Research On The Role And Mechanism Of MARVELD1 In Mouse Stem Cell Differetiation And Somatic Cell Reprogramming

Epithelial-mesenchymal transition?EMT?and mesenchymal-epithelial transformation?MET?are closely related to"stemness"in development and cancer,and EMT plays a key role in ESCs differentiation in vitro.In addition,the generation of iPSCs from mouse fibroblasts requires a mesenchymal-to-epithelial transition?MET?.And the MET process has an important influence on efficiency of induced pluripotent stem cells.Recent studies has shown that MARVELD1 played an important role in EMT and MET.Therefore,in this study we focus on MARVELD1 and its function on ESCs differentiation and ipsc induction.In this article,the expression of MARVELD1 in R1/E,Eph4-Ev,NIH/3T3 and MEF cells is detected by real-time quantitative PCR,and it is found that MARVELD1had low expression in embryonic stem cell line-R1/E and epithelial cell line-Eph4-Ev,rather than high expression in fibroblasts-NIH/3T3 and MEF.The expression of MARVELD1 is associated with E/M and stemness.The effect of MARVELD1 on cell proliferation was detected by MTT assay.It was found that the down regulation of MARVELD1 inhibited the proliferation of MEF cells.The effect of MARVELD1 on the migration and movement of fibroblasts was detected by scratch assay.MARVELD1 was found to inhibit the migration of MEF cells.The differences in MET phenotype and efficiency between MARVELD1^-/-MEF cells and MARVELD1^+/+MEF cells by OKSM induction were detected by cell phenotypic comparison and AP staining.MARVELD1inhibited cell reprogramming of iPSCs.The effect of MARVELD1 on single factor-induced phenotype and molecular changes in MEF cells showed that MARVELD1 had the greatest effect on early MET of Klf4-infected MEF cells.In the study of the effect of MARVELD1 on mouse embryonic stem cell differentiation,real-time quantitative PCR was used to detect the expression level of MARVELD1 during the random differentiation of ESCs induced in vitro,and it was found that MARVELD1 was up-regulated during this process.Using immunofluorescence,staining of embryoid bodies under ES medium culture conditions revealed that down-regulation of MARVELD1 resulted in down-regulation of epithelial marker molecules,and had no significant effect on cell pluripotency related markers.MARVELD1 had significant effect on EMT in ESCS cells rather than cell pluripotency.The in vitro induction model of ESCs differentiation to mesendoderm was constructed,and the MARVELD1 overexpressed ESCS cell line was constructed.The morphological study of the embryoid body showed that the up-regulation of MARVELD1 did not significantly affect the differentiation.Molecular detection also confirmed this conclusion.The study of EMT marker molecules during differentiation revealed that MARVELD1 inhibits the process of epithelial-mesenchymal transition in ESCS to mesendoderm differentiation.In the research of the mechanism of MARVELD1 on reprogramming,the viral infection technique was used to construct MARVELD1 overexpressed MARVELD1^+/+and MARVELD1^-/-MEF cells,and the.The level of histone H3 modification above four cell lines was detected by Western Blot.MARVELD1 was found to inhibit histone H3K4me3 and H3K27me3 modifications in MEF cells.The results of component separation experiments showed that MARVELD1 is in both cytoplasm and nucleus.The Co-IP technique showed that MARVELD1 had no direct interaction with histone H3.Using ChIP-seq experiments and bioinformatics technology,MARVELD1 knockout promoted the binding of H3K4me3 and H3K27me3 to DNA,which had the most significant influence on the H3K27me3 modification on Klf4 binding site,MARVELD1inhibited the balance of H3K4me3 and H3K27me3 on E-cadherin,SNAIL and other MET related genes.