Effects Of MiR-24 On Hydrogen Peroxide-induced Apoptosis And Lumen Formation Of Human Umbilical Vein Endothelial Cells

Objective:To investigate the effects of miRNA-24（miR-24）on the apoptosis and lumen formation of human umbilical vein endothelial cells（HUVECs）induced by hydrogen peroxide（H₂O₂）and to explore its molecular mechanism.Methods:MTT was used to select the appropriate concentration of H₂O₂ to establish a cellular oxidative stress model.q RT-PCR was used to detect the expression of miR-24 in H₂O₂-induced injuried HUVECs.Construction of miR-24 high expression,anti-miR-24 and its negative control lentiviral plasmids,and transfected into HUVECs to establish stable transfected cell lines.q RT-PCR was used to detect the expression level of miR-24 and then treated with H₂O₂,the cells were divided into normal control group,H₂O₂ group,miR-24 group,anti-miR-24 group and miR-NC group.The content of ROS,MDA,SOD activity and NO concentration were detected by DHE fluorescence probe,MDA,SOD activity test kit and nitric acid reduction method respectively.MTT assay and flow cytometry were used to detect the proliferation of the cells in each group;the scratch test and transwell were used to detect the ability of migration;Hoechst33258 staining and flow cytometry were used to detect the apoptosis;the artificial basal glue（Matrigel）was used to detect the ability of tube formation in vitro.The m RNA and protein levels of apoptotic genes Bax,Bcl-2and Caspase-3 in,and the level of Akt/e NOS phosphorylation were detected by q RT-PCR and immunocytochemistry and Western blotting respectively.Results:1 The HUVECs damage induced by H₂O₂ was concentration-dependent and the expression of miR-24 in injured HUVECs increased（P<0.05）.The oxidative damage model was established with the concentration of IC₅₀ at 500μmol×L^-1and 12 h.Construction of a lentivirus stable cell line,compared with miR-NC group,the expression level of miR-24 was increased in miR-24 group,anti-miR-24 group decreased（all P<0.05）,and the cell lines were successfully constructed.2 Compared with miR-NC group,the ROS content and MDA activity in miR-24 group were increased（P<0.05）,SOD activity and NO concentration decreased（P<0.05）.3 Compared with miR-NC group,the proliferation and migration rate of HUVECs in miR-24 group were significantly decreased（P<0.05）,and the apoptosis rate increased（P<0.05）,and no obvious lumen structure was formed.4 Compared with miR-NC group,the m RNA and protein expression of apoptosis genes Bax,Caspase-3 in miR-24 group increased（P<0.05）,while Bcl-2 m RNA and protein expression decreased（P<0.05）,and the ratio of p-Akt/Akt decreased（P<0.05）.Conclusions:1 miR-24 significantly increased the content of ROS and MDA in HUVECs,decreased SOD activity and NO concentration,and further promoted oxidative stress damage.2 miR-24 significantly promoted HUVECs apoptosis under oxidative stress and inhibited cell proliferation,migration and tube formation,Down-regulated the expression of miR-24 can exert its effect of antioxidant damage,inhibit cell apoptosis,and enhance the tubulogenesis ability of HUVECs in vitro.3 miR-24 can significantly promote the apoptosis of HUVECs under oxidative stress,and the mechanism may be related to increase the expression of Bax and caspase-3 protein and inhibit bcl-2 expression.At the same time,miR-24 can inhibit the Lumen forming of HUVECs under oxidative stress may be related to decrease phosphorylation of Akt/e NOS protein.