目的：探讨miRNA-147靶向调控VEGF对人视网膜色素上皮细胞增殖和凋亡及迁移的影响，并初步研究其分子机制。方法：选择人视网膜色素上皮细胞株(ARPE-19细胞)，将细胞分为7组：空白对照组(不处理)、无义miRNA组(转染mimic NC)、miRNA-147模拟物组(转染miRNA-147 模拟物)、抑制剂阴性对照组(转染shRNA NC)、VEGF抑制剂组(转染VEGF抑制剂)、miRNA-147模拟物+空载病毒载体组(转染miRNA-147模拟物和空载体)和miRNA-147模拟物+VEGF过表达组(转染miRNA-147模拟物和VEGF过表达)。使用RT-qPCR检测各组细胞miRNA-147及VEGF mRNA表达水平； 双荧光素酶实验验证miRNA-147与VEGF靶向关系； Western blot检测VEGF蛋白表达水平； MTT法检测细胞增殖； 流式细胞术检测细胞凋亡水平及细胞周期的改变； 细胞划痕实验检测细胞迁移。结果：与空白对照组和无义miRNA组相比，miRNA-147模拟物组miRNA-147表达水平显著升高，VEGF mRNA及蛋白表达水平显著降低(P<0.05)； 与抑制剂阴性对照组相比，VEGF抑制剂组VEGF mRNA及蛋白表达水平显著降低(P<0.05)； 与miRNA-147模拟物+空载病毒载体组相比，miRNA-147模拟物+VEGF过表达组VEGF mRNA表达水平显著升高(P<0.05)。双荧光素酶报告显示VEGF是miRNA-147的靶基因。转染miRNA-147 模拟物和VEGF抑制剂均可降低ARPE-19细胞增殖和迁移水平，促进细胞凋亡(P<0.05)。转染VEGF过表达可逆转miRNA-147 mimic对ARPE-19细胞增殖、迁移和凋亡的影响(P<0.05)。结论：miRNA-147可通过靶向VEGF抑制ARPE-19细胞增殖和迁移，促进细胞凋亡。

AIM: To investigate the effect of miRNA-147 targeted regulation of vascular endothelial growth factor(VEGF)on the proliferation, apoptosis and migration of human retinal pigment epithelial cells, and to explore its molecular mechanism. METHODS: Human retinal pigment epithelial(ARPE-19)cells were selected and divided into 7 groups: blank control group(untreated), nonsense miRNA group(transfected with mimic NC), miRNA-147 simulant group(transfected with miRNA-147 mimic), inhibitor negative control group(transfected with shRNA NC), VEGF inhibitor group(transfected with shRNA VEGF), miRNA-147 simulant+empty viral vector group(transfected with miRNA-147 mimic and pcDNA3.1)and miRNA-147 simulant+VEGF overexpression group(transfected with miRNA-147 mimic and pcDNA3.1 VEGF). RT-qPCR was used to detect the expression of miRNA-147 and VEGF mRNA. Dual luciferase experiments were used to verify the targeting relationship between miRNA-147 and VEGF. Western blot was used to detect the expression of VEGF protein. MTT method was used to detect the proliferation. Flow cytometry to detect the apoptosis level and cell cycle changes. Cell scratch test to detect the level of cell migration. RESULTS: Compared with the blank control group and the nonsense miRNA group, the expression level of miRNA-147 in miRNA-147 simulant group was significantly increased, while the expression levels of VEGF mRNA and protein were significantly reduced(P<0.05). Compared with the inhibitor negative control group, the expression levels of VEGF mRNA and protein in the VEGF inhibitor group were significantly reduced(P<0.05). Compared with the miRNA-147 simulant+empty viral vector group, the expression level of VEGF mRNA in the miRNA-147 simulant+VEGF overexpression group was significantly increased(P<0.05). The dual luciferase report shows that VEGF is the target gene of miRNA-147. Transfection of miRNA-147 mimic and shRNA VEGF can reduce the proliferation and migration of ARPE-19 cells and promote apoptosis can reduce the proliferation and migration of ARPE-19 cells and promote apoptosis(P<0.05). Transfection VEGF overexpression reverses the effect of miRNA-147 mimics on proliferation, migration and apoptosis of ARPE-19 cells(P<0.05). CONCLUSION: miRNA-147 can inhibit ARPE-19 cell proliferation, migration and promote cell apoptosis by targeting VEGF.

遂宁市中心医院科研课题项目(No.2019y44)