miR-204靶向SIRT1基因调控高糖作用下视网膜色素上皮细胞增殖的分子机制

Molecular mechanism of miR-204 targeting SIRT1 gene regulating proliferation of retinal pigment epithelial cells under high glucose in vitro

目的探讨miR-204通过靶向沉默信息调节因子1(SIRT1)对高糖作用下视网膜上皮细胞ARPE-19细胞增殖的调控作用及分子机制。方法以30 mmol/L葡萄糖体外处理ARPE-19细胞,采用脂质体转染法将miR-204 inhibitor和NC-inhibitor转染至ARPE-19细胞,将ARPE-19细胞分为正常葡萄糖组(NG组)、高糖组(HG组)、阴性对照组(anti-NC组)和miR-204转染组(anti-miR-204组)。采用实时荧光定量PCR(qRT-PCR)检测ARPE-19细胞中miR-204的表达水平,蛋白免疫印迹(Western blot)法检测细胞中ARPE-19细胞中增殖细胞核抗原(PCNA)和SIRT1蛋白表达,噻唑蓝(MTT)法检测细胞增殖能力。通过TargetScan等数据库预测miR-204与SIRT1互补结合位点,采用双荧光素酶报告基因实验验证miR-204和SIRT1的靶向关系。结果与NG组比较,HG组ARPE-19细胞中miR-204的表达水平明显升高(P<0.05),PCNA和SIRT1蛋白表达明显下调(P<0.05),细胞增殖能力明显降低(P<0.05)。与HG组和anti-NC比较,anti-miR-204组ARPE-19细胞中miR-204的表达水平明显降低(P<0.05),PCNA和SIRT1蛋白表达明显上调(P<0.05),细胞增殖能力明显升高(P<0.05)。生物信息学预测结果显示miR-204与SIRT1存在互补结合位点。双荧光素酶报告基因实验检测结果显示miR-204和SIRT1能够靶向结合。结论高糖作用下视网膜上皮细胞中miR-204的表达上调,抑制miR-204的表达可靶向SIRT1逆转高糖诱导的ARPE-19细胞增殖抑制。

Objective To investigate the regulatory effects and molecular mechanism of miR-204 on the proliferation of ARPE-19 cells in retinal epithelial cells induced by high glucose by targeting silencing regulator 1(SIRT1).Methods ARPE-19 cells were treated with 30mmol/L glucose in vitro.The miR-204 inhibitor and NC-inhibitor were transfected into ARPE-19 cells by lipofection.The ARPE-19 cells were divided into normal glucose group(NG group),high glucose group(HG group),negative control group(anti-NC group)and miR-204 transfection group(anti-miR-204 group).Real-time quantitative PCR(qRT-PCR)was used to detect the expression of miR-204 in ARPE-19 cells.The expression levels of proliferating cell nuclear antigen(PCNA)and SIRT1 protein in ARPE-19 cells were detected by Western blot.The cell proliferation ability was measured by the thiazolyl blue(MTT)method.The complementary binding sites of miR-204 and SIRT1 were identified by a database such as TargetScan.Moreover the dual luciferase reporter gene assay was used to verify the targeting relationship between miR-204 and SIRT1.Results As compared with those in NG group,the expression levels of miR-204 in ARPE-19 cells of HG group were significantly increased(P<0.05),however,the expression levels of PCNA and SIRT1 protein were down-regulated(P<0.05),and the cell proliferation ability was significantly decreased(P<0.05).As compared with those in HG group and anti-NC,the expression levels of miR-204 were significantly decreased in anti-miR-204 ARPE-19 cells(P<0.05),and the expression levels of PCNA and SIRT1 protein were significantly up-regulated(P<0.05).Bioinformatics prediction results showed that miR-204 had a complementary binding site with SIRT1.The dual luciferase reporter assay showed that miR-204 and SIRT1 were able to target binding.Conclusion The expression levels of miR-204 are up-regulated in retinal epithelial cells under high glucose.The inhibition of miR-204 expression can target SIRT1 to reverse the inhibition of high glucose-induced ARPE-19 cell proliferation.