miR-486促人脐静脉内皮细胞内皮间质转化的机制研究

Mechanism of miR-486 promoting endothelial-mesenchymal transition in human umbilical vein endothelial cells

目的 探讨微小RNA-486(miR-486)对人脐静脉内皮细胞(HUVECs)内皮间质转化(End MT)的影响及其调控机制。方法 常规培养HUVECs,并转染miR-486模拟物(mimic)及阴性对照(NC)质粒,实时荧光定量PCR测定miR-486表达;Western blot检测血管内皮细胞钙黏蛋白(VE Cadherin)、Ⅲ型胶原蛋白(CollagenⅢ)、血小板-内皮细胞黏附分子(CD31)、α-平滑肌肌动蛋白(α-SMA)、成纤维细胞特异性蛋白1(FSP1)、纤连蛋白(FN)表达;免疫荧光染色检测细胞中CD31与α-SMA表达;Western blot检测磷酸酶及张力蛋白同源物(PTEN)与磷脂酰肌醇3激酶/蛋白激酶B(PI3K/AKT)信号通路相关蛋白表达。使用PI3K/AKT信号通路抑制剂LY294002与miR-486 mimic共作用于HUVECs,Western blot检测VE Cadherin、CollagenⅢ、CD31、α-SMA、FSP1、FN、PTEN及PI3K/AKT信号通路相关蛋白表达。通过生物信息学在线软件预测miR-486与PTEN的靶向结合位点,构建PTEN 3'UTR野生型(WT)及突变型(MUT)质粒载体,双荧光素酶报告基因实验检测miR-486对PTEN的靶向作用。结果 与Control组和miR-486 NC组比较,miR-486 mimic组HUVECs中miR-486表达升高(P<0.05),VE Cadherin、CD31表达降低(P<0.05),CollagenⅢ、α-SMA、FN表达升高(P<0.05),FSP1表达未发生变化(P>0.05)。免疫荧光结果显示,与Control组和miR-486 NC组比较,miR-486 mimic组HUVECs中CD31表达明显减弱(P<0.05),α-SMA表达明显增强(P<0.05);与Control组和miR-486 NC组比较,miR-486 mimic组HUVECs中PTEN蛋白表达显著降低(P<0.05),p-PI3K和p-AKT蛋白表达显著升高(P<0.05)。与miR-486 mimic组比较,miR-486 mimic+LY294002组PTEN蛋白表达显著升高(P<0.05),p-PI3K和p-AKT蛋白表达显著降低(P<0.05)。与miR-486 mimic组比较,miR-486mimic+LY294002组VE Cadherin、CD31表达显著升高(P<0.05),且CollagenⅢ、α-SMA、FN表达显著降低(P<0.05),FSP1表达未发生变化(P>0.05)。双荧光素酶报告基因分析结果显示,与转染miR-486 NC比较,转染miR-486 mimic能够有效抑制含PTEN原序列的PTEN WT 3'UTR的荧光素酶活性(P<0.05),而对敲除了结合位点的PTEN MUT 3'UTR则无明显抑制作用(P>0.05)。结论miR-486通过抑制PTEN表达促进HUVECs的End MT进程,该机制与激活PI3K/AKT信号通路有关。

Objective To explore the effect of microRNA-486(miR-486)on endothelial-mesenchymal transition(End MT)in human umbilical vein endothelial cells(HUVECs)and its regulatory mechanism.Methods HUMECs were conventionally cultured and transfected with miR-486 mimics and negative control(NC)plasmids.Real-time fluorescence quantitative PCR was used to detect the expression of miR-486.Western blot was used to detect the expression of vascular endothelial cadherin(VE Cadherin),CollagenⅢ,platelet-endothelial cell adhesion molecule-1(CD31),α-smooth muscle actin(α-SMA),fibroblast specific protein-1(FSP1)and fibronectin(FN).Immunofluorescence staining was used to detect the expression of CD31 andα-SMA in cells.Western blot was used to detect the expression of phosphatase and tensin homologues(PTEN)and phosphatidylinositol 3-kinase/serthreonine protein kinase(PI3K/AKT)signaling pathway related proteins.The PI3K/AKT signaling pathway inhibitor LY294002 and miR-486 mimic were used to co-act on HUVECs.Western blot was used to detect the expression of VE Cadherin,CollagenⅢ,CD31,α-SMA,FSP1,FN,PTEN and PI3K/AKT signaling pathway related proteins.The target binding sites of miR-486 and PTEN were predicted by bioinformatics online software,the wild type(WT)and mutant(MUT)plasmid vectors of the 3'untranslated region of PTEN gene were constructed,and dual luciferase reporter gene assay was used to detect the targeting effects of miR-486 and PTEN.Results Compared with the Control group and the miR-486 NC group,the expression of miR-486 of HUVECs in the miR-486 mimic group was increased(P<0.05),and the expression levels of VE Cadherin and CD31 were decreased(P<0.05),the expression levels of CollagenⅢ,α-SMA and FN were increased(P<0.05),and the expression level of FSP1 was not changed(P>0.05).The result of immunofluorescence staining showed that compared with the Control group and the miR-486 NC group,the expression of CD31 of HUVECs in the miR-486 mimic group was significantly weakened(P<0.05),and the expression ofα-SMA was significantly enhanced(P<0.05).Compared with the Control group and the miR-486 NC group,the expression of PTEN protein of HUVECs in the miR-486 mimic group was significantly decreased(P<0.05),and the expression levels of p-PI3K and p-AKT protein were significantly increased(P<0.05).Compared with the miR-486 mimic group,the expression of PTEN protein in the miR-486 mimic+LY294002 group was significantly increased(P<0.05),and the expression levels of p-PI3K and p-AKT protein were significantly decreased(P<0.05).Compared with the miR-486 mimic group,the expression levels of VE Cadherin and CD31 in the miR-486 mimic+LY294002 group were significantly increased(P<0.05),the expression levels of CollagenⅢ,α-SMA and FN were significantly decreased(P<0.05),and the expression level of FSP1 was not changed(P>0.05).The results of dual luciferase reporter gene assay showed that compared with transfecting miR-486 NC,transfecting miR-486 mimic could effectively inhibit the luciferase activity of PTEN WT 3'UTR containing the original sequence of PTEN(P<0.05),but has no significant inhibitory effect on PTEN MUT 3'UTR which knocked out the binding site(P>0.05).Conclusion miR-486 promotes the End MT process in HUVECs by inhibiting the expression of PTEN,which is related to the activation of PI3K/AKT signaling pathway.