ABSTRACT
Objective To investigate the differences in eNOS gene expression,activity and its metabolites before and after human bone marrow mesenchymal stem cells (hBMSCs) are induced into vascular endothelial cells.Methods hBMSCs were induced into vascular endothelial cells.The morphological changes of the cells were observed under inverted microscope.Transwell assay was used to detect the cells' migration ability.The protein expression of eNOS was detected by immunofluorescence and Western blot.The activity of eNOS was detected by ELISA and the content of NO in cell culture supernatant was determined by nitrate reduction method.Results Compared with those in the undifferentiated group,the morphological changes of the differentiated cells were obvious.Cell migration ability increased by 238.10％ (73.000±7.002 vs.21.000±4.359,P＜0.05).The expression of eNOS protein increased by 114.72％ (0.423±0.011 vs.0.197±0.079,P＜0.05).The activity of eNOS was enhanced by 157.49％ (4.967±0.073 vs.1.929±±0.103,P＜0.05).The synthesis and release of NO increased by 155.67％ (184.909±1.853 vs.72.323±0.426,P＜0.05).Conclusion After hBMSCs are induced into endothelial cells,the expression of eNOS gene increases,their activities increase,synthesis and release of the metabolite NO increase.It may provide a basis for prevention and treatment of cardiovascular diseases with stem cells.
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.
ABSTRACT
Objective: To investigate the effects of miR-24 on endothelial nitric oxide synthase (eNOS) gene expression with regulation and endothelial cell proliferation, migration, tube formation in human umbilical vein endothelial cells (HUVECs). Methods: Constructed high expression plasmid of miR-24 and miR-24 antisense sequence were introduced into HUVECs and the cells included in 3 groups: Control group, miR-24 group and miR-24 inhibitor group. HUVEC proliferation was detected by MTT test, migration was measured by Scratching and Transwell methods, tube formation was examined by Matrigel assay; mRNA and protein expressions of eNOS and Sp1were determined by RT-PCR and Western blot analysis respectively. Results:①Compared with Control group, miR-24 group had decreased cell proliferation by 45.45% as (0.36 ± 0.04) vs (0.66 ± 0.08),P<0.05; miR-24 group had lower speed of cell migration, decreased number of cell migration by 74.75% as (30.25±3.78) vs (119.80±10.94),P<0.01 and there was no obvious tube formation.②Compared with Control group, miR-24 group showed reduced eNOS mRNA expression by 46.2% as (0.49±0.02) vs (0.91±0.01),P<0.05, reduced protein expression by 49.07% as (0.55±0.05) vs (1.08±0.05),P<0.05; meanwhile, decreased Sp1 mRNA expression by 44.9% as (0.49±0. 01) vs (0. 89±0.02)P<0.05, decreased protein expression by 54.90% as (0.46±0.02) vs (1.02±0.04),P<0.05. In miR-24 inhibitor group, the above indexes were lower than Control group but higher than miR-24 group, the amount of tube formation and the length of tubes were similar between Control group and miR-24 inhibitor group. Conclusion: MiR-24 may inhibit HUVECs proliferation, migration, tube formation and suppress eNOS expression; Sp1 might be one of the important regulators.