摘要
目的:探讨妊娠期糖尿病(GDM)患者胎盘组织中微小RNA-508-3p(miR-508-3p)与肝细胞生长因子(HGF)的表达及两者的相互作用,初步阐明miR-508-3p介导滋养细胞胰岛素抵抗(IR)的相关分子机制。方法:选取GDM患者及正常妊娠孕产妇各15例,分别为GDM组和正常对照组。采用实时定量PCR(RT-PCR)法、免疫组织化学染色法和免疫印迹法检测2组孕产妇胎盘组织中miR-508-3p表达水平和HGF蛋白表达水平,分析两者的相关性。应用生物信息学技术筛选HGF作为miR-508-3p的目标靶基因,并通过双荧光素酶报告基因实验及免疫印迹法进行验证。体外培养人滋养细胞HTR-8/Svneo,建立HTR-8/Svneo IR细胞模型(HTR-8/Svneo-IR),采用脂质体瞬时转染法对HTR-8/Svneo-IR细胞转染miR-508-3p-inhibitor(miR-508-3p-inhibitor-IR组)和miR-NC(miR-NCIR组),RT-PCR法检测其转染效率,免疫印迹法检测各组细胞中HGF及磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路中磷酸化PI3K(p-PI3K)和磷酸化AKT(p-AKT)蛋白表达水平。结果:与正常对照组比较,GDM组患者胎盘组织中miR-508-3p表达水平明显升高(P<0.05),HGF蛋白表达水平明显降低(P<0.01),且GDM组患者胎盘组织中HGF蛋白表达水平与miR-508-3p表达水平呈负相关关系(r=-0.542,P<0.05)。与HTR-8/Svneo组比较,HTR-8/Svneo-IR组细胞中miR-508-3p表达水平明显升高(P<0.05);与HTR-8/Svneo-IR组比较,miR-508-3p-inhibitor-IR组细胞中miR-508-3p表达水平明显降低(P<0.05)。与HTR-8/Svneo组比较,HTR-8/Svneo-IR组细胞中HGF、p-PI3K和p-AKT蛋白表达水平均明显降低(P<0.05);与HTR-8/Svneo-IR组比较,miR-508-3p-inhibitor-IR组细胞中HGF、p-PI3K和p-AKT蛋白表达水平明显升高(P<0.05)。结论:在GDM患者胎盘组织中miR-508-3p可靶向抑制HGF的表达,并通过PI3K/AKT信号通路促进滋养细胞的IR,继而参与GDM的发生发展。
Objective:To investigate the expressions of microRNA-508-3p(miR-508-3p)and hepatocyte growth factor(HGF)in placenta tissue of the patients with gestational diabetes mellitus(GDM)and their relationship,and to clarify the related molecular mechanism of miR-508-3p in mediating trophoblast insulin resistance(IR).Methods:A total of 15 GDM patients and 15 normal pregnant women were selected and used as GDM group and normal control group.The expression levels of miR-508-3p and HGF protein in placenta tissue of the patients in two groups were detected by RT-PCR,immunohistochemistry and Western blotting methods,and their relationship was analyzed.HGF was screened as the target gene of miR-508-3p with bioinformatics,which was verified by double luciferase reporter gene assay and Western blotting method.The human trophoblast cells HTR-8/Svneo were cultured in vitro to establish the HTR-8/Svneo IR cell model(HTR-8/Svneo-IR).The HTR-8/Svneo-IR cells were transfected with miR-508-3p-inhibitor(miR-508-3p-inhibitor-IR grouop)and miR-NC(miR-NCIR group)by liposome transient transfection method.The transfection efficiency was detected by RTPCR;Western blotting method was used to detect the expression levels of HGF and phosphorylated phosphatidylinosital 3 kinase(p-PI3K)and phosphorylated protein kinse B(p-AKT)proteins in PI3K/AKT signaling pathway in the cells in various groups.Results:Compared with normal control group,the expression levels of miR-508-3p in placenta tissue of the patients in GDM group was significantly increased(P<0.05),while the HGF protein expression level was significantly decreased(P<0.01),and there was a negative correlation between the expression level of HGF protein and the expression level of miR-508-3p in placenta tissue of the patients in GDM group(r=-0.542,P<0.05).Compared with HTR-8/Svneo group,the expression level of miR-508-3p in the cells in HTR-8/Svneo-IR group was significantly increased(P<0.05);compared with HTR-8/Svneo-IR group,the expression level of miR-508-3p in the cells in miR-508-3p-inhibitor-IR group was significantly decreased(P<0.05).Compared with HTR-8/Svneo group,the expression levels of HGF,p-PI3K and p-AKT proteins in the cells in HTR-8/Svneo-IR group were significantly decreased(P<0.05);compared with HTR-8/Svneo-IR group,the expression levels of HGF,p-PI3K and p-AKT proteins in the cells in miR-508-3p-inhibitor-IR group were significantly increased(P<0.05).Conclusion:MiR-508-3p can inhibit the expression of HGF in placenta tissue of the GDM patients,and promote IR of trophoblasts through PI3K/AKT signaling pathway,and then participate in the occurrence and development of GDM.
作者
黄好
贾虹
王晓霜
张鹭
段亚亭
HUANG Hao;JIA Hong;WANG Xiaoshuang;ZHANG Lu;DUAN Yating(Department of Gynecology and Obstetrics,Chongqing Hospital of Traditional Chinese Medicine,Chongqing 400011,China;Department of Ultrasound,Chongqing Hospital of Traditional Chinese Medicine,Chongqing 400011,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2021年第1期187-195,共9页
Journal of Jilin University：Medicine Edition
基金
国家中医药管理局全国名老中医药专家传承工作室建设项目(20140814)。
