下调miR-25对高糖诱导视网膜色素上皮细胞系ARPE-19细胞Smad7和VEGF表达的影响

Effect of down-regulation of miR-25 on expressions of Smad7 and VEGF in high glucose induced retinal pigment epithelial cell line ARPE-19 cells

目的研究miR-25在高糖条件下人视网膜色素上皮细胞系ARPE-19细胞中表达情况,及下调miR-25对ARPE-19细胞中Smad7和血管内皮生长因子(VEGF)表达的影响。方法体外培养ARPE-19细胞,采用葡萄糖(30 mmol/L)处理细胞模拟高糖状态,实时荧光定量PCR(qRT-PCR)检测细胞中miR-25表达水平;采用Lipofectamine TM3000试剂盒进行转染,将ARPE-19细胞分为miR-25低表达(antagomir)组、阴性对照(NC)组、高糖(HG)组及正常葡萄糖(NG)组。转染后48 h,光学显微镜观察细胞形态,CKK-8法检测细胞增殖情况,实时荧光定量PCR(qRT-PCR)检测ARPE-19细胞E-钙黏蛋白(E-cad)、α-平滑肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(COL-Ⅰ)mRNA表达;蛋白免疫印记(WB)检测E-cad、α-SMA、COL-Ⅰ、VEGF、Smad7蛋白表达情况。结果与NG组比较,HG组ARPE-1细胞miR-25表达显著升高,差异有统计学意义(P<0.05);转染miR-25 antagomir后,与HG组和NC组比较,ARPE-19细胞miR-25表达显著下调(P<0.05)。与NG组比较,HG组、NC组ARPE-19细胞变长呈纺锤形细胞,细胞中α-SMA、COL-Ⅰ、VEGF表达上调(P<0.05),E-cad、Smad7表达下调(P<0.05);与HG组、NC组比较,antagomir组纺锤形ARPE-19细胞数量减少,细胞中α-SMA、COL-Ⅰ、VEGF表达下调(P<0.05),E-cad、Smad7表达上调(P<0.05)。结论高糖条件下,下调miR-25可抑制VEGF表达,促进Smad7表达,抑制人视网膜色素上皮细胞纤维化。

Objective To investigate the expression of miR-25 in human retinal pigment epithelial cell line ARPE-19 cells under the condition of high glucose and the effects of down-regulation of miR-25 on the expressions of Smad7 and VEGF in ARPE-19 cells. Methods ARPE-19 cells were cultured in vitro,glucose（ 30 mmol/L） was used to simulate the condition of high glucose. Real-time fluorescence quantitative PCR（ qRT-PCR） was used to detect the expression level of miR-25 in cells. Transfect the ARPE-19 cells with Lipofectamine TM 3000 Kit,and then they were divided into miR-25 low expression（ antagomir） group,negative control（ NC） group,high glucose（ HG） group and normal glucose（ NG） group. After 48 h of transfection,the morphology of cells was observed by optical microscopy; CKK-8 assay was used to detect cell proliferation; real-time fluorescence quantitative PCR（ qRT-PCR） was used to detect the expressions of E-cadherin（ E-cad）,α-smooth muscle actin（ α-SMA）,and type I collagen（ COL-I） mRNA in ARPE-19 cells. Western blot（ WB） was used to detect the expressions of E-cad,α-SMA,COL-I,VEGF and Smad7 proteins. Results Compared with NG group,the expression of ARPE-19 cells miR-25 in HG group was increased significantly,the difference was statistically significant（ P〈0. 05）. After transfection with miR-25 antagomir,the expression of ARPE-19 cells miR-25 was significantly lower than that in HG group and NC group（ P〈0. 05）. Compared with the NG group,the ARPE-19 cells in HG group and NC group grew to spindle shaped cells,and the expressions of α-SMA,COL-I and VEGF in the cells were up-regulated（ P〈0. 05）,and the expressions of E-cad and Smad7 were down-regulated（ P〈0. 05）. Compared with HG group and NC group,the number of spindle ARPE-19 cells in group antagomir was decreased,the expressions of α-SMA,COL-I and VEGF in cells were down-regulated（ P〈0. 05）. The expressions of E-cad and Smad7 were up-regulated（ P〈0. 05）. Conclusion Under the condition of high glucose,down-regulation of miR-25 can inhibit the expression of VEGF,promote the Smad7 expression,and inhibit human retinal pigment epithelial cell fibrosis.