MiR-204降调Caveolin-1表达降低HRGECs对大分子蛋白的通透性

miR-204 decrease the permeability of human glomerular endothelial cells to macromolecular protein through down-regulating of caveolin-1

目的:探讨miR-204对肾小球内皮细胞(HRGECs)通透性及Caveolin-1(CAV1)表达的影响。方法:利用生物信息学方法预测以CAV1为靶基因的miRNA,miR-204为CAV1表达的调节miRNA。分别用子痫前期(PE)患者血清和正常血清处理HRGECs,q PCR法检测miR-204表达水平。将体外培养肾小球内皮细胞(HRGECs)分为4组:过表达组(转染miR-204 mimic),降表达组(转染miR-204 inhibitor),转染miR-204mimic NC(mimic阴性对照组)和转染miR-204 inhibitor NC(inhibitor阴性对照组)。转染48h后,荧光定量PCR及Western blot法检测miR-204及CAV1 mRNA和蛋白表达水平,Evans-blue检测HRGECs单层细胞对大分子蛋白的通透性改变。结果:PE血清可抑制HRGEC中miR-204表达(P<0.05)。荧光定量PCR和Western blot结果显示,与阴性对照组比较,miR-204过表达组中CAV1 mRNA表达量无明显差异,CAV1蛋白表达明显降低,差异有统计学意义(P<0.05);miR-204降表达组中CAV1 mRNA表达量无明显差异,CAV1蛋白表达量明显升高,差异有统计学意义(P<0.05)。与阴性对照组比较,miR-204过表达组中HRGECs对大分子蛋白的通透性降低,miR-204降表达组中HRGECs对大分子蛋白的通透性提高,差异均有统计学意义(P<0.05)。结论:miR-204可能通过抑制CAV1转录后翻译从而调节其靶基因CAV1表达,进而影响肾小球内皮细胞通透性,miR-204可能参与PE患者肾小球内皮细胞尿蛋白形成的调节。

Objective：To explore the effects of microRNA-204 on caveolin-1 expression and permeability of human glomerular endothelial cells（HRGECs）.Methods：The putative miR-204 targeted on CAV1 were predicted by means of the bioinformatics methods.HRGECs were cultured in vitro and 2 groups were devided.Control group was treated with the serum of normal pregnant and PE group was treated with the preeclampsia patients serum.Then we transfected HRGECs with exogenous miRNAs.HRGECs were separately transfected with miR-204 mimics,miR-204 inhibitor,miR-204 mimic NC sequence and miR-204 inhibitor NC sequence and cultured for 48 hours.The expressions of miR-204 and CAV1 mRNA were detected by qPCR.Western blot was used to detect the quantity of caveolin-1 expression.The permeability of HRGECs was tested by Evans-blue conjuncted BSA in transwell chambers.Result：QPCR results showed that the expression of miR-204 displayed a marked decrease after treated with PE serum while increase after transfected with miR-204 mimics in HRGECs and in miR-204 down-regulated group,the expression of miR-204 was reduced （P〈0.05）.The caveolin-1 expression was significantly inhibited when treated with miR-204 mimics and was enhanced in miR-204 inhibited group （P〈0.05）.The expression of CAV1 mRNA had no noted differences in both group.MiR-204 inhibited the permeability of HRGECs more obvious in miR-204 overexpressed group than that in control groups（P〈0.05）.Conclusion：MiR-204 may regulate the expression of caveolin-1 and the permeability of HRGECs by inhibiting the process of translation of CAV1.MiR-204 may participate in proteinuria formation in preeclampsia patients.