复发性流产患者绒毛组织中miR-27a表达对滋养细胞增殖和凋亡的影响及其作用机制  

Expression of miR-27a in villi tissue of patients with recurrent abortion and its effects on trophoblast cell proliferation and apoptosis and their mechanisms

作　　者：周立花 胡英[1] 邹晖[1] ZHOU Lihua;HU Ying;ZOU Hui(Department of Reproductive Medicine,Second Affiliated Hospital,Hainan Medical College,Haikou 570311,China)

机构地区：[1]海南医学院第二附属医院生殖科,海南海口570311

出　　处：《吉林大学学报（医学版）》2022年第4期1018-1027,共10页Journal of Jilin University：Medicine Edition

基　　金：海南省卫健委卫生健康行业科研项目(19A200038)。

摘　　要：目的:探讨复发性流产(RM)患者绒毛组织中miR-27a的表达,阐明其对滋养细胞增殖和凋亡的影响及其作用机制。方法:收集17例RM患者(RM组)和正常妊娠妇女(健康对照组)的绒毛组织,实时荧光定量PCR(RT-qPCR)法检测绒毛组织中miR-27a表达水平。体外培养人绒毛滋养细胞HTR-8/SVneo,利用脂质体瞬时转染技术将miR-27a模拟物(miR-27a mimic)、miR-27a抑制剂(miR-27ainhibitor)和miR-27a阴性对照(miR-NC)序列转染入滋养细胞中,同时设不转染的对照组,RT-qPCR法检测转染效率后,分别采用CCK-8法和EdU实验检测各组细胞增殖活性和EdU阳性细胞率,流式细胞术检测各组不同细胞周期细胞百分率,AnnexinⅤ-FITC/PI法检测各组细胞凋亡率。应用生物信息学分析miR-27a的作用靶基因,筛选周期蛋白D1(Cyclin D1)和胰岛素样生长因子1(IGF1)进行验证,采用荧光素酶靶基因报告实验、RT-qPCR法和Western blotting法检测miR-27a与Cyclin D1和IGF1的调控关系。采用RT-qPCR法和免疫组织化学染色检测RM组和健康对照组受试者绒毛组织中Cyclin D1和IGF1 mRNA及蛋白表达水平,采用Pearson相关分析法对miR-27a与Cyclin D1和IGF1 mRNA表达水平的相关性进行分析。结果:与健康对照组比较,RM组患者绒毛组织中miR-27a表达水平明显升高(P<0.01)。与对照组比较,miR-27a mimic组细胞中miR-27a表达水平明显升高(P<0.01),miR-27a inhibitor组细胞中miR-27a表达水平明显降低(P<0.01),miR-NC组细胞中miR-27a表达水平差异无统计学意义(P>0.05)。与对照组比较,miR-27a mimic组细胞增殖活性和S期细胞百分率均明显降低(P<0.05),G0/G1期细胞百分率和细胞凋亡率均明显升高(P<0.05);而miR-27a inhibitor组细胞增殖活性和S期细胞百分率均明显升高(P<0.05),G0/G1期细胞百分率和细胞凋亡率均明显降低(P<0.05);miR-NC组细胞中上述各指标差异无统计学意义(P>0.05)。荧光素酶靶基因报告实验、RT-qPCR法和Western blotting法检�Objective:To investigate the expression of miR-27a in villus tissue of the patients with recurrent abortion(RM),and to clarify its effects on trophoblast cell proliferation and apoptosis and their mechanisms.Methods:The villus tissues of 17 RM patients(RM group)and normal pregnant women(healthy control group)were collected.The expression levels of miR-27a in villus tissue of the subjects were detected by real-time fluorescence quantitative PCR(RT-qPCR).The human chorionic trophoblasts HTR-8/SVneo were cultured in vitro.The miR-27a mimic or inhibitor or negative control sequence(miRNC)were transfected into the trophoblasts by liposome transient transfection;at the same time,control group(without transfection)was set up.After RT-qPCR method was used to detect the transfection efficiency,CCK-8 method and EdU experiment were used to detect the proliferation activities of the cells and the rates of EdU positive cells in various groups,and flow cytometry was used to detect the percentages of trophoblasts at different cell cycles in various groups;Annexin Ⅴ-FITC/PI method was used to detect the apoptotic rates of cells in various groups.The target genes of miR-27a were analyzed by bioinformatics,Cyclin D1 and insulin-like growth factor 1(IGF1)were screened for verification,and luciferase target gene reporting experiment,RT-qPCR method and Western blotting method were used to detect the regulatory relationship between miR-27a and Cyclin D1 IGF1;RT-qPCR method and Immunohistochemistry were used to detect the expressions of Cyclin D1 and IGF1 mRNA and proteins in villi tissue of subjects in RM group and healthy control group.Pearson’s correlation analysis was used to analyze the correlation between miR-27a and the expression levels of Cyclin D1 and IGF1 mRNA.Results:Compared with healthy control group,the expression level of miR-27a in villus tissue of the patients in RM group was increased significantly(P<0.01).Compared with control group,the expression level of miR-27a in the cells in miR-27a mimic group was signific

关 键 词：复发性流产 微小RNA-27a 周期蛋白D1 胰岛素样生长因子1 滋养细胞 

分 类 号：R714.21[医药卫生—妇产科学]