<0.05)。结论 miR-195-5p可能通过靶向CLDN6调控PCOS大鼠卵巢颗粒细胞的增殖和凋亡。Objective To investigate the regulatory mechanism of microRNA-195-5p(miR-195-5p)/CLDN6 on the proliferation and apoptosis of ovarian granulocyte cells in rats with polycystic ovary syndrome(PCOS).Methods Ten SD rats were divided into normal group and PCOS group with 5 rats in each group.Construct the PCOS model.Isolation and culture of PCOS rat ovarian granulosa cells and divided into Con group,miR-NC group,miR-195-5p group,si-NC group,si-CLDN6 group,miR-195-5p+pcDNA group and miR-195-5p+pcDNA-CLDN6 group.Dual luciferase reporting experiment verified the targeting relationship between miR-195-5p and CLDN6.The proliferative activity of ovarian granulosa cells in PCOS rats was detected by MTT assay.The cloning ability of ovarian granulosa cells in PCOS rats was detected by plate cloning assay.The apoptosis rate of ovarian granulosa cells in PCOS rats was determined by flow cytometry.The expression levels of CLDN6,activated cysteine protease 9(cleaved-caspase 9),activated cysteine protease 3(cleaved-caspase 3),and B-lymphoblastoma 2(Bcl-2)associated X protein(Bax)were detected by Western blot.Results The expression level of miR-195-5p in ovarian granulosa cells of PCOS rats was lower than that of normal rats,and the expression level of CLDN6 was higher then that of normal rat ovarian granulosa cells(P<0.05).MiR-195-5p can target the expression of CLDN6 protein.Overexpression of miR-195-5p or interference with CLDN6 expression inhibited the proliferation of ovarian granulosa cells in PCOS rats and promoted cell apoptosis(P<0.05).The apoptosis rate and the expression levels of cleaved-caspase 3,cleaved-caspase 9 and Bax protein were significantly increased(P<0.05).Up-regulation of CLDN6 partially reversed the effects of overexpression of miR-195-5p on proliferation of ovarian granulosa cells in PCOS rats(P<0.05).Conclusion MiR-195-5p may regulate the pro-liferation and apoptosis of ovarian granulosa cells in PCOS rats by targeting CLDN6.

关 键 词：多囊卵巢综合征 卵巢颗粒细胞 微小RNA-195-5p 紧密连接蛋白6 

分 类 号：R711.75[医药卫生—妇产科学]