天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (11): 1121-1125.doi: 10.11958/20210996

• 细胞与分子生物学 •    下一篇

miR-199a在妊娠糖尿病中的表达及参与胰岛素抵抗的作用机制研究

马双玲，李国芸，杨颖，李辞妹，周芳芳，张新宁，王茜   

  1. 1新乡市中心医院（新乡医学院第四临床学院）妇产科（邮编453000）；2天津医科大学总医院，天津市女性生殖健康与优生重点实验室
  • 收稿日期:2021-04-25 修回日期:2021-07-02 出版日期:2021-11-15 发布日期:2021-11-19
  • 基金资助:
    天津市卫生局科技基金项目（2014KZ095）

The expression of miR-199a in gestational diabetes mellitus and its mechanism in insulin resistance

MA Shuang-ling, LI Guo-yun, YANG Ying, LI Ci-mei, ZHOU Fang-fang, ZHANG Xin-ning, WANG Xi   

  1. 1 Department of Obstetrics and Gynecology, Xinxiang Central Hospital / the Fourth Clinical College of Xinxiang Medical College, Xinxiang 453000, China; 2 General Hospital of Tianjin Medical University, Tianjin Key Laboratory of Female Reproductive Health and Eugenics
  • Received:2021-04-25 Revised:2021-07-02 Published:2021-11-15 Online:2021-11-19

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摘要： 目的　探究微小RNA（miR）-199a在妊娠糖尿病（GDM）中的表达及调控沉默信息调节因子1（SIRT1）/叉头盒转录因子O1（FOXO1）通路参与胰岛素抵抗（IR）的机制。方法　GDM孕妇56例为GDM组，正常孕妇60例为正常孕妇组，实时荧光定量PCR（qPCR）检测2组胎盘中miR-199a水平；计算胰岛素抵抗指数（HOMA-IR）；Pearson相关分析miR-199a与HOMA-IR的相关性。体外培养人绒毛膜滋养层细胞HTR-8/SVneo并建立IR模型。miR-199a功能验证：细胞依处理方式不同分为模型组、miRNA inhibitor NC组、miR-199a inhibitor组、miRNA mimic NC组、miR-199a mimic组；qPCR检测各组细胞中miR-199a表达情况，蒽酮法检测细胞内葡萄糖吸收量，试剂盒检测超氧化物歧化酶（SOD）、丙二醛（MDA）水平，Western blot检测SIRT1、FOXO1、乙酰化（Ac）-FOXO1蛋白水平，双荧光素酶报告基因实验鉴定miR-199a与SIRT1的靶向关系。结果　与正常孕妇组比较，GDM组胎盘中miR-199a、HOMA-IR水平升高（P＜0.05）；GDM组miR-199a水平与HOMA-IR呈正相关（P＜0.05）。棕榈酸、胰岛素共同作用细胞24 h成功建立IR模型。与模型组和miRNA inhibitor NC组比较，miR-199a inhibitor组miR-199a、MDA水平降低，葡萄糖吸收量、SOD水平、SIRT1蛋白水平升高（P＜0.05）。与模型组和miRNA mimic NC组比较，miR-199a mimic组miR-199a和Ac-FOXO1蛋白表达升高、MDA水平升高，葡萄糖吸收量、SIRT1蛋白水平降低（P＜0.05）。Tarbase数据库分析显示，miR-199a与SIRT1存在互补的结合位点，并经双荧光素酶实验证实。结论　GDM患者胎盘中miR-199a高表达，升高的miR-199a可通过靶向下调SIRT1而促进FOXO1乙酰化，导致IR。

关键词: 糖尿病, 妊娠, 微RNAs, 叉头框蛋白O1, 微小RNA-199a, 沉默信息调节因子1/叉头盒转录因子O1通路, 胰岛素抵抗

Abstract: Objective　To investigate the expression of microRNA (miR)-199a in gestational diabetes mellitus (GDM) and the mechanism of silent mating type information regulator 2 homolog 1/forkhead box transcription factor O1 (SIRT1/FOXO1) pathway involved in insulin resistance (IR). Methods　A total of 56 GDM pregnant women were selected as the research objects (GDM group), and 60 normal pregnant women were selected as the control group. Real-time fluorescence quantitative PCR (qPCR) was used to detect the level of miR-199a in placenta. The insulin resistance index (HOMA-IR) was calculated. Pearson method was used to analyze the correlation between miR-199a and HOMA-IR. Human beings chorial trophcytes HTR-8/SVneo cells were cultured in vitro, and IR model was made. The function of miR-199a was verified. Cells were divided into the model group, the miRNA inhibitor NC group, the miR-199a inhibitor group, the miRNA mimic NC group and the miR-199a mimic group. The expression levels of miR-199a were detected by qPCR in each group. The intracellular glucose uptake was measured by anthrone method. The levels of superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by kit. The protein levels of SIRT1, FOXO1 and acely (Ac)-FOXO1 were detected by Western blot assay. The double luciferase reporter gene assay was used to identify the targeting relationship between miR-199a and SIRT1. Results　Compared with the normal pregnant women, the levels of miR-199a and HOMA-IR in placenta were higher in GDM patients (P＜0.05), and there was a positively correlation between miR-199a level and HOMA-IR (P＜0.05). The combined action of palmitic acid and insulin on cells for 24 h could establish an IR model. Compared with the model group and the miRNA inhibitor NC group, the levels of miR-199a and MDA were decreased in the miR-199a inhibitor group, while the levels of glucose uptake, SOD and SIRT1 protein were increased (P＜0.05). Compared with the model group and the miRNA mimic NC group, the levels of miR-199a, Ac-FOXO1 protein and MDA were increased in the miR-199a mimic group, while the levels of glucose uptake and SIRT1 protein were decreased (P＜0.05). Tarbase database analysis showed that miR-199a and SIRT1 had complementary binding sites, which were verified by double luciferase. Conclusion　The level of miR-199a in placenta is highly expressed in GDM patients. The increased miR-199a can promote FOXO1 acetylation by targeting down-regulation of SIRT1, relulting in IR.

Key words: diabetes, gestational, microRNAs, Forkhead box protein O1, microRNA-199a, silent information regulator 1/forkhead transcription factor O1 pathway, insulin resistance