Histone Demethylase KDM5A Maintains The Stability Of Mouse Embryonic Stem Cells

Background and PurposeEmbryonic stem cells(ESCs)are derived from the inner cell mass in the blastocyst stage.Due to their unlimited self-renewal ability in vitro,ESCs can differentiate into all types of adult cells,so they are one of the main cell sources for stem cell therapy.At present,stem cell therapy has made some remarkable progress in cardiovascular diseases,diabetes,spinal cord injury and other diseases,but there are many problems that need to be solved:such as genetic instability,tumorigenicity,poor differentiation efficiency of ESCs,high risk of immune rejection,etc.,which seriously hinder the clinical transformation of ESCs.Therefore,a thorough exploration of the molecular mechanism of ESCs maintaining stability is of great significance for the clinical transformation and application of ESCs.Recent studies have shown that post-translational modifications of histones,especially histone demethylation,play an indispensable role in maintaining the stability of ESCs.Among them,histone demethylase KDM2B(Lysine-specific histone demethylase 2B,KDM2B),KDM4B,KDM4C have all been proven to help maintain the stability of embryonic stem cells,and whether KDM5A plays a role in maintaining the stability of embryonic stem cells has not been clarified.Based on the similar structure and function between the members of the same protein family,we speculated that histone demethylase KDM5 A might help maintain the stability of embryonic stem cells,thus starting the research of this subject.This study aims to explore the mechanism of histone demethylase KDM5A in maintaining the stability of mouse embryonic stem cells,and to provide ideas and theoretical basis for the clinical transformation of stem cells.Method1.Verify whether KDM5A is expressed in mESCsCollect mouse embryonic fibroblasts and embryonic stem cells(E14TG2a cells in this study),and compare the expression differences of KDM5 A in the two kinds of cells by Western Blotting experiment.2.Explore the expression change of KDM5A during the spontaneous differentiation of mESCsmESCs were cultured in differentiation medium without leukemia inhibitory factors to induce their spontaneous differentiation,and then cells at different differentiation time points were collected.Western Blotting was used to detect the protein expression changes of KDM5A and pluripotency factors during the spontaneous differentiation of mESCs.QRT-PCR(Quantitative Real-time PCR)experiment was used to detect changes in mRNA expression of pluripotency factors and lineage-specific genes.3.Explore the influence of KDM5A on the stability of mESCsThe KDM5A knockdown mESCs line was constructed to observe whether its morphology has changed,and then a recovery experiment was performed to observe whether its morphology has recovered.At the same time,the alkaline phosphatase staining experiment was used to detect whether mESCs differentiated.In addition,qRTPCR experiments were used to detect changes in the mRNA expression of pluripotency factors and lineage-specific genes after KDM5A knockdown,to explore the effects of KDM5A on the pluripotency of mESCs.After KDM5A knockdown or overexpression in mESCs,Western Blotting experiments were used to detect the expression changes of cyclinDl and cyclinD2,to explore the influence of KDM5A on the cell cycle of mESCs.Through the clone formation experiment,it was detected whether the clone formation ability of mESCs would change with the knockdown of KDM5A.4.Explore proteins that interact with KDM5ABioinformatics software was used to predict the proteins that could interact with KDM5A,and the prediction results were verified by Co-Immunoprecipitation experiments.Result1.KDM5A is highly expressed in mESCsThe protein expression level of KDM5A in mouse embryonic stem cells was significantly higher than that in fibroblasts.2.KDM5A is down-regulated during the spontaneous differentiation of mESCsDuring the spontaneous differentiation of mESCs,the lineage-specific genes GATA2,GATA4,Foxa2 were activated;the expression of pluripotency factors Oct4,Nanog,and Sox2 was down-regulated;the expression of KDM5A was down-regulated.3.KDM5A plays an important role in maintaining the stability of mESCsKDM5A knockdown affected the morphology of mESCs,and a distinct differentiated phenotype appeared;the recovery experiment re-expressed KDM5A,and mESCs also restored their normal morphology.When KDM5A was knocked down,the alkaline phosphatase staining of mESCs was negative;after KDM5A was re-expressed,the alkaline phosphatase staining of mESCs returned positive.These results indicated that KDM5A affected the differentiation potential of mESCs cells.In addition,knockdown of KDM5A significantly down-regulated the expression of pluripotency factors Oct4,Nanog,Sox2,and Esrrb.Endoderm marker genes GATA4,Col4a2,HNF4A,Foxa2,and Sox7,mesendoderm marker genes SMAD4,SMAD2,and Nodal,mesoderm marker genes GATA2 and Eomes,as well as ectoderm marker gene Krt18 were significantly up-regulated,indicating that knockdown of KDM5A would promote mESCs to differentiate into three germ layers.At the same time,after knocking down KDM5A,the expression of cyclinDl and cyclinD2 in mESCs was significantly down-regulated;while KDM5A was overexpressed,and the expression of cyclinDl and cyclinD2 was significantly up-regulated,indicating that KDM5A affects the cell cycle of mESCs.In addition,when KDM5A was knocked down,the cloning ability of mESCs was significantly reduced.The above results indicate that KDM5 A affects the morphology,pluripotency,cell cycle and clone formation ability of mESCs,therefore playing an important role in maintaining the stability of mESCs.4.KDM5A interacts with EZH2 and Suz12,two important members of the polycomb group proteinConclusionHistone demethylase KDM5A plays an important role in maintaining the stability of mouse embryonic stem cells and interacts with EZH2 and Suz12,the important members of PcG protein..