Effects Of 27nt-miRNA On Tube Formation Of Vecs And Angiogenesis In Cam And Its Underlying Mechanisms

ObjectivesOur previous study suggested that the 27nt-miRNA derived from the 27 th base repeat of the fourth intron of the endothelial nitric oxide synthase can negatively regulate the transcription and protein expression levels of the host gene eNOS,thereby regulating the proliferation of endothelial cells.This study aimed to establish a human umbilical vein endothelial cell line with high expression of 27nt-miRNA by lentiviral transfection,and explore the effects of 27nt-miRNA on proliferation,migration and lumen formation of HUVECs and its molecular mechanism,and further explore the effect of 27nt-miRNA on angiogenesis of chicken chorioallantoic membrane,and provide a theoretical basis for elucidating the role of 27nt-miRNA in cardiovascular disease.Methods1 Culture and lentivirus transfection of HUVECsThe Shanghai Jikai Gene Company was commissioned to construct 27nt-miRNA,anti-27nt-miRNA and control vector according to the corresponding sequenceand and analyze whether the vector was successfully constructed by PCR and DNA sequencing.Finally,the corresponding vector was packaged with lentivirus to form 27nt-miRNA,anti-27nt-miRNA and control lentivirus.The lentivirus were transfected into HUVECs and divided into 27nt-miRNA high expression group,anti-27nt-miRNA group and control group.The transfection efficiency and cell line establishment were detected by microscopic observation and flow cytometry.2 To investigate the effects of 27nt-miRNA on proliferation,migration and lumen formation of HUVECsThe 27nt-miRNA,anti-27nt-miRNA and control lentiviral vector were constructed and transfected into HUVECs and divided into 27nt-miRNA high expression group,anti-27nt-miRNA group and control group.MTT assay was used to detect the proliferative capacity of each group.The scratch test was used to detect the migration ability of each group.The artificial basement membrane(Matrigel)was used to detect the tube formation ability of each group.3 To explore the effect of 27nt-miRNA on eNOS and Sp1 expression in HUVECsThe 27nt-miRNA,anti-27nt-miRNA and control lentiviral vector were constructed and transfected into HUVECs and divided into 27nt-miRNA high expression group,anti-27nt-miRNA group and control group.The expression of eNOS and Sp1 mRNA were detected by RT-PCR.The expression of eNOS and Sp1 protein were detected by immunocytochemistry and Western blotting,determination of NO concentration by One-step method.4 To investigate the effect of 27nt-miRNA on angiogenesis in chicken chorioallantoic membraneThe 27nt-miRNA,anti-27nt-miRNA and control lentiviral vector were transfected into the CAM mode,respectively.The effect of 27 nt-miRNA on angiogenesis was evaluated by the percentage of vascular area to CAM area.Results1 Under the fluorescence microscope,the green fluorescence of the transfected cells was strong.The lentiviral transfection efficiency of the 27nt-miRNA group,the anti-27nt-miRNA group and the control group was more than 90%,indicating that the cell lines were constructed.2 Compared with the control group,the proliferation activity of HUVECs in the 27nt-miRNA group was significantly decreased(P<0.05),the migration ability was decreased(P<0.05),and no tube-like structure was observed.3 Compared with the control group,the mRNA and protein expressions of eNOS and Sp1 of HUVECs in 27nt-miRNA group were significantly decreased(P<0.05),and the production of NO was reduced(P<0.05).4 Compared with the control group,the angiogenesis area of CAM in the 27nt-miRNA group was decreased(P<0.05).Conclusions1 27nt-miRNA significantly inhibits the proliferation,migration and tube formation of HUVECs,which is related to the regulation of eNOS expression.2 27nt-miRNA significantly suppresses the mRNA and protein expression of eNOS and Sp1 in HUVECs.27nt-miRNA may cooperate with Sp1 to regulate the expression of eNOS,thereby affecting angiogenesis.3 27nt-miRNA significantly inhibits angiogenesis of CAM.