目的 探索微小RNA-22(miR-22)对妊娠糖尿病(GDM)患者糖代谢的影响。方法 收集2018-01至2019-01在武警广东总队医院就诊的75例妊娠糖尿病孕妇组的胎盘组织作为观察组(GDM组),75例正常妊娠孕妇作为对照组(HC组)。培养人绒毛膜HTR8/Svneo细胞建立细胞模型,检测胎盘绒毛组织和细胞的miR-22表达。用人工合成的miR-22类似物和抑制物(反义寡核苷酸)转染HTR8/Svneo细胞,葡萄糖氧化酶法检测体外细胞摄取葡萄糖能力,Western blot检测细胞胰岛素信号通路上的PI3K、AKT、IRS、萄糖转运体4(GLUT4)的表达。设计构建含目的基因野生型和突变型的质粒,双荧光素酶报告基因系统分析miR-22与靶基因的关系。结果 GDM胎盘组织和高糖组细胞中miR-22表达均明显低于对照组(P＜0.05),miR-22表达与胰岛素抵抗指数呈负相关。GDM组织的IRS、PI3K、AKT、Glut4蛋白表达水平明显低于对照组;miR-22表达时,HTR8/Svneo细胞摄糖升高,Glut4表达升高,miR-22表达抑制时,细胞摄糖减少,GLUT4表达下降;miR-22过表达或抑制时,IRS、PI3K、AKT表达明显降低。双荧光素酶报告基因系统实验显示SLC2A4基因(编码GLUT4)是miR-22调节靶点。结论 发生胰岛素耐受时,胎盘绒毛组织和细胞miR-22表达下降,miR-22过表达可以提高体外细胞摄取糖水平,miR-22可能通过调节GLUT4而参与GDM的发病机制。
Abstract
Objective To explore the effects of microRNA-22 (miR-22) on glucose metabolism in patients with gestational diabetes mellitus.Methods The placental tissues of 75 gestational diabetic patients(GDM group) and 75 healthy pregnant people (control group) treated in Guangdong Provincial Corps Hospital of Chinese People's Armed Police Force from January 2018 to January 2019 were collected. A cell model was established by culturing human chorionic villous HTR8/Svneo cells with gradient high glucose, and the expression of cellular miR-22 was analyzed. Synthetic miR-22 mimics or antisense oligonucleotides (suppressors) were transfected into HTR8/Svneo cells. Glucose utilization of cells in vitro was determined by glucose oxidase method. Western blot was used to detect PI3K, AKT, IRS, and Glut4 proteins on the insulin signaling pathway in HTR8/Svneo cells, GDM group and normal control tissues. Plasmids containing wild-type and mutant versions of the target genes were designed and constructed; and the relationship between miR-22 and the target genes was analyzed using a dual luciferase reporter gene system.Results miR-22 expression was significantly lower in GDM placental tissues and cells in the high glucose treatment group than in the control group(P＜0.05), and miR-22 expression was negatively correlated with insulin resistance index. The expression levels of IRS, PI3K, AKT and Glut4 in GDM were significantly lower than those in control group. When miR-22 was expressed, glucose uptake of HTR8/Svneo cells increased and Glut4 expression increased; when miR-22 was inhibited, glucose uptake of HTR8/SVneo cells decreased and GLUT4 expression decreased. When miR-22 was overexpressed or inhibited, the expressions of IRS, PI3K and AKT were significantly decreased. The dual luciferase reporter gene system assay showed that SLC2A4 gene (encoding GLUT4) was the regulatory target of miR-22.Conclusions When insulin tolerance occurs, the expression of miR-22 in placental villi tissues and cells is decreased, and the overexpression of miR-22 can improve the level of sugar uptake in vitro. miR-22 may participate in the pathogenesis of GDM by regulating GLUT4.
关键词
妊娠糖尿病 /
微小RNA-22 /
葡萄糖转运体4 /
双荧光素酶报告基因
Key words
gestational diabetes mellitus /
miR-22 /
GLUT4 /
dual luciferase reporter gene
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