Role Of β-catenin In The BMP9-induced Differentiation Of C3H10T1/2 Cells Into Cardiomyocyte-like Cells

PART ONE:EXPRESSION OF B-CATENIN IN THE BMP9-INDUCED DIFFERENTIATION OF C3H10T1/2CELLS INTO CARDIOMYOCYTE-LIKE CELLSObjective: To investigate the expression of β-catenin, the key factors of canonical Wnt signaling pathway,in the bone morphogenetic protein 9(BMP9) induced C3H10T1 / 2 cells into cardiomyocytes-like cells.Methods: The cells named HEK293 were used to amplified recombinant adenovirus GFP and BMP9.C3H10T1/2 cells were transfected with the Ad-BMP9 for up to 7 days.24 hours after transfection,the inverted microscope was used to observed the morphology of C3H10T1/2,and the efficacy of recombinant adenovirus in C3H10T1/2 cells were examined by fluorescence microscope and flow cytometry.The expression of β-catenin were evaluated using Real-time quantitative PCR(q RT-PCR), Western blot,and immunofluorescence staining.Results: 1.Compared with the control group,the C3H10T1/2 cell morphology of Ad-BMP9 group arranged orderly and gradually changed into long spindle.2.The transfection was successful with over 44% of the cells positive for all the transfection groups(Ad-BMP9, Ad-GFP) 24 hours after transfection.3.Although the expression of β-catenin in the m RNA levels were increased in Ad-BMP9 group,there were no significant difference between Blank group,Ad-GFP group and Ad-BMP9 group.4.Western blot and immunofluorescence staining showed thatβ-catenin can be excessively activated and their expressions were significantly increased in the protein levels.Conclusion: β-catenin can be actived in protein levels by BMP9 in the differentiation of C3H10T1/2 cell into cardiomyocytes-like cells.PART TWO:THE BMP9-INDUCED DIFFERENTIATION OF C3H10T1/2 CELLS INTO CARDIOMYOCYTE-LIKE CELLS AFTER SPECIFIC INHIBITION OF Î'-CATENINObjective: To investigate the role of β-catenin in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells induced by bone morphogenetic protein 9(BMP9).Methods: C3H10T1/2 cells were infected with the recombinant adenovirus for BMP9 and differentiated into cardiomyocytes in vitro for up to 21 days. The actived levels of β-catenin after cultivated with BMP9 and different concentrations of β-catenin specific inhibitor XAV-939 were detected by Western blotting. Real-time quantitative PCR(q RT-PCR) was performed to evaluate the expression of cardiac specific gene myocyte enhancer factor 2C(MEF2C) and guanine-adenine-thymine-adenine binding protein 4(GATA4) after one week induced by BMP9 and different concentrations of XAV-939. Three weeks after transfection,the expressions of myocardium specific proteins connexin 43(CX43) and cardiac troponin T(c Tn T) were analyzed by using Western blotting, and immunofluorescence staining was used to observe the locations of CX43 in the cells.Results: 1.The expression of β-catenin were significantly increased in the protein levels,while the activation rate could be decreased with the concentration of inhibitor XAV939 increased.2.After XAV-939 inhibited the activity of β-catenin, the expressions of MEF2 C, GATA4, CX43,c Tn T of C3H10T1/2 cells induced by BMP9 was significantly suppressed.Conclusion: The β-catenin can be actived by BMP9 and the activation of β-catenin plays an important role in the differentiation of C3H10T1/2cell into cardiomyocytes-like cells induced by BMP9.