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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 investigate the effect of miR-204 on the permeability of glial endothelial cells (HRGECs) and the expression of Caveolin-1 (CAV1). Methods: Bioinformatics methods were used to predict the miRNAs that target CAV1 and miR-204 was the regulator of CAV1 expression. Respectively with preeclampsia (PE) serum and normal serum HRGECs treatment, q PCR method to detect miR-204 expression levels. The cultured glioma endothelial cells (HRGECs) were divided into 4 groups: overexpression group (transfected with miR-204 mimic), decreased expression group (transfected with miR-204 inhibitor), transfected with miR-204mimic NC (mimic negative control Group) and transfected with miR-204 inhibitor NC (inhibitor negative control group). Forty-eight hours after transfection, the mRNA and protein levels of miR-204 and CAV1 were detected by real-time PCR and Western blot. The permeability of monolayers of HRGECs to macromolecular proteins was detected by Evans-blue. Results: PE serum inhibited the expression of miR-204 in HRGEC (P <0.05). Fluorescent quantitative PCR and Western blot results showed that compared with the negative control group, the expression of CAV1 mRNA in miR-204 overexpression group was not significantly different, but the expression of CAV1 protein was significantly decreased (P <0.05); miR-204 There was no significant difference in the expression of CAV1 mRNA between the two groups. The expression of CAV1 protein was significantly increased (P <0.05). Compared with the negative control group, the permeability of HRGECs to macromolecular proteins in miR-204 overexpression group was decreased, and the permeability of HRGECs to macromolecular proteins in miR-204 down-regulated group was increased, the differences were statistically significant (P <0.05). CONCLUSION: miR-204 may regulate the expression of CAV1, a target gene of CAV1, and thus affect the permeability of glomerular endothelial cells. MiR-204 may be involved in the regulation of urinary protein formation in glomerular endothelial cells of PE patients.