沉默TCF7L2对胰岛素抵抗HepG2细胞胰岛素降解酶表达的调控作用观察

Regulation effect of TCF7L2 gene silence on the expression of insulin degrading enzyme in insulin resistant HepG2 cells

目的探讨沉默转录因子7类似物2(TCF7L2)对胰岛素抵抗(IR)Hep G2细胞胰岛素降解酶(IDE)表达的调控作用及可能机制。方法将Hep G2细胞分为空白组、TCF7L2干扰组、空载体组、IR组、IR+TCF7L2干扰组、IR+空载体组。采用高浓度胰岛素(5×10-6mol/L)持续作用24h诱导IR模型(IR-Hep G2细胞)生成。以人TCF7L2 m RNA编码序列为干扰靶点构建TCF7L2特异性小干扰RNA慢病毒载体(LV-TCF7L2-si RNA)转染空白组及IR组细胞,空载体病毒转染空载体组及IR+空载体组细胞。q RT-PCR法检测各组细胞TCF7L2及IDE m RNA的表达,Western blotting检测各组细胞TCF7L2、IDE、胰岛素刺激后蛋白激酶B(AKT)、磷酸化蛋白激酶B(p-AKT)蛋白表达的变化,流式细胞术检测各组2-脱氧-D-葡萄糖(2-NBDG)荧光葡萄糖摄取率。结果与空白组比较,IR组细胞葡萄糖消耗量及2-NBDG摄取率均明显降低(P<0.01),证明IR细胞模型建立成功。q RT-PCR及Western blotting结果显示,IR组TCF7L2及IDE m RNA种蛋白表达水平均明显低于空白组(P<0.05),TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较空白组、空载体组明显下降,IR+TCF7L2干扰组TCF7L2、IDE m RNA和蛋白表达水平较IR组、IR+空载体组均明显下降(P<0.05)。生理剂量胰岛素刺激后,IR组、IR+TCF7L2干扰组p-AKT蛋白水平较空白组明显下降(P<0.01),各组总AKT水平差异无统计学意义。TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较空白组和空载体组明显下降,IR+TCF7L2干扰组2-NBDG荧光葡萄糖摄取率较IR组、IR+空载体组明显下降(P<0.01)。结论 TCF7L2联合IDE致肝细胞IR,其机制可能与减少胰岛素信号通路关键酶p-AKT蛋白的表达有关。

Objective To evaluate the effects of transcription factor 7-like 2 （TCF7L2） silence on the expression of insulin degrading enzyme （IDE） in insulin resistance （IR） model HepG2 cells and its possible mechanism. Methods The HepG2 cells were divided into blank group, TCF7L2 interference group, empty vector group, IR group, IR＋TCF7L2 interference group and IR＋empty vector group. IR-HepG2 cell model was induced by in vitro cultivation of the cells in high concentration of insulin （5 × 10.6 mol/L） for 24 hours; GOD-POD and 2-NBDG method was used to verify successful reproduction of IR-cell model. TCF7L2 specific siRNA lentivirus vector （LV-TCF7L2-siRNA） was constructed with TCF7L2 mRNA coding sequence as the interference target, and it was used to transfect the cells in blank group and IR group. Empty vector virus was used to transfect the cells in empty vector group and IR＋empty vector group. The expressions of TCF7L2 and IDE mRNA were detected by qRT-PCR, and the changes in the expression of TCFTL2, IDE, insulin stimulated protein kinase B（AKT） and phosphorylated protein kinase B（p-AKT） were detected by Western blotting. The uptake rate of 2-deoxy-D-glucose （2-NBDG） was analyzed by flow cytometry. Results Compared with that in control group, the glucose consumption and the uptake rate of 2-NBDG significantly decreased in IR group （P〈0.01）, proving that the IR cell model had been reproduced successfully. Western blotting and qRT-PCR revealed that the expression levels of TCF7L2 and IDE mRNA and protein were obviously decreased in IR group compared with that in blank group （P〈0.05）, in TCF7L2 interference group than in blank group and empty vector group, and in IR＋TCF7L2 interference group than in blank group and IR＋empty vector group （P〈0.05）. After physiological insulin stimulation, the expression levels of p-AKT protein decreased more significantly in IR group and IR＋TCF7L2 interference group than in blank group （P〈0.01）, while no statistically significant difference in the total AKT protein level was found among all the groups. 2-NBDG uptake rate was significantly decreased in TCF7L2 interference group as compared with that in blank group and empty vector group, and also in IR＋TCF7L2 interference group than in IR group and IR＋empty vector group, respectively P〈0.01. Conclusion The mechanism of IR induced by the interaction of TCF7L2 and IDE might be related to the decreased expression of the insulin signaling pathway key enzyme p-AKT protein.