miR-223靶向NLRP3影响高糖诱导的肾小管上皮细胞EMT发生的机制

目的:探讨miR-223对高糖诱导的肾小管上皮(renal tubule epithelia,NRK-52E)细胞上皮间质转化(epithelial-mesenchymal transition,EMT)的影响及其机制。方法:将体外培养的NRK-52E细胞分为对照组(5 mmol·L^-1 D-葡萄糖)、高糖组(30 mmol·L^-1 D-葡萄糖)、高糖+模拟物对照组(转染miR-223模拟物对照后,30 mmol·L^-1 D-葡萄糖处理)和高糖+模拟物组(转染miR-223模拟物后,30 mmol·L^-1 D-葡萄糖处理),采用RT-PCR检测各组细胞中miR-223、NLRP3、IL-1β和IL-18 mRNA的表达,Western blot检测NLRP3蛋白和EMT相关蛋白α-SMA、E-cadherin的表达,双荧光素酶报告基因实验检测miR-223和NLRP3的靶向关系。另外,构建NLRP3过表达的NRK-52E细胞株,观察NLRP3过表达对miR-223过表达的NRK-52E细胞EMT进展的影响。结果:与对照组比较,高糖刺激后可引起miR-223和E-cadherin蛋白的表达降低,而NLRP3、IL-1β和IL-18 mRNA以及NLRP3、α-SMA蛋白表达升高(P<0.05)。与高糖组比较,转染miR-223模拟物后高糖环境下NRK-52E细胞中miR-223和E-cadherin蛋白的表达水平显著升高,NLRP3、IL-1β和IL-18 mRNA以及NLRP3、α-SMA蛋白的表达水平显著降低(P<0.05);但转染miR-223模拟物阴性对照后对高糖环境下的NRK-52E细胞无显著影响(P>0.05)。双荧光素酶报告基因实验证实NLRP3是miR-223的靶基因。转染pcDNA3.1-NLRP3过表达质粒成功上调NLRP3表达后,miR-223过表达对EMT进程抑制作用得以部分逆转。结论:miR-223可通过靶向NLRP3抑制高糖诱导的NRK-52E细胞EMT的发生。

The Effect of Mir-223 Targeting NLRP3 on EMT of Renal Tubular Epithelial Cells Induced by High Glucose

Objective:To investigate the effect and mechanism of mir-223 on the epithelial-mesenchymal transition(EMT)of renal tubular epithelial(NRK-52E)cells induced by high glucose.Method:NRK-52E cells were divided into control group(5 mmol·L^-1 D-glucose),high glucose group(30 mmol·L^-1 D-glucose),high glucose+simulant control group(30 mmol·L^-1 D-glucose after mir-223 simulant control)and high glucose+simulant group(30 mmol·L^-1 D-glucose after mir-223 simulant control).The expression of mir-223,NLRP3,IL-1βand IL-18 mRNA,the expression of NLRP3 protein,EMT related proteinα-SMA and E-cadherin were detected by RT-PCR,and the targeting relationship between mir-223 and NLRP3 was detected by double luciferase reporter gene assay.In addition,NRK-52E cell lines overexpressed with NLRP3 were constructed to observe the effect of NLRP3 overexpression on EMT progression of mir-223 overexpressed NRK-52E cells.Results:Compared with the control group,the ex-pression of mir-223 and E-cadherin protein decreased,but the expression of NLRP3,IL-1βand IL-18 mRNA and NLRP3,α-SMA protein increased(P<0.05).Compared with the high glucose group,the expression level of mir-223 and E-cadherin protein in NRK-52E cells was significantly increased,and the expression levels of NLRP3,IL-1βand IL-18 mRNA,NLRP3 andα-SMA pro-tein were significantly decreased(P<0.05);however,the negative control of mir-223 had no significant effect on NRK-52E cells in high glucose environment(P>0.05).Double luciferase reporter gene experiment confirmed that NLRP3 was the target gene of mir-223.After transfection of pcDNA3.1-NLRP3,the inhibition of mir-223 on EMT process was partially reversed.Conclusion:Mir-223 can inhibit EMT of NRK-52E cells induced by high glucose by targeting NLRP3.