| BackgroundmicroRNAs (miRNAs) was discovered in 2003, a class of the length of the short chain oligonucleotide about 22 nucleotides, a number of studies have made it clear that the main function is to regulate the expression of target genes by way of sequence specificity. Recent studies show that, miRNAs involved in the regulation of specific genes expression of vascular endothelial cells (VECs), its level has obvious changed during in VECs normal physiological processes such as differentiation, proliferation, apoptosis and angiogenesis.Angiogenesis contain two main important reactions, respectively are nascent angiogenesis and vasculogenesis, both are closely related with VECs. In recent research, angiogenesis as a normal physiological processes involved in maintaining steady state of multiple organs. However, its dysfunction is closely associated with cardiovascular disease.Endothelial nitric oxide synthase (eNOS) is expressed mainly in endothelial cells, its mediated generate nitric oxide (NO) is one of the important regulatory factor to maintain vascular homeostasis. Our previously reported that eNOS gene could regulated by intronic 27-nucleotide (nt) miRNA, miR-21 and miR-24.Several transcription factors (for example Spl) which are involved in the regulation of eNOS, contribute to the regulation of VECs proliferation and apoptosis. Because miR-24 is closely related to cell apoptosis and angiogenesis,make its role hot in cancer and myocardial infarction. However, the studies of miR-24 are still in the initial state.We recently observed that miR-24 have difference expression amongst usual person and myocardial infarction or hypertension patients plasma, and this change may involve the regulation of miR-24 of VECs pathological and physiological process. Our previous research suggests miR-24 on endothelial cell proliferation regulation related to its inhibition of the expression of eNOS and Spl. However, this process whether to participate in the regulation of angiogenesis, and to further participate in the occurrence and development of cardiovascular disease, is still not clear.ObjectiveTo verify again the molecular mechanism of eNOS gene expression regulated by miR-24, and discuss its influence on endothelial cell proliferation.To study the molecular mechanism miR-24 of for regulation of eNOS gene expression of endothelial cells and its influence on in vitro tube formation.Further to study the molecular mechanism of miR-24 for regulation of endothelial cell differentiation and its influence on blood vessel formation.Discuss the role of miR-24 on the cardiovascular system development, and relationship to cardiovascular disease.Methods1 To test whether miR-24 influences Human umbilical vein endothelial cells eNOS and Spl expression.Constructed high expression plasmids, using X-ray tremeGENE HP DNA transfection reagent introduce into Human umbilical vein endothelial cells(HUVECs). According to transfection plasmid, HUVECs were divided into miR-24 high expression group, miR-24 interference (anti-miR-24) group and control group. The expression of eNOS and Spl at mRNA and protein levels were examined by real-time PCR and Western blotting.2 To test whether miR-24 influences HUVECs proliferation, migration, and tube formation.Constructed high expression plasmids, using X-ray tremeGENE HP DNA transfection reagent introduce into HUVECs. According to transfection plasmid,HUVECs were divided into miR-24 high expression group, miR-24 interference(anti-miR-24) group and control group. HUVECs proliferation ability was exam by tetramethyl azo azole salt (MTT) at 24, 48 and 72 h after transfection.HUVECs migration was exam by Transwell assay. Tube formation was exam by Matrigel.3 To test whether miR-24 influences mesenchymal stem cells differentiated into endothelial cells.Constructed high expression plasmids, using X-ray tremeGENE HP DNA transfection reagent introduce into Human bone marrow mesenchymal stem cells (HBMSCs). According to transfection plasmid, HBMSCs were divided into miR-24 high expression group, miR-24 interference (anti-miR-24) group andcontrol group. VEGF and b-FGF were used to induce HBMSCs differentiation to endothelial cells. The morphological changes of HBMSCs during differentiation period were observed by microscope. The NO concentration of culture medium supernatant was measured by Nitrate reductive enzymatic assay at 1, 3, 5, 7, 9 day during differentiation period. The expression of eNOS and Spl at protein levels were examined by Immunocytochemistry. Tube formation ability of HBMSCs after differentiation was exam by artificial basement membrane (Matrigel).4 To test whether miR-24 influences Vasculogenesis of chicken chorioallantoic membrane.Collect HBMSCs conditioned medium, and using chicken embryo villus allantoic membrane (chicken chorioallantoic membrane, CAM) model to exam the influence of miR-24 on Vasculogenesis.Results1 Compared with control group, the expression of eNOS and Spl at mRNA and protein levels in miR-24 high expression group were both significant decreased (P<0.05), respectively.2 Compared with the control group, the cell proliferation and migrated number in miR-24 high expression group were both significant decreased(P<0.05), respectively. And matrigel assay showed very few capillaries.3 Compared with the control group, cells in miR-24 high expression group didn’t formed a endothelial-like morphology during differentiation period. NO concentration of culture medium supernatant were decreased at five time points(P<0.05). The fluorescence intensity of eNOS and Spl were significant decreased. And matrigel assay showed very few capillaries.4 Compared with the control group, the blood vessel number and angiogenesis area ratio of CAM were both significant decreased (P<0.05),respectively.Conclusions1 MiR-24 significantly suppresses HUVECs eNOS expression at mRNA and protein, the transcription factor Spl may contribute to this inhibitory effect.2 MiR-24 play an important role in regulation of eNOS expression,therefore contributing to the suppression of HUVECs proliferation, migration and tube formation ability.3 MiR-24 play an important role in regulation of eNOS expression,therefore contributing to suppression of HBMSCs differentiated into endothelial and tube formation ability after differentiation.4 MiR-24 significantly suppresses the angiogenesis of CAM, and relate to its suppression of HBMSCs differentiation. |
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