Mechanism of miR-486 promoting endothelial-mesenchymal transition in human umbilical vein endothelial cells
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 and α-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,Collagen Ⅲ,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 Collagen Ⅲ,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 of α-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 Collagen Ⅲ,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.
microRNA-486human umbilical vein endothelial cellsendothelial-mesenchymal transitionphosphatase and tensin homologues
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