OCT4 Maintains Self-renewal And Inhibits Senescence In Human Hair Follicle Mesenchymal Stem Cells Through DNMTs/p21 Signal

Due to the abundance of supplies,safe access and low immunogenicity,human hair follicle mesenchymal stem cells（hHFMSCs）are considered to be a promising source of cells for the rapidly emerging field of regenerative medicine and to be particularly beneficial for personalized cell therapy.However,hHFMSCs lost the self-renwel ability and enter into a state of replicative senescence after a certain period of cell culture.And it will greatly limit the application of hHFMSCs.Thus,fully understanding the molecular mechanisms involved in the regulation of hHFMSC self-renewal would provide a great advancement in the application of these cells.The key to maintaining self-renewal of stem cells is to maintain the proliferation and multipotential differentiation,and inhibit cell differentiation and senescence.The regulation of stem cell self-renewal and its properties resides in conserved transcriptional regulatory networks and epigenetic modifications,such as DNA methylation,that work together to repress developmental genes and activate stemness genes.OCT4,one of the most critical transcription factors,plays a pivotal role in maintaining self-renewal.OCT4 regulates multiple pathways though different target genes to maintain self-renewal.OCT4 also regulates G1/S transition,the checkpoint of cell cycle progression,which is necessary for stem cells self renewal,to maintain self-renewal.As the checkpoint of cell cycle progression,G1/S transition is regulated by G1regulatory molecules.Among these regulatory molecules,cyclin-dependent kinase inhibitors（CDKI）play a key role in regulating G1/S transformation.p21 is a member of CDKIs and plays critical roles in the regulation of the G1/S transition.The expression of p21 can be regulated by OCT4-targeted DNA methyltransferases（DNMTs）,which play distinct roles in gene regulation and maintaining pluripotency properties.We speculate that OCT4 maintains hHFMSCs self-renewal through the downregulation of p21 by DNMTs.AimTo discuss the role and mechanism of OCT4 in maintaining self-renewal and inhibiting senescence in hHFMSCs.Methods1.The effects of OCT4 on self-renewal and senescence in hHFMSCsa.Establishment of the hHFMSC cell line with ectopic expression of OCT4(hHFMSCs^OCT4)To determine the role of OCT4 in the maintenance of hHFMSC stem cell properties,hHFMSCs were infected with lentiviruses encoding OCT4 or GFP（a scramble control）.Hereafter,hHFMSCs infected with scrambled lentiviruses are referred to as hHFMSCs^EGFP and hHFMSCs transduced with lentiviruses encoding OCT4 are referred to as hHFMSCs^OCT4.RT-qPCR,western bolt and flow cytometry were used to detect the expression of OCT4 in hHFMSCs^OCT4.Immunofluorescence was performed to test the expression and location of OCT4 in hHFMSCs^OCT4.b.The effects of OCT4 on self-renewal in hHFMSCsCell proliferation,population doubling time,colony formation assays,and sphere formation assay were performed to detect hHFMSCs proliferation.Flow cytometry was used to detect cell cycle.Western bolt was ultilized to test the expressions of PCNA and cyclin D1.Immunofluorescence was used to test the expressions of Ki67.Osteogenic differentiation assay was performed to demonstrate hHFMSCs osteogenic differentiation potential.RT-qPCR was ultilized to test the expression of development genes.c.The effects of OCT4 on senescence in hHFMSCsTransmission electron microscopy was used to detect the influence on cell ultrastructure.SA-β-gal staining assay was ultilized to demonstrate the expression ofβ-galactosidase.RT-qPCR was performed to test the expressions of p21,p16 and hTERT.2.The mechanism of OCT4 maintains self-renewal and inhibits senescence in hHFMSCsa.Identification of differentially expressed genes in hHFMSCs^OCT4 by next-generation sequencingNext-generation sequencing（NGS）was utilized to illustrate the potential molecular mechanisms by which OCT4 promotes self-renewal and reverses senescence in hHFMSCs.GO enrichment was used to the function of enrichment genes.KEGG was proformed to analyze the enrichment signaling pathways.b.OCT4 maintains self-renewal and inhibits senescence through downregulation of p21Lentiviruses were used to overexpress p21 in hHFMSCs^OCT4,and the cells were verified by qRT-PCR and western blot.CCK8 and colony formation assays were performed to detect hHFMSCs^OCT4 proliferation.Western bolt was ultilized to test the expressions of PCNA and cyclin D1.Immunocytochemistry was used to test the expressions of Ki67.Osteogenic differentiation assay was performed to demonstrate hHFMSCs^OCT4 osteogenic differentiation potential.SA-β-gal staining assay was ultilized to demonstrate the expression ofβ-galactosidase.RT-qPCR was used to test the expressions of p16 and hTERT.c.Mechanism of OCT4 regulate the expression of p21RT-qPCR and western blot were used to test the different expressions of DNMT1,DNMT3a and DNMT3b in hHFMSCs^OCT4.ChIP assay was performed to detect whether the transcriptional factor OCT4 bound to the promoter regions of DNMTs in hHFMSCs.MSP assay was ultilized to illustrate DNA methylation state of the p21promoter region.d.OCT4 maintains self-renweal and inhibits senescence through DNMTs/p21 signal in hHFMSCsTo further characterize the role of DNMTs in the stem cell phenotypes maintained by OCT4 in hHFMSCs,the DNMT inhibitor 5-aza-dC and zebularine were applied to hHFMSCsOCT4.Western blot was used to determine the optimal concentration and time of 5-aza-dC and zebularine suppression of DNMT activities.And the optimal concentration and time were performed in the following experiments.Western blot was used to test the expression of DNMT1,DNMT3a,DNMT3b and p21.MSP assay was performed to illustrate the methylation state of the p21 promoter region.CCK8 and colony formation assays were ultilized to detect hHFMSCs^OCT4 proliferation.Flow cytometry was used to detect cell cycle.Western blot was used to test the expressions of PCNA and cyclin D1.Immunocytochemistry was performed to test the expression and location of Ki67.Osteogenic differentiation assay was ultilized to demonstrate osteogenic differentiation potential.RT-qPCR was ultilized to test the expression of development genes.Transmission electron microscopy was used to detect the effect on cell ultrastructure.SA-β-gal staining assay was performed to demonstrate the expression ofβ-galactosidase.RT-qPCR was ultilized to test the expressions of p21,p16 and hTERT.Results1.The effects of OCT4 on self-renewal and senescence in hHFMSCsa.Establishment of the hHFMSC cell line with ectopic expression of OCT4(hHFMSCs^OCT4)The hHFMSCs cell line with ectopic expression of OCT4 was established successfully.The expression of OCT4 was markedly higher in hHFMSCs^OCT4 than in control cells,and OCT4 was located in the nuclei of the cells.b.The effects of OCT4 on self-renewal in hHFMSCsExpression of OCT4 leaded to the increased proliferative capacity,shorter population doubling time,and higher colony formation and sphere formation efficiency.OCT4 leaded to a shortened G1 phase and an extended G2 and S phases,and promoted the cell cycle progression.The expressions of PCNA,cyclin D1 and Ki67 were upregulated.OCT4 promoted osteogenic differentiation abillity in hHFMSCs.The expressions of development genes were downregulated.c.The effects of OCT4 on senescence in hHFMSCsIn hHFMSCs^OCT4,the cells were enriched in microvilli throughout the entire cell surface,and the nuclei were regular in shape,containing a single nucleolus or multiple nucleoli.Euchromatin was abundant in the nucleus.The cytoplasm was enriched with rough endoplasmic reticulum,mitochondria and glycogen granules.These data indicate that the vigorous proliferative status of hHFMSCs^OCT4 was maintained.OCT4 inhibited the expression of SA-β-gal.OCT4 inhibited the expressions of p21and p16,and upregulated the expression of hTERT.2.The mechanism of OCT4 maintains self-renewal and inhibitssenescence in hHFMSCsa.Identification of differentially expressed genes in hHFMSCs^OCT4 by next-generation sequencingGO analysis results showed that the upregulated DEGs were significantly enriched in“G1/S transition of mitotic cell cycle”.KEGG analysis showed that the upregulated DEGs were enriched in“cell cycle”.b.OCT4 maintains self-renewal and inhibits senescence through downregulation of p21Following p21 overexpression,cells with p21 overexpression became enlarged and flat.Proliferation and colony formation abillities of the cells were suppressed,PCNA and Ki67 were downregulated,but the expression of cyclin D1 was not influenced in hHFMSCs^OCT4.The osteogenic differentiation capacity was inhibited with p21 overexpression compared with that of the control.SA-β-gal staining was increased when p21 was overexpressed in hHFMSCs^OCT4.p16 was also upregulated,and hTERT was downregulated.c.Mechanism of OCT4 regulate the expression of p21DNMT1,DNMT3a and DNMT3b were upregulated in hHFMSCs^OCT4 at both the mRNA and protein levels.The result of ChIP assay confirmed that OCT4 was interacts with the promoter regions of DNMT1,DNMT3a,and DNMT3b.MSP assay showed that the methylation level of the p21 promoter region was markedly higher and the amount of the promoter that was not methylated was lower in hHFMSCs^OCT4than in hHFMSCs^EGFP.d.OCT4 maintains self-renweal and inhibits senescence through DNMTs/p21 signal in hHFMSCsFollowing the treatment of 5-aza-dC/zebularine,the expressions of DNMT1,DNMT3a and DNMT3b were downregulated,while the expression of p21 was downregulated.The methylation of the p21 promoter region was downregulated and that the amount of the promoter that was not methylated was upregulated.Proliferative rate and colony formation capacity were decreased in hHFMSCs^OCT4.These cells also displayed a G1 phase arrest and a low PI.The expression of cyclin D1,PCNA,and Ki67 were downregulated.Additionally,the osteogenic differentiation capacity of hHFMSCs^OCT4CT4 was also blocked.The expressions of development genes were upregulated.Electron microscopy showed that the cells manifested in a senescent state and that the level of positive SA-β-gal staining was high when DNMTs were inhibited in hHFMSCs^OCT4.p21 and p16 were upregulated,and hTERT was downregulated.Conclusion1.OCT4 maintains self-renewal ability and inhibits senescence in hHFMSCs.2.OCT4 upregulates DNMT1,DNMT3a,and DNMT3b through interacting with their promoter regions,then promotes the methylation level of the p21 promoter region to inhibit the p21 expresssion,and then maintains self-renewal ability and inhibits senescence in hHFMSCs.