miR-338-3p/TCF4对脉络膜微血管内皮细胞增生及凋亡的影响

Effects of miR-338-3p/TCF4 on choroidal microvascular endothelial cell proliferation and apoptosis

目的探讨微小RNA-338-3p(miR-338-3p)对脉络膜微血管内皮细胞增生和凋亡的影响及其调控机制。方法体外培养人脉络膜微血管内皮细胞,将培养的细胞分为血管内皮生长因子(VEGF)组和正常对照组,正常对照组细胞常规培养,VEGF组细胞应用VEGF处理24 h;将VEGF组培养的细胞分为4个亚组,分别将阴性对照(miR-NC)、miR-338-3p拟似物、miR-338-3p拟似物+pcDNA、miR-338-3p拟似物+pcDNA-TCF4转染至脉络膜微血管内皮细胞后用VEGF处理24 h。采用实时荧光定量PCR法(qRT-PCR)检测细胞中miR-338-3p和TCF4相对表达量;采用MTT法检测细胞增生率以评估细胞活力;流式细胞术检测细胞凋亡率;行双荧光素酶报告实验验证miR-338-3p与TCF4的靶向关系;采用Western blot法检测细胞中增生标记蛋白细胞增生核抗原-67(Ki-67)、增生细胞核抗原(PCNA)、B淋巴细胞瘤-2相关蛋白(bax)、B淋巴细胞瘤-2(bcl-2)蛋白相对表达量。结果与正常对照组比较,VEGF组细胞中miR-338-3p mRNA表达水平显著降低,TCF4 mRNA表达水平显著升高,细胞增生率显著升高,细胞凋亡率显著降低,细胞中Ki-67、PCNA、bcl-2蛋白表达水平显著升高,bax蛋白水平显著降低,差异均有统计学意义(均P<0.05);与VEGF+miR-NC组比较,VEGF+miR-338-3p组细胞增生率显著降低,细胞凋亡率显著升高,细胞中Ki-67、PCNA、bcl-2蛋白表达水平显著降低,bax蛋白表达水平显著升高,差异均有统计学意义(均P<0.05);双荧光素酶报告实验证实miR-338-3p可靶向结合TCF4;与VEGF+miR-338-3p+pcDNA组比较,VEGF+miR-338-3p+pcDNA-TCF4组细胞增生率显著升高[(56.48±13.20)%vs.(96.24±16.24)%],细胞凋亡率显著降低[(30.59±3.57)%vs.(12.36±1.29)%],细胞中Ki-67、PCNA、bcl-2蛋白表达水平显著升高(0.41±0.11 vs.0.96±0.19;0.44±0.10 vs.0.97±0.20;0.55±0.12 vs.0.98±0.15),bax蛋白表达水平显著降低(0.87±0.13 vs.0.42±0.11),差异均有统计学意义(t=5.700、14.408、7.516、7.111、6.715、7.927,均P<0.01)。结论miR-338-3p过表达可负向调控TCF4在细胞中的表达量,从而抑制脉络膜微血管内皮细胞的增生及诱导细胞凋亡。

Objective To investigate whether microRNA-338-3p(miR-338-3p)affects the proliferation and apoptosis of choroidal microvascular endothelial cells by regulating the expression of T cell factor 4(TCF4).Methods Human choroidal microvascular endothelial cells were cultured in vitro,and they were divided into vascular endothelial growth factor(VEGF)group and Normal control group.The Cultured cells in the VEGF group were divided into four subgroups,and were transfected with miR-NC,miR-338-3p mimics,miR-338-3p mimics+pcDNA,miR-338-3p mimics+pcDNA-TCF4 before VEGF treatment,respectively.Quantitative real-time PCR(qRT-PCR)was used to detect the expression of miR-338-3p and TCF4.MTT assay was used to detect cell proliferation.Flow cytometry was used to detect the apoptosis rate.The double luciferase report experiment verified the targeting relationship of miR-338-3p and TCF4.Western blot was used to detect the expression of Ki-67,PCNA,bax,and bcl-2.Results After VEGF treatment,the expression level of miR-338-3p was significantly reduced,the expression level of TCF4 mRNA was significantly increased,the cell viability was significantly increased,and the apoptosis rate was significantly decreased,the levels of Ki-67,PCNA,bcl-2 protein were increased significantly,and the level of bax protein was decreased significantly(all at P<0.05).After miR-338-3p overexpression,the cell viability was significantly reduced,the apoptosis rate was significantly increased,and the levels of Ki-67,PCNA,and bcl-2 proteins were significantly reduced,the level of bax protein was increased significantly(all at P<0.05).Double luciferase reporting experiments confirmed that miR-338-3p targeted TCF4.Compared with the VEGF+miR-338-3p+pcDNA group,the cell viability of the VEGF+miR-338-3p+pcDNA-TCF4 group was significantly increased([56.48±13.20]%vs.[96.24±16.24]%),and the apoptosis rate was significantly reduced([30.59±3.57]%vs.[12.36±1.29]%),the levels of Ki-67,PCNA and bcl-2 protein were significantly increased(0.41±0.11 vs.0.96±0.19;0.44±0.10 vs.0.97±0.20;0.55±0.12 vs.0.98±0.15),and the levels of bax protein were significantly decreased(0.87±0.13 vs.0.42±0.11)(t=5.700,14.408,7.516,7.111,6.715,7.927;all at P<0.01).Conclusions Overexpression of miR-338-3p can negatively regulate TCF4 expression,thereby inhibite choroidal microvascular endothelial cell proliferation and induce apoptosis.