The Expression An Function Of MicroRNA-424 In Cardiac Microvascular Endothelial Cell Angiogenesis In Diabetes

Objective:Diabetes induced vascular angiogenesis impairment is the main reason for diabetic patients complicated with myocardial infarction suffering from severe stenosis and having poor clinical outcome.In the process of tissue repair in ischemic diseases,mi R-424 is considered to promote angiogenesis and tissue repair.However,it is not clear whether mi R-424 plays a similar physiological role in diabetic patients with myocardial ischemia.In this study,we investigate the effect of mi R-424 on angiogenesis in diabetes mellitus complicated with myocardial infarction.Methods: 1.Hypoxia/reoxygenation model was established in human cardiac microvascular endothelial cells(HCMECs)and cells were divided into four groups.All groups were undergone hypoxia(H)for 6h and reoxygenation(R)for 2h except the NG group and HG group:(1)NG group: HCMECs were cultured under normal conditions;(2)HG group: cultured in high glucose medium culture;(3)NG+H/R group;(4)HG+H/R group.We used q PCR to detect the expression levels of mi R-424,CUL2 and HIF-1? genes and Western blot was applied to detect the expression levels of CUL2 and HIF-1?.2.mi R-424 mimic or inhibitor was transfected into HCMECs and cells were divided into four groups.All groups were undergone hypoxia(H)for 6h and reoxygenation(R)for 2h except the NG group:(1)NG group;(2)HG+H/R group;(3)HG+H/R+mi R-424 mimic group: mi R-424 mimic was transfected into cells before hypoxia;(4)HG+H/R+mi R-424 inhibitor group: mi R-424 inhibitor was transfected into cells before hypoxia.We used q PCR to detect the expression levels of mi R-424 after transfecting mimic or inhibitor.Western blot was applied to detect the expression levels of CUL2,HIF-1?and p-Akt.Cell proliferation ability in vitro was measured by CCK-8 assay.Tube formation on ECMatrix gel was used to assess the capacity for vasculogenesis.Results: 1.The expression level of mi R-424 in high glucose and hypoxia condition is higher than that in NG group(P<0.05).2.Compared with NG group,the CUL2 gene transcription level of HG+H/R group was decreased,and the activity of HIF-1 was increased(P< 0.05).3.Western blot test showed that both the expression of CUL2 in NG+H/R group and HG+H/R group was significantly lower than that NG group(P< 0.05),and the level in HG+H/R group was significantly lower than that in the NG+H/R group(P< 0.05).NG group had no HIF-1? expression.Compared with HG group,HIF-1? had higher expression in NG+H/R and HG+H/R groups(P<0.05),and HG+H/R group increased more significantly than that in NG+H/R group.4.The results of q PCR showed that the expression of mi R-424 in the HG+H/R group was significantly increased after transfection with mi R-424 mimic.The expression of mi R-424 in the HG+H/R group was downregulated after transfection with mi R-424 inhibitor.5.Compared with HG+H/R group,the expression of CUL2 protein further reduced after adding mi R-424 mimic,and the expression of HIF-1 increased(P<0.05).Compared with HG+H/R group,the expression of CUL2 increased and HIF-1 decreased after adding mi R-424 inhibitor(P< 0.05).Compared with the NG group,p-Akt in HG+ H/R group increased(P< 0.05),but the levels of p-Akt increased after adding mi R-424 mimic and further decreased after adding mi R-424 inhibitor(P< 0.05).6.Under high glucose and hypoxia condition,HCMECs proliferation rate and angiogenesis ability in vitro reduced,however,the proliferation rate and angiogenesis ability increased after adding mi R-424 mimic and further reduced after adding mi R-424 inhibitor.Conclusion: Under high glucose and hypoxia condition,the expression of mi R-424 was increased,which probably promoted the transcription of HIF-1? gene via inhibiting the expression of Cullin-2 protein.Furthermore,mi R-424 overexpression can enhance the proliferation of diabetic cardiac microvascular endothelial cells and the phosphorylation of Akt maybe be involved in the process of promoting angiogenesis in vitro.In conclusion,mi R-424 plays an important role in promoting angiogenesis in diabetes mellitus complicated with myocardial ischemiaprobablythrough enhancing the transcriptional activity of HIF-1? and increasing the levels of phosphorylation of Akt.