Effect Of ?-catenin Pathway On Pleripotency Maintenance And Cardiomyocyte Differentiation Of MSCs

Mesenchymal stem cells(MSCs)are a promising option for cell-based therapies of myocardial injury diseases to replace current invasive techniques.Complete differentiation of MSCs into cardiomyocytes has not been achieved to date.Wnt/ ?-Catenin signaling plays essential roles in cardiomyocyte differentiation,but the mechanism governing this effect is largely unknown.Therefore,this study focuses on the role of ?-catenin in the cardiocarcytic process of C3H10T1 / 2 cells and its mechanisms.In this study,a C3H10T1/2 cell model with knockdown of ?-Catenin was established,cell morphological changes were observed by microscopy;immunofluorescence,QPCR,Westernblot were used to detect changes in dry factors and cardiac markers;FCS detected cell metabolism.Experiments found:(1)As the tapping period is prolonged,the cells have gradually appear in myocardial pattern,and the level of myocardial transcription factors and myocardial markers are gradually enhanced,which is the most significant in day 7.Note that the C3H10T1/2 cells will induce C3H10T1/2 cells to differentiate to myocardial direction after knocking ?-catenin,and the induced differentiation effect is most significant in the descending of the descent;(2)By detecting the mitochondrial film potential,mitochondrial amount and mitochondrial ROS level,mitochondrial calcium The ionic level,the ATP content found that after knocking down ?-catenin,the cell energy consumption was significantly increased;(3)After overexpressed ?-catenin,the pleripotency factors was significantly up-regulated,and the myocardial markers were significantly reduced.Clarified ?-catenin has a key role in the C3H10T1 / 2 cell cardiac transformation.The preliminary study found that Islet1 is an important role in C3H10T1/2 cell cardiac transformation,which can be combined with acetyltransferase(GCN5)to enhance its transcriptional activity,and activate myocardial transcription factor expression.At the same time,it has been found that MLIP is combined with islet1 to significantly inhibit its transcriptional activity.Therefore,we detect the expression of Islet1,GCN5,and MLIP through Co IP,isotope trace and Ch IP.After knocking down ?-catenin,GCN5 has no significant change in transcription or translation level,and Islet1 and MLIP changes significantly in translation levels,and the isotope trace results show that significant changes are mainly due to degradation of two proteins.The experimental results suggest that this may be related to the ubiquitinization modification of the protein,so we found key E3 ubiquitinization enzyme UBE3 C and WWP1,which are related to MLIP and Islet1 ubiquitization modifications through the Ubibrowser ubiquitization database,and the role and mechanism verification.The experimental results found that:(1)Knockdown of ?-Catenin significantly upregulated the expression of the E3 ubiquitination ligase UBE3 C,thereby modifying MLIP ubiquitination and reducing its expression;(2)Knockdown of ?-Catenin upregulated Islet1 by inhibiting the expression of the E3 ubiquitinating ligase WWP1;(3)Through the Ch IP and luciferase reporter system,it was found that when MLIP binds to Islet1,it significantly inhibits the transcriptional activity of Islet1;(4)Knockdown of ?-Catenin was observed to significantly downregulate the expression of MLIP and reduce the binding of MLIP to Islet1,thereby further promoting the binding of Islet1 and GCN5 and increasing Islet1 transcription activity,inducing the differentiation of MSCs into cardiomyocytes.In summary,our results show that decreasing ?-catenin regulates the ubiquitination of Islet-1 and MLIP,affecting their expression,reducing the amount of Islet-1 binding to MLIP and increasing the amount of binding to GCN5 in the nucleus.Therefore,the transcriptional activity of Islet-1 is significantly activated,inducing C3H10T1/2 cells to differentiate into myocytes.We have focused on the function of ?-Catenin in cardiogenesis.We also discussed possible relationships between ?-Catenin and Islet-1 in the regulation of cardiomyocyte differentiation.Further knowledge of biochemical pathways,including molecular signaling pathways,can provide more insights into the myocardial differentiation mechanism of mesenchymal stem cells(MSCs).This study provide the groundwork of application of MSCs in clinical treatment of heart disease.