The Role Of Microrna-24 In The Regulationof Enos Expressionand Vascular Endothelial Cellproliferationandunderlying Mechanisms

ObjectiveThis study is to investigate whether miRNA-24 is involved in regulating endothelial nitric oxide synthase (eNOS) expression and vascular endothelial cell proliferation and to discuss the potential its molecular mechanism underlying the processes.Methods1.Roles of miRNA-24 in regulating vascular endothelial cell proliferationConstructed high expression miRNA-24 plasmids, Vacant Plasmids and high expression anti-miRNA-24 Plasmids were introduced into human umbilical vein endothelial cells (HUVECs) respectively by liposome in RPMI-1640 medium for 4h. Then cell medium was changed by RPMI-1640 with 10% FBS and HUVECs were cultured for 12h,24h,48h and 72h. Cell numbers were counted by a Hemocytometer and cell proliferation was detected by MTT assay. Migration ability of cell was measured by scratch wound model in vitro. NO production was measured by Griess Reaction in cell culture supernatants.2.Roles of miRNA-24 in regulating endothelial nitric oxide synthase expressionConstructed high expression miRNA-24 plasmids,Vacant Plasmids and high expression anti-miRNA-24 Plasmids were introduced into human umbilical vein endothelial cells (HUVECs) respectively by liposome in RPMI-1640 medium for 4h.Then cell medium was changed by RPMI-1640 with 10% FBS and HUVECs were cultured for 24h. eNOS mRNA expressions were detected by Real-time PCR. The protein expressions of eNOS were determined by Western blot and immunohistochemistry.3. Roles of miRNA-24 in regulating SP-1 expressionConstructed high expression miRNA-24 plasmids,Vacant Plasmids and high expression anti-miRNA-24 Plasmids were introduced into human umbilical vein endothelial cells (HUVECs) respectively by liposome in RPMI-1640 medium for 4h.Then cell medium was changed by RPMI-1640 with 10%FBS and HUVECs were cultured for 24h.SP-1 mRNA expressions were detected by Real-time PCR.The protein expressions of SP-1 were determined by Western blot and immunohistochemistry.4. Confirmed eNOS and SP-1 are miRNA-24 target genesIn order to prove that miRNA-24 regulates the expression of eNOS and SP-1 by directly targeting its 3’-UTR,the luciferase reporter vectors pMIR-Report with both wild-type or mutated eNOS-3’UTR and SP-1-3’UTR were constructed and named as pGL3-eNOS-wt,pGL3-eNOS-mut, pGL3-Sp-1-wt,pGL3-Spl-mut,respectively.HUVECs were seeded in 24-well Plates 24h prior to transfection. In each well, cells were transfected with reporter plasmids and pRL-TK vector containing Renilla luciferase and miR-24mimics or NC. Transfection was performed using Lipofectamine 2000 for 4h. Luciferase activity assay was carried out by the dualluciferase reporter assay system.Results1.Compared with the control group, the cell number in the miRNA-24 group was significantly reduced (P<0.01). Cell proliferation and migration were significantly decreased (P<0.01).The release of nitric oxide was significantly decreased (P<0.01)2.Compared with the control group, the expression of eNOS at mRNA and protein levels in miRNA-24 group was significantly decreased (P<0.05).3.Compared with the control group, the expression of SP-1 at mRNA and protein levels in miRNA-24 group was significantly decreased (P<0.05).4. compared with control group, miRNA-24 significantly decreased the activity when co-transfected with the pGL3-eNOS-wt reporter plasmid and pGL3-Sp-1-wt reporter plasmid(P<0.01)Conclusions1.miRNA-24 significantly inhibits the proliferationand migration of the HUVECs and NO expression.2.miRNA-24 regulating the level of expression of eNOS mRNA and protien. eNOS is the target gene of miRNA-24.3.miRNA-24 regulating the level of expression of SP-1 mRNA and protien. SP-1 is the target gene of miRNA-24.4.miRNA-24 and SP-1 articipates in the process of cell proliferation in vascular endothelial cell through co-regulate eNOS expression.