ABSTRACT
AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs).METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs.Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion.The cell viability was measured by MTT assay.The migration ability was detected by scratch assay.The mRNA and protein expression of SM22αwas determined by RT-PCR, immunocytochemical staining and Western blot.RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05) , the migration ability was increased (P<0.05) and the expression of SM22αat mRNA and protein level was decreased (P<0.05).Compared with negative control lentiviral group, the cell viability in 27ntmiRNA over-expression group was decreased (P<0.05) , the migration ability was decreased (P<0.05) , and the mRNA and protein expression of SM22αwas increased (P<0.05).While in anti-27nt-miRNA group, the cell viability was increased (P<0.05) , the migration ability was increased (P<0.05) , and the mRNA and protein expression of SM22αwas decreased (P<0.05).CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.
ABSTRACT
Objective To further investigate the molecular mechanism of microRNA(miRNA)-24 on endothelial nitric oxide synthase(eNOS)gene expression, activity and the impact on its metabolites. Methods The plasmid of miRNA-24 was constructed and transfected to human umhilical vein endothelial cells(HUVECs). The proliferation of cells was detected by MTT test. Migration ability was measured by scratch wound model. The mRNA transcription and protein expressions of eNOS were determined by RT-PCR and Western blot respectively. The eNOS activity was detected by ELISA. The metabolite NO production in cell culture supernatant was detected by nitrate reduction. Results Compared with control group, the proliferation of endothelial cells in the miRNA-24 group was decreased by 54.32%;the migration of HUVECs was reduced by 48.62%;the expressions of eNOS mRNA and protein were de? creased by 43.92% and 42.71%, respectively. miRNA-24 suppressed eNOS activity by 73.20%. Meanwhile, the NO production was decreased by 55.29%. Conclusion miRNA-24 inhibits the synthesis and release of metabolite NO, which may be a molecular target of prevention and treatment in cardiovascular diseases.