miR-30c靶向Wnt/β-catenin信号对高糖诱导的人视网膜血管内皮细胞增殖、凋亡的影响

Effect of miR-30c targeting Wnt/β-catenin signal on the proliferation and apoptosis of human retinal endothelial cells induced by high glucose

目的探讨miR-30c靶向Wnt/β-catenin信号对高糖诱导的人视网膜血管内皮细胞(human retinal endothelial cells,HRECs)增殖、凋亡的影响。方法培养HRECs细胞,分别给予正常浓度(对照组)和高浓度葡萄糖(高糖组)。分别转染miR-30c模拟物、阴性对照(miR-NC)、Wnt1过表达载体(pcDNA3.1-Wnt1)和空载体(pcDNA3.1)。RT-PCR法检测各组细胞miR-30c的表达水平。Western blot法检测各组细胞Wnt1、β-catenin和GSK-3β蛋白的表达水平。双荧光素酶实验验证miR-30c对Wnt1的靶向关系。噻唑蓝法检测各组细胞的增殖活性。流式细胞术检测各组细胞的凋亡水平。结果与对照组比较,高糖组细胞中miR-30c的表达明显降低[(0.94±0.11)vs(0.32±0.06),P<0.001],细胞增殖活性显著降低,细胞凋亡率显著增加[(0.75±0.08)vs(0.13±0.04),(3.53±0.29)%vs(14.89±0.94)%,P<0.001];与高糖+miR-NC组比较,高糖+miR-30c组细胞增殖活性显著升高,细胞凋亡率降低[(0.14±0.04)vs(0.64±0.06),(14.14±0.86)%vs(6.28±0.45)%,P<0.001]。与miR-NC组比较,miR-30c组共转染WT-Wnt1后的荧光素酶活性显著降低[(0.97±0.09)vs(0.26±0.03),P<0.001]。与对照组比较,高糖组细胞Wnt1、β-catenin和GSK-3β蛋白表达均显著升高[(0.43±0.05)vs(1.02±0.09),(0.25±0.04)vs(0.82±0.10),(0.39±0.04)vs(0.76±0.11),P<0.001];与高糖+miR-NC组比较,高糖+miR-30c组细胞Wnt1、β-catenin和GSK-3β蛋白表达均显著降低[(1.04±0.10)vs(0.68±0.06),(0.79±0.09)vs(0.34±0.05),(0.74±0.12)vs(0.48±0.06),P<0.001]。与高糖+miR-30c组比较,高糖+miR-30c+pcDNA3.1-Wnt1组细胞增殖活性显著降低,细胞凋亡率明显升高[(0.66±0.07)vs(0.31±0.05),(4.26±0.57)%vs(9.75±0.85)%,P<0.001]。结论miR-30c可能通过负调控Wnt/β-catenin信号通路,促进细胞增殖,抑制高糖引起的HRECs细胞凋亡。

Objective To investigate the protective effect and mechanism of miR-30c targeting Wnt/β-catenin signal on the proliferation and apoptosis of human retinal endothelial cells(HRECs)induced by high glucose.Methods Human retinal endothelial cells(HRECs)were cultured and given normal concentration(control group)and high concentration glucose(high glucose group)respectively.According to the experimental design,miR-30c mimic,negative control(miR-NC),Wnt1 overexpression vector(pcDNA3.1-Wnt1)and empty vector(pcDNA3.1)were transfected respectively.RT-PCR was used to detect the expression level of miR-30c in each group.Western blot was used to detect the expression levels of Wnt1,β-catenin and GSK-3βprotein in each group.The dual luciferase experiment verified the targeting relationship of miR-30c to Wnt1.Thiazole blue method was used to detect the proliferation activity of each group.Flow cytometry was employed to detect the level of apoptosis of each group of cells.Results Compared with the control group,the expression of miR-30c in the high glucose group was significantly reduced[(0.94±0.11)vs(0.32±0.06),P<0.001];compared with the control group,the cell proliferation activity of the high glucose group was significantly reduced,and the apoptosis rate was significantly increased[(0.75±0.08)vs(0.13±0.04),(3.53±0.29)%vs(14.89±0.94)%,P<0.001];compared with the high glucose+miR-NC group,the cell proliferation activity of the high glucose+miR-30c group was significantly increased,and the apoptosis rate was reduced[(0.14±0.04)vs(0.64±0.06),(14.14±0.86)%vs(6.28±0.45)%,P<0.001];compared with the miR-NC group,the luciferase activity of the miR-30c group co-transfected with WT-Wnt1 was significantly reduced[(0.97±0.09)vs(0.26±0.03),P<0.001];compared with the control group,the protein expression of Wnt1,β-catenin and GSK-3βin the high glucose group were significantly increased[(0.43±0.05)vs(1.02±0.09),(0.25±0.04)vs(0.82±0.10),(0.39±0.04)vs(0.76±0.11),P<0.001];compared with the high glucose+miR-NC group,the protein expression of Wnt1,β-catenin and GSK-3βin the high glucose+miR-30c group were significantly reduced[(1.04±0.10)vs(0.68±0.06),(0.79±0.09)vs(0.34±0.05),(0.74±0.12)vs(0.48±0.06),P<0.001];compared with the high glucose+miR-30c group,the cell proliferation activity was significantly reduced in the high glucose+miR-30c+pcDNA3.1-Wnt1 group,and the apoptosis rate was significantly increased[(0.66±0.07)vs(0.31±0.05),(4.26±0.57)%vs(9.75±0.85)%,P<0.001].Conclusion miR-30c may negatively regulate the Wnt/β-catenin signaling pathway,promote cell proliferation,and inhibit cell apoptosis induced by high glucose.