ABSTRACT
Objective:To verify DNA methyltransferase 1(DNMT1) is the direct target of miR-148a and explore the internal mechanism by which miR-148a regulates the cardiac differentiation of mesenchymal stem cells(MSCs)by directly targeting DNMT1.Methods:miRNA microarray was used to screen out the abnormally expressed miRNAs in MSCs before and after 5′-azacytidine(5′-aza) treatment.Target scan was uesd to predict the target of miR-148a.miR-148a mimics and DNMT1-wt,scramble and DNMT1-wt,miR-148a and DNMT1-mut,scramble and DNMT1-mut were co-transfected in MSCs cells and luciferase activity were detected by single photon.MSCs cells were transfected with miR-148a lentivirus plasmid.Respectively extract DNA,RNA and protein 1,7,14 and 28 days after transfection.The CpG methylation level on DNA regulatory sequences of Gata-4 upstream gene was detected by methylation detection,the mRNA and protein expressions of myosin heavy chain(MHC),cardiac troponin T(cTnT),CD90,CD29,Nkx2.5 and Gata-4 were detected by qRT-PCR and Western blot.Results:miRNA Microarray screened out the abnormally expressed miRNAs in MSCs before and after 5′-aza-induced,including miR-146a-5p,miR-148a,miR-539,etc.Among which miR-148a was remarkably upregulated.Targetscan,Luciferase Reporter Gene and qRT-PCR verified that DNMT1 was the direct target of miR-148a.By infected MSCs cells with miR-148a lentivirus plasmid,we confirmed that the methylation level of Gata-4 gene upstream was changed,and with prolong of transfection,the methylation level of Gata-4 was decreased,the expression of MHC and cTnT were increased,while CD90 and CD2 were continually decreased,the mRNA expression of Nkx2.5 and Gata-4 were increased MSCs cells started myocardial differentiation.Conclusion:miR-148a can regulate the cardiac differentiation of MSCs by directly targeting DNMT1.
ABSTRACT
Objective To explore the relationship between bronchial pneumonia in children and the level of myocardial enzyme , C-reactive protein (CRP) and platelet count .Methods 1 000 cases of children with bronchial pneumonia from August 2010 to De-cember 2010 were retrospectively analyzed (bronchial pneumonia group) ,According to the clinical symptom severity ,the children wwer divided into mild bronchial pneumonia group and severe bronchial pneumonia .1 000 healthy children were recruited in control group(control group) ,and the changes of myocardial enzyme ,platelet count and CRP were tested .Results The myocardial enzyme , including aminotransferase (AST ) ,lactate dehydrogenase (LDH ) ,a- hydroxybutyric acid dehydrogenase (alpha HBDH ) ,serum creatine kinase (CK) ,CK isoenzyme (CK-MB) ,platelet count and CRP in bronchial pneumonia group were all higher than that of control group (P<0 .05);In the severe bronchial pneumonia group ,the increased rate of AST ,LDH ,α-HBDH ,CK ,CK-MB ,plate-let count and CRP indicators were significant higher than that in the mild pneumonia group (P<0 .05) .Conclusion The determina-tion of myocardial enzyme ,platelet count and CRP would be useful and helpful for the diagnosis of children bronchial pneumonia ,in particular the severe bronchial pneumonia .