miR-20a-5p通过下调BMPR2促进人脑微血管内皮细胞增殖和抑制凋亡

miR-20a-5p promotes human brain microvascular endothelial cell proliferation and inhibits apoptosis by down-regulating BMPR2

目的探讨微小RNA-20a-5p(miR-20a-5p)是否通过下调骨形成蛋白2型受体(BMPR2)基因调控人脑微血管内皮细胞增殖和抑制凋亡。方法在永生化人脑微血管内皮细胞(hCMEC/D3)中分别转染miR-20a-5p mimics,BMPR2小干扰RNA(si-BMPR2)或过表达质粒(pcDNA-BMPR2)。实时定量PCR(qRT-PCR)测定miR-20a-5p表达,免疫印迹(Western Blot)检测BMPR2、细胞周期蛋白D1(CyclinD1)、活化的的天冬氨酸特异性半胱氨酸蛋白酶3(Cleaved-caspase-3)、磷酸化-磷脂酰肌醇-3激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)蛋白水平,噻唑蓝(MTT)检测细胞增殖,流式细胞仪检测细胞凋亡。生物信息学预测与荧光素酶报告基因检测分析miR-20a-5p和BMPR2之间的关系。共转染miR-20a-5p mimics和pcDNA-BMPR2,利用上述方法评估其对细胞增殖、凋亡和PI3K/AKT信号通路的影响。结果过表达miR-20a-5p明显增加hCMEC/D3的细胞活性,CyclinD1、p-PI3K、p-AKT蛋白表达水平,显著减少细胞凋亡率和Cleaved-caspase-3蛋白表达水平(P<0.05)。低表达BMPR2明显提高hCMEC/D3的细胞活性、CyclinD1蛋白表达水平,显著减少细胞凋亡率和Cleaved-caspase-3蛋白表达水平(P<0.05),高表达BMPR2时具有相反的效果。miR-20a-5p靶向BMPR2,并调控BMPR2蛋白的表达。高表达BMPR2可以逆转miR-20a-5p促hCMEC/D3增殖、PI3K/AKT信号通路活性和抑凋亡的作用。结论miR-20a-5p通过靶向BMPR2激活PI3K/AKT信号通路,促进人脑微血管内皮细胞的增殖,并抑制凋亡。

Objective To investigate whether microRNA-20a-5p(miR-20a-5p)regulates the proliferation and apoptosis of human brain microvascular endothelial cells by down-regulating the BMPR2 gene.Methods MiR-20a-5p mimics,BMPR2 small interfering RNA(si-BMPR2)or overexpression plasmid(pcDNA-BMPR2)were transfected into immortalized human brain microvascular endothelial cells(hCMEC/D3).qRT-PCR was used to determine the expression of miR-20a-5p.Western Blot detected the expression of BMPR2,CyclinD1,Cleaved-caspase-3,p-PI3K,p-AKT protein,thiazole blue(MTT)to assay cell proliferation,and flow cytometry to detect cell apoptosis.Bioinformatics prediction and luciferase reporter gene detection analyzed the relationship between miR-20a-5p and BMPR2.Co-transfected miR-20a-5p mimics and pcDNA-BMPR2,using the above method to evaluate their effects on cell proliferation,apoptosis and PI3K/AKT signaling pathway.Results Overexpression of miR-20a-5p significantly increased hCMEC/D3 cell activity,CyclinD1,p-PI3K,and p-AKT protein expression,and greatly decreased the apoptosis rate and Cleared-caspase-3 protein expression(P<0.05).Low expression of BMPR2 obviously enhanced hCMEC/D3 cell activity,CyclinD1 protein expression level,dramatically reduced the apoptosis rate and the level of Cleaved-caspase-3 protein expression(P<0.05),while high expression of BMPR2 showed the opposite effects.miR-20a-5p targets BMPR2 and regulates the expression of BMPR2 protein.Overexpression of BMPR2 can reverse the effects of miR-20a-5p on promoting hCMEC/D3 proliferation,PI3K/AKT signaling pathway activity and inhibiting apoptosis.Conclusion miR-20a-5p activates PI3K/AKT signaling pathway by targeting BMPR2,promotes the proliferation of human brain microvascular endothelial cells,and inhibits apoptosis.