Effect Of 27nt-miRNA On ENOS Expression,NO Production And Angiogenesis And The Related Mechanisms

Objectives: Our previous study found that,27nt-miRNA was produced by the 27-base repeats of the fourth intron of endothelial NOS(eNOS)and negatively regulated the transcription level and protein expression of eNOS.This research is to further investigate the regulation of 27nt-miRNA on endothelial nitric oxide synthase expression,activity and the effect of its NO production,the tube formation of endothelial cells and the related molecular mechanism.1)To investigate the regulation of 27nt-miRNA expression on eNOS gene expression in vascular endothelial cells and its related molecular regulation mechanism.2)To investigate the effect of 27nt-miRNA on eNOS activity and its metabolite NO production and its molecular mechanism in vascular endothelial cells.3)To investigate the molecular mechanism of 27 ntmiRNAs on cell migration and angiogenesis,and to provide theoretical basis for the treatment of cardiovascular disease.Methods: 1)Wild-type and mutant-type gene sequence of 27nt-miRNA high expression plasmid was constructed and transfected with X-treme GENE HP DNA liposomes.Human umbilical vein endothelial cells(HUVECs)were divided into three groups according to the transfection plasmid: Blank plasmid control group(Control group),Wild-type 27-nt-miRNA plasmid group(Wt-27-nt-miRNA group)and mutant 27-nt-miRNA plasmid group(Mut-27-nt-miRNA group).2)To investigate the effect of 27nt-miRNA on eNOS gene expression: The blank plasmid,Wt-27-nt-miRNA plasmid and Mut-27-nt-miRNA plasmid were transfected into HUVECs respectively.The changes of eNOS mRNA and nuclear transcription factor AP-1 mRNA transcription were detected by RT-PCR.The changes of eNOS protein and nuclear transcription factor AP-1 protein expression were detected by immunocytochemistry and western blotting.3)To investigate the effect of 27nt-miRNA on eNOS activity and its metabolites NO: The blank plasmid,Wt-27-nt-miRNA plasmid and Mut-27-nt-miRNA plasmid were transfected into HUVECs respectively.The changes of eNOS activity in cell culture medium were measured by enzyme-linked immunosorbent assay(ELISA).The changes of NO production in supernatant of different time were measured by nitric acid reduction method.4)To detect the effect of 27 ntmiRNA on the proliferation of HUVECs: The blank plasmid,Wt-27-nt-miRNA plasmid and Mut-27-nt-miRNA plasmid were transfected into HUVECs respectively.Cells were transfected and digested in 96-well plates,the proliferation of HUVECs at 24,48 and 72 h three time points was detected by MTT assay.5)To investigate the effect of 27nt-miRNA on the migration,invasion and lumen formation of HUVECs: The blank plasmid,Wt-27-ntmiRNA plasmid and Mut-27-nt-miRNA plasmid were transfected into HUVECs respectively.Cells were transfected and digested in 96-well plates,the migration ability of each group was detected by scratch test and Transell test.The tube formation ability of each group was detected by artificial basement membrane(Matrigel).Results: 1)Compared with the control group,eNOS mRNA and AP-1 mRNA were significantly decreased in 27nt-miRNA overexpression group(P <0.05),and the expression of eNOS and AP-1 protein was significantly decreased(P <0.05).2)Compared with the control group,the activity of eNOS in the supernatant of 27nt-miRNA overexpression group was significantly decreased(P <0.05)and the NO production of metabolites was significantly decreased(P <0.05).3)Compared with the control group,the cell proliferation ability of the 27nt-miRNA high expression group was significantly decreased(P <0.05),the migration ability decreased significantly(P <0.05),and the rate of angiogenesis was decreased(P <0.05).Conclusions: 1)27nt-miRNA significantly inhibits the expression of eNOS mRNA and protein in HUVECs,significantly inhibited the transcription of nuclear transcription factor AP-1 mRNA and protein expression.The involvement of AP-1 may be one of the important molecular mechanisms of this regulatory process.2)27nt-miRNA significantly inhibits the activity of eNOS and the production of its metabolites NO in vascular endothelial cells,and may be related to the regulation of eNOS gene expression.3)27nt-miRNA significantly inhibits the proliferation,migration and lumen formation of vascular endothelial cells,which may be related to its regulation of eNOS gene expression,and eNOS/NO may be one of the relevant pathways for its regulation.