Effect Of Anti-miR-21 On ENOS Expression And Angiogenesis And Its Related Mechanisms

Objective: Previous studies have shown that miR-21 antisense oligodeoxynucleotides(AS-miR-21)can significantly inhibit the expression of AP1 and eNOS proteins,and transcription factor AP1 plays an important role in the regulation of AS-miR-21 for eNOS expression.Further,to study the molecular mechanism of anti-miR-21 in the regulation of the expression of Ang?-induced endothelial nitric oxide synthase and the effect of its angiogenesis in vitro.1 To study the molecular mechanism of anti-miR-21 in the regulation of the expression of Ang?-induced endothelial nitric oxide synthase.2 To investigate the mechanism of anti-miR-21 inhibits the proliferation and migration of Ang ?-induced endothelial cells and the effect of its angiogenesis in vitro.3 To elucidate the molecular mechanism of endothelial cell proliferation,migration and angiogenesis,nd to explore the role of anti-miR-21 in cardiovascular system development and cardiovascular disease.Methods:1 To investigate the effect of anti-miR-21 on the expression of eNOS and Ap1 in Ang?-induced human umbilical vein endothelial cellsConstructed high expression plasmids of anti-miR-21,the anti-miR-21 plasmid was transfected into human umbilical vein endothelial cells(HUVECs)by X-tremeGENE HP DNA transfection reagent.According to the experimental purpose,HUVECs were divided into 3 groups: control group,Ang? group and anti-miR-21 + Ang?.The NO concentration in the supernatant was measured by nitrate reductase method.The mRNA expressions of eNOS and AP1 were observed by RT-PCR.The protein expressions of eNOS and AP1 were analyzed by Western blot and immunohistochemical staining,respectively.2 To test the effect of anti-miR-21 on the migration,proliferation,and tube formation in Ang?-induced human umbilical vein endothelial cellsConstructed high expression plasmids of anti-miR-21,the anti-miR-21 plasmid was transfected into human umbilical vein endothelial cells(HUVECs)by X-tremeGENE HP DNA transfection reagent.According to the experimental purpose,HUVECs were divided into 3 groups: control group,Ang? group and anti-miR-21 + Ang?.The effects of anti-miR-21 on HUVECs proliferation,migration,apoptosis and tube formation were analyzed by MTT method,wound-healing assay,Annexin ?-FITC/PI staining and Matrigel method,respectively.3 To detect the molecular mechanism of the effect of anti-miR-21 on the migration,proliferation,and tube formation in Ang?-induced human umbilical vein endothelial cellsConstructed high expression plasmids of anti-miR-21,the anti-miR-21 plasmid was transfected into human umbilical vein endothelial cells(HUVECs)by X-tremeGENE HP DNA transfection reagent.According to the experimental purpose,HUVECs were divided into 3 groups: control group,Ang? group and anti-miR-21 + Ang?.The mRNA expressions of miR-21 was detected by RT-PCR.The protein expressions of CD31 and VEGFR2 were analyzed by Western blot.Results:1 Compared with control group,Ang?-induced group were significantly increased the mRNA and protein expression of eNOS and AP1(P<0.05),while anti-miR-21 significantly decreased the eNOS and AP1 mRNA and eNOS and AP1 protein levels of HUVECs(P<0.05).2 Compared with the control group,Ang ? markedly increased the proliferation(P<0.05),migration(P<0.05),apoptotic rate(P<0.05),and the formation of luminal was enhanced.while anti-miR-21 inhibited the proliferation,migration,apoptotic rate and the formation of luminal of HUVECs.3 Compared with control group,Ang?-induced 6h group and Ang?-induced 24 h group were significantly increased the mRNA expression of miR-21(P<0.01),but the expression of miR-21 mRNA decreased with the increase of induction time(P<0.01).Ang?-induced group were significantly increased the protein expression of CD31 and VEGFR2(P<0.05),while anti-miR-21 inhibited the CD31 and VEGFR2 protein expression of HUVECs(P<0.05).Conclusions:1 Anti-miR-21 significantly inhibite the eNOS mRNA and eNOS protein of Ang?-induced HUVECs,the transcription factor AP1 may contribute to this inhibitory effect.2 Anti-miR-21 play an important role in regulation of eNOS expression,therefore may contribut to the inhibition of proliferation,migration and tube formation of Ang?-induced HUVECs.3 miR-21 may be played an important role in regulation of endothelial cell angiogenesis by AngII induced.