IFN-α调控miR-214-5p/MFGE8通路对人脑血管外膜成纤维细胞增殖、凋亡的影响

Effect of IFN-αon Proliferation and Apoptosis of Human Cerebral Vascular Adventitial Fibroblasts by Regulating miR-214-5p/MFGE8 pathway

目的探讨干扰素α(IFN-α)调控miR-214-5p/乳脂肪球表皮生长因子8(MFGE8)通路对人脑血管外膜成纤维细胞(HBVAFs)增殖、凋亡的影响。方法将HBVAFs细胞分为NC组、IFN-α组、IFN-α+anti-miR-con组、IFN-α+anti-miR-214-5p组、IFN-α+pcDNA组、IFN-α+pcDNA-MFGE8组、IFN-α+anti-miR-214-5p+si-con组和IFN-α+anti-miR-214-5p+si-MFGE8组。实时荧光定量PCR(qRT-PCR)检测miR-214-5p和MFGE8 mRNA的表达;四甲基偶氮唑蓝(MTT)法检测细胞活力;流式细胞术检测细胞凋亡;蛋白质印记(Western blot)法检测MFGE8、活化的半胱氨酸天冬氨酸蛋白酶3(Cleaved caspase-3)、B细胞淋巴瘤/白血病-2(Bcl-2)和Bcl-2相关X蛋白(Bax)的表达水平。双荧光素酶报告基因实验和Western blot法验证miR-214-5p和MFGE8的靶向调控关系。结果与NC组比较,IFN-α组HBVAFs细胞miR-214-5p的表达显著升高,MFGE8和Bcl-2的表达显著降低,Cleaved caspase-3和Bax的表达显著升高,细胞活力显著降低,细胞凋亡率显著升高(P<0.05)。与IFN-α+anti-miR-con组比较,IFN-α+anti-miR-214-5p组HBVAFs细胞miR-214-5p、Cleaved caspase-3和Bax的表达显著降低,Bcl-2表达显著升高,细胞活力显著降低,细胞凋亡率显著升高(P<0.05)。miR-214-5p靶向负性调控MFGE8表达。与IFN-α+pcDNA组比较,IFN-α+pcDNA-MFGE8组HBVAFs细胞Cleaved caspase-3和Bax的表达显著降低,MFGE8和Bcl-2表达显著升高,细胞活力显著降低,细胞凋亡率显著升高(P<0.05)。与IFN-α+anti-miR-214-5p+si-con组比较,IFN-α+anti-miR-214-5p+si-MFGE8组HBVAFs细胞Cleaved caspase-3和Bax的表达显著升高,MFGE8和Bcl-2表达显著降低,细胞活力显著升高,细胞凋亡率显著降低(P<0.05)。结论IFN-α通过调控miR-214-5p/MFGE8通路抑制人脑血管外膜成纤维细胞增殖并促进细胞凋亡。

Objective To investigate the effect of interferonα(IFN-α)on proliferation and apoptosis of human cerebral vascular adventitial fibroblasts(HBVAFs)by regulating miR-214-5p/milk fat globule epidermal growth factor 8(MFGE8)pathway.Methods HBVAFs cells were divided into NC group,IFN-αgroup,IFN-α+anti-miR-con group,IFN-α+anti-miR-214-5p group,IFN-α+pcDNA group,IFN-α+pcDNA-MFGE8 group,IFN-α+anti-miR-214-5p+si-con group and IFN-α+anti-miR-214-5p+si-MFGE8 group.Real-time quantitative PCR(qRT-PCR)was used to detect the expression of miR-214-5p and MFGE8 mRNA.Cell viability was detected by MTT assay.Apoptosis was detected by flow cytometry.Expression levels of MFGE8,Cleaved caspase-3,Bcl-2 and Bax proteins were detected by Western blot.The dual luciferase reporter gene assay and Western blot were used to verify the targeted and regulatory relationship between miR-214-5p and MFGE8.Results Compared with NC group,the expression of miR-214-5p was significantly increased in IFN-αgroup,the expression of MFGE8 and Bcl-2 was significantly decreased,the expression of Cleaved caspase-3 and Bax was significantly increased,cell viability was significantly decreased,and the apoptotic rate was significantly increased(P<0.05).Compared with IFN-α+anti-miR-con group,the expression of miR-214-5p,Cleaved caspase-3 and Bax in HBVAFs cells of IFN-α+anti-miR-214-5p group was significantly decreased,and the expression of Bcl-2 was significantly increased,cell viability was significantly decreased,and apoptosis rate was significantly increased(P<0.05).miR-214-5p targets negatively regulated the expression of MFGE8.Compared with IFN-α+pcDNA group,the expression of Cleaved caspase-3 and Bax in HBVAFs cells of IFN-α+pcDNA-MFGE8 group was significantly decreased,the expression of MFGE8 and Bcl-2 was significantly increased,the cell viability was significantly decreased,and the apoptosis rate was significant increased(P<0.05).Compared with IFN-α+anti-miR-214-5p+si-con group,the expression of Cleaved caspase-3 and Bax in HBVAFs cells of IFN-α+anti-miR-214-5p+si-MFGE8 group was significantly increased,the expression of MFGE8 and Bcl-2 was significantly decreased,cell viability was significantly increased,and apoptosis rate was significantly decreased(P<0.05).Conclusion IFN-αinhibits proliferation and promotes apoptosis of human cerebral vascular adventitial fibroblasts by regulating the miR-214-5p/MFGE8 pathway.