TCF7L2基因RNA干扰对胰岛素原水平的影响及机制

Effects of TCF7L2 RNA interference on proinsulin levels and its potential mechanism

摘要:

目的：研究转录因子7类似物2（transcription factor 7 like 2，TCF7L2）表达下调对胰岛素原水平的影响，进一步探讨TCF7L2通过前蛋白转化酶（proprotein convertase，PC）基因影响胰岛素原水平的机制。方法：在体外培养Beta-TC-6细胞株，通过构建TCF7L2的RNA干扰慢病毒转染细胞，下调TCF7L2的表达，实验分为3组，分别为空白对照组、乱序对照组和TCF7L2干扰组，采用qRT-PCR和Western blot检测TCF7L2和PC基因mRNA及蛋白水平的变化；收集上清液，采用ELISA方法测定基础和葡萄糖刺激状态下胰岛素和胰岛素原水平的变化。结果：慢病毒感染Beta-TC-6细胞48 h后，在荧光显微镜下观察慢病毒感染效率达95%以上，与空白对照组比较，TCF7L2 mRNA的干扰率约为75%（0.25±0.03 vs. 0.97±0.11，P=0.000），蛋白质水平的干扰率约为50%（0.29±0.08 vs. 0.57±0.09，P=0.008）。与乱序对照组比较，TCF7L2干扰组葡萄糖刺激后胰岛素水平明显降低（7.47±0.21） mU/L vs. （10.53±0.38） mU/L（P=0.000），而胰岛素原水平却明显增加（10.73±0.51） pmol/L vs. （7.81±0.29） pmol/L（P=0.001），且PC1和PC2的mRNA和蛋白表达水平均明显降低（P<0.05）。结论：TCF7L2基因的RNA干扰可能通过降低PC基因的表达，进而减少该酶对胰岛素原的加工，从而影响胰岛素原的水平。

Abstract:

Objective：To investigate the effects of transcription factor 7 like 2（TCF7L2） down-regulation on proinsulin levels and to explore the mechanism of TCF7L2 regulating proinsulin levels though proprotein convertase（PC）. Methods：Beta-TC-6 cells were cultured in vitro. TCF7L2 expression was down regulated by siRNA. There were three groups：blank control，scrambled control，and TCF7L2 interference group. QRT-PCR and Western blot were used to detect the mRNA and protein levels of TCF7L2，PC1 and PC2. Cell culture medium was collected and changes in insulin and proinsulin levels under basic and glucose stimulated conditions were detected by ELISA. Results：At 48 h after lentivirus infection of Beta-TC-6 cells，the infection rate achieved more than 95% under fluorescence microscopy. Compared with those of blank control group，the interference efficiency of mRNA was about 75%（0.25±0.03 vs. 0.97±0.11，P=0.000） and the interference efficiency of protein level was 50% in TCF7L2 interference group（0.29±0.08 vs. 0.57±0.09，P=0.008）. Compared with those of scrambled control，the TCF7L2 interference group had significantly lower levels of in－sulin（7.47±0.21） mU/L vs. （10.53±0.38） mU/L（P=0.000），but higher levels of proinsulin（10.73±0.51） pmol/L vs. （7.81±0.29） pmol/L（P=0.001） and lower mRNA and protein levels of PC1 and PC2（P<0.05）. Conclusions：TCF7L2 interference might affect the process and levels of proinsulin by decreasing the expression of PC.