AIM:To observe the expression of Snail1 and insulin-like growth factor-1 (IGF-1) in NRK-52E cells induced by high glucose,and to investigate the relationship of Snail1 and IGF-1 in the mechanism of epithelial to mesenchymal transition (EMT) in diabetic kidney disease (DKD).METHODS:The NRK-52E cells were treated with Snail1 siRNA and IGF-1 siRNA after cultured with high glucose medium for 72 h,and divided into control group,high glucose group,non-targeting (NT) siRNA group,Snail1 RNAi group and IGF-1 RNAi group.The cells were harvested at 48 h and 72 h.Real-time PCR was used to detect the mRNA expression of Snail1,IGF-1,E-cadherin and fibronectin (FN),and the protein levels were determined by immunofluorescence staining.RESULTS:Compared with control group,the expression of E-cadherin at mRNA and protein levels declined after stimulation with high glucose (P ＜ 0.01),while that of FN was elevated (P ＜0.01).Meanwhile,the mRNA and protein levels of Snail1 and IGF-1 were markedly increased (P ＜0.01).The expression of E-cadherin at mRNA and protein levels was improved in Snail1 RNAi group as compared with high glucose group (P ＜ 0.01),while that of FN,IGF-l and Snail1 was significantly down-regulated (P ＜ 0.01).The same changes were observed in IGF-1 RNAi group (P ＜0.01).The protein expression of each factor in NT group had no significant change as compared with high glucose group (P ＞ 0.05).Pearson correlation analysis showed a close positive relationship between the expression of Snail1 and IGF-1 protein (r =0.852,P ＜ 0.01).CONCLUSION:Snail1 may facilitate DKD development by regulating IGF-1 in the process of EMT.%目的:观察高糖诱导大鼠肾小管上皮细胞NRK-52E中锌指转录因子Snail1和胰岛素样生长因子1(insulin-like growth factor-1,IGF-1)的表达变化,并初步探讨Snail1与IGF-1在糖尿病肾脏病(diabetic kidney disease,DKD)上皮-间充质转化(epithelial to mesenchymal transition,EMT)过程中的关系.方法:高糖培养大鼠近端肾小管上皮细胞系NRK-52E 72 h后,给予Snail1 siRNA和IGF-1 siRNA处理,分为高糖组、non-targeting (NT) siRNA组、Snail1 RNAi组和IGF-1 RNAi组,并设置对照组.于转染后48和72 h两个时点收获细胞.分别用实时荧光定量PCR检测细胞Snail1、IGF-1、E-钙黏蛋白(E-cadherin)和纤维连接蛋白(fibronectin,FN)的mRNA表达,用免疫荧光方法检测各蛋白的表达.结果:高糖诱导NRK-52E细胞E-cadherin的mRNA和蛋白表达明显降低(P＜0.01),FN的mRNA和蛋白表达明显升高(P＜0.01);同时,Snail1和IGF-1的mRNA和蛋白表达也明显升高(P＜0.o1).Snail1 RNAi组与高糖组比较,细胞中E-cadherin的mRNA和蛋白表达明显升高(P＜0.01),FN、Snail1和IGF-1的mRNA和蛋白表达明显降低(P ＜0.01),Snail1的mRNA表达减少62.8％.与高糖组比较,IGF-1 RNAi组细胞IGF-1的mRNA表达减少61.1％,E-cadherin的mRNA和蛋白表达明显升高(P＜0.01),FN的mRNA和蛋白表达明显降低(P＜0.01).NT组E-cadherin、FN、Snail1及IGF-1的mRNA和蛋白表达与高糖组比较差异无统计学显著性.Pearson相关性分析显示,NRK-52E细胞中Snail1与IGF-1蛋白的表达呈显著正相关(r=0.852,P＜0.01).结论:Snail1及IGF-1的mRNA和蛋白在高糖诱导的肾小管上皮细胞EMT过程中表达升高,且沉默Snail1基因,IGF-1表达随之减少,提示Snail1/IGF-1可能促进DKD时肾小管上皮细胞EMT.
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