The Mechanism Of OCT4 Promoted The Self-renewal Of HHFMSC Through Remodeling The Cytoskeleton Based On ZO-1

Human hair follicle mesenchymal stem cell(hHFMSC)derived from hair follicles and had multidirectional differentiation potential.After transduction of OCT4,the cell morphology and adhesion of hHFMSC changed and two subpopulations emerged including adherent cells and floating cells,and floating cells were induced to transdifferentiate into erythrocytes by a series of hematopoietic factors.Objective:The expression and localization of OCT4 in adherent cells and floating cells were detected,and the changes in cell morphology,cytoskeleton,adhesion and proliferation ability were observed.The transcriptome sequencing was used to analyze the difference in dedifferentiation between the two subpopulations,and to verify the ability of OCT4 to remodeling cytoskeleton and self-renewal through the molecular pathway of ZO-1/Actin/?-catenin.Thus,it was revealed that OCT4 may affect the skeleton and adhesion of hHFMSC through ZO-1,thus promoting the molecular pathway of reprogramming hHFMSC,and laying the experimental foundation for obtaining a large number of hematopoietic precursor cells that can be expanded in vitro.Methods:Based on the cell model of OCT4 transducing hHFMSC established earlier,the following studies were carried out:(1)The expression and localization of OCT4 in adherent cells and floating cells were detected by Immunofluorescence,and the expression of OCT4 in the two subpopulations was further detected by qPCR and Western blot(2)The morphological changes of adherent cells and floating cells were observed by Wright-Giemsa staining,the cytoskeleton was observed by Coomassie brilliant blue staining,and the morphology and distribution of F-actin were observed by phalloidin staining.Adhesion changes were compared by cell adhesion assay and cell separation assay.(3)Tissue specific gene expression profiles and proliferation-related gene expression profiles were analyzed by transcriptome sequencing,and GO function enrichment analysis was performed.(4)CCK8 assay was used to detect the proliferation ability of cells.The rate of cell clone formation was detected by two dimensional clone formation assay.Ki67 proliferation index was detected by Immunocytochemistry and the expressions of PCNA were detected by qPCR.(5)The expression of ZO-1,Act N2,E-cadherin and ?-catenin were detected by qPCR and Western blot,and the expression of Cyclin D1 was detected by qPCR.(6)si RNA knockdown ZO-1 contradicts the above experimental results.Results:(1)OCT4 was localized in the nuclei of two subpopulations,OCT4 was significantly overexpressed in the two subpopulations,and was higher in the floating cells.(2)After transduction of OCT4,the morphology and cytoskeleton of hHFMSC were changed from long spindles to short spindles or polygonal adherent subpopulation and small and round floating subpopulation.The microfilaments of cytoskeleton changed from relatively parallel fibrous to irregular network,and the morphology of filamentous actin changed from parallel bundle to coagulated block.The adhesion of hHFMSC was decreased after transduction of OCT4,and the adhesion of suspension cells was lower than that of adherent cells.(3)The down-regulated genes in the adherent cells and the floating cells were significantly enriched in the GO terms related to hair follicle and skin development,including KRT15,COL17A1,FZD3,FOXQ1,FGF7 and ALX4.The up-regulated genes in adherent cells and floating cells were significantly enriched in proliferation-related biological processes,including regulation of mitotic cell cycle,negative regulation of cell cycle arrest,regulation of cell growth,G1/S transformation of cell cycle,and regulation of stem cell proliferation.(4)The self-renewal ability and clone formation rate of cells were significantly improved after OCT4 transduction,and the proliferation ability and clone formation rate of adherent cells were significantly higher than that of floating cells.However,the positive percentage of Ki67 in adherent cells was lower than that in floating cells,and the m RNA expression of PCNA was also lower than that in floating cells.(5)The m RNA expressions of ZO-1,ACTN2,?-catenin and Cyclin D1 in the floating cells after OCT4 transduction were significantly higher than those in the adherent cells,but there was no significant difference in the expression of E-cadherin.(6)After ZO-1 silencing,the m RNA and protein expressions of ZO-1,ACTN2 and?-catenin in floating cells were significantly down-regulated,while the expression levels of E-cadherin and Cyclin D1 were not significantly changed.After ZO-1 silencing,the floating cytoskeleton was slightly extended,and the F-actin interleaved in a ring-like grid,and the tendency of aggregation around the nucleus was more obvious.Conclusion:OCT4 may remodeling cytoskeleton and adhesion through the molecular pathway of ZO-1/Actin/?-catenin,and may further regulate the expression of proliferation-related genes to affect the self-renewal of hHFMSC,and different levels of overexpression of OCT4 have different effects on the self-renewal of hHFMSC.Significance:The aim of this study was to explore the role of OCT4 in regulating the cytoskeletal and adhesion of hHFMSC through the ZO-1/Actin/?-catenin molecular pathway,and to further promote the self-renewal and reprogramming of hHFMSC.This study lays an important experimental foundation for the further study of the molecular mechanism of OCT4 regulating the balance between pluripotency and hematopoietic differentiation by remodeling cytoskeleton and adhesion,and provides a new research strategy for obtaining a large number of hematopoietic precursor seed cells that can be expanded in vitro.