miR-33a-5p靶向SMAD7对血管平滑肌细胞增殖、凋亡、迁移及细胞外基质降解的影响

Effect of miR-33a-5p on proliferation,apoptosis,migration and degradation of extracellular matrix of vascular smooth muscle cells by targeting SMAD7

目的:探究miR-33a-5p靶向SMAD7对血管平滑肌细胞增殖、凋亡、迁移及细胞外基质降解的影响。方法:生物信息学预测靶向关系,荧光素实验进一步验证靶向关系,RT-PCR检测miR-33a-5p和SMAD7的表达,CCK-8法检测VSMC的增殖,流式检测细胞凋亡,划痕实验检测细胞迁移,Western blot检测Ki67、PCNA、Caspase-3、Caspase-9、MMP-2和MMP-9蛋白表达水平。结果:PDGF-bb处理能够增强血管平滑肌细胞增殖,抑制miR-33的表达;miR-33a-5p与SMAD7在3′UTR区存在结合位点,miR-33a-5p直接靶向作用于SMAD7,且miR-33a-5p过表达抑制SMAD7的表达;miR-33a-5p靶向SMAD7抑制PDGF-bb诱导的血管平滑肌细胞增殖、下调增殖标记蛋白PCNA和Ki67表达,促进细胞凋亡、上调凋亡标记蛋白Caspase-3和Caspase-9的表达,降低伤口愈合率、抑制细胞迁移,下调MMP-2和MMP-9的表达(P<0.01)。结论:miR-33a-5p靶向SMAD7抑制PDGF-bb诱导的血管平滑肌细胞增殖、迁移和细胞外基质的降解,并促进凋亡。

Objective:To investigate the effect of miR-33a-5p on proliferation,apoptosis,migration and degradation of extracellular matrix of vascular smooth muscle cells by targeting SMAD7.Methods:The target relationship was predicted by bioinformatics,the targeting relationship was further verified by fluorescein experiments,and the expressions of miR-33a-5p and SMAD7 were detected by RT-PCR.The proliferation of VSMC was detected by CCK-8 method,and apoptosis was detected by flow cytometry.The cell migration was detected by scratch test.The expression levels of Ki67,PCNA,Caspase-3,Caspase-9,MMP-2 and MMP-9 protein were detected by Western blot.Results:PDGF-bb treatment could enhance the proliferation of vascular smooth muscle cells and inhibit the expression of miR-33;miR-33a-5p and SMAD7 had binding sites in the 3′UTR region,miR-33a-5p directly targets SMAD7,and miR-33a-5p overexpression inhibited SMAD7 expression;miR-33a-5p inhibited proliferation of vascular smooth muscle cells and down-regulated the expression of proliferating marker proteins PCNA and Ki67 by targeting SMAD7.miR-33a-5p promoted apoptosis and up-regulated the apoptotic marker proteins Caspase-3 and Caspase-9 by targeting SMAD7.In expression,miR-33a-5p reduced wound healing rate and inhibited cell migration by targeting SMAD7,and miR-33a-5p down-regulated MMP-2 and MMP-9 expression by targeting SMAD7(P<0.01).Conclusion:miR-33a-5p inhibits PDGF-bb-induced vascular smooth muscle cell proliferation,migration and degradation of extracellular matrix,and promotes apoptosis by targeting SMAD7.