MTA Family Genes Regulate The Steady State Of Human Embryonic Stem Cells Through DYRK Signaling

The emergence of embryonic stem cells opens a new door in the field of life science research.The establishment of mouse and human embryonic stem cells provides infinite possibilities for the prospect of stem cell translational medicine.However,the development of human embryonic stem cells is faced with some problems,such as the difficulty to maintain stem cell homeostasis in vitro,the imperfect existing culture system,the low efficiency of differentiation into somatic cells and the unclear differentiation mechanism.In order to achieve the safe and effective application of stem cells in the future,it is urgent to solve these problems.Exploring and analyzing the function of specific genes in human embryonic stem cells is an important way to solve the maintenance of the pluripotency of human embryonic stem cells in vitro,the differentiation of stem cells into specific lineages and the application of stem cells in clinical treatment,which is very important for the early application of stem cellsMTA family genes(Metastasis-associated family gene)are a class of metastasisrelated genes,whose family members were first found in mouse breast cancer cells.MTA family genes play an important role in regulating the proliferation,growth,invasion,and metastasis of many tumor cells,and their expression has a certain correlation with the survival rate of cancer patients.MTA family genes include three main members: MTA1,MTA2 and MTA3.Although these three genes are homologous,they have some differences in physiological function.Besides,some studies have found that MTA family genes are involved in the formation of NURD(Nucleosome remodeling and histone deacetylation)complex and thus play multiple functions.In recent years,the function of MTA family genes has been gradually discovered in tumor cells.A clear understanding of their regulatory mechanisms is conducive to the development of anti-tumor drugs and has a positive help to cure cancer.The functional diversity of the MTA gene has been reported more frequently in the maintenance of mouse embryonic stem cell self-renewal.Previouly,we found that MTA1 plays an important role in the maintenance of the undifferentiated state of mouse embryonic stem cells,but the role of MTA family genes in the maintenance of human embryonic stem cell steady state has not been reported.Although both human and mouse embryonic stem cells are pluripotent stem cells,there are great differences in their biological characteristics,such as culture conditions and gene expression profiles.Therefore,our project will focus on the function of MTA family genes in maintaining the homeostasis of human embryonic stem cells.First,we used the PLKO.1 lentivirus system to construct shRNA,targeting three memebers of human MTA genes,and packaged the virus and infected normal human embryonic stem cells.The expression levels of MTA1,MTA2 and MTA3 in human embryonic stem cells was successfully down-regulated.Inhibition of MTA1 gene expression did not affect the state of human embryonic stem cells,but the original pluripotent homeostasis of human embryonic stem cells was destroyed when the transcriptional levels of MTA2 and MTA3 were suppressed.The clonal morphology of stem cells was destroyed and became flate.In addition,the alkaline phosphatase activity was gradually lost,indicating that these stem cells differentiated.Next,based on the above experimental phenotype,we used quantitative RT-PCR to detect the expression levels of the maker genes of endoderm,mesoderm and ectoderm to determine the effects of the MTA genes on the differentiation fate of human embryonic stem cells.The results showed that compared with the control cells,knockdown of the transcript of MTA2 or MTA3 genes significantly reduced the transcription levels of the pluripotent genes such as NANOG and OCT4.On the contrary,the expression levels of the marker genes of mesoderm(GATA4,GATA6,SOX17,FOXA2)and endoderm(GATA4,GATA6,SOX17,FOXA2)was higher,while the marker genes(NESTIN,OTX2,SOX1)of ectoderm was not changed,indicating that the loss of MTA2 and MTA3 expression mainly induced the emergence of endoderm and mesoderm in human embryonic stem cells.At the same time,we also overexpressed the expression of human MTA2 and MTA3 genes in human embryonic stem cells,but each gene could not maintain the undifferentiated state without addition of exogenous self-renewal maintenance factors Activin A and bFGF.These results suggest that MTA2 and MTA3 genes are necessary for human embryonic stem cells to maintain pluripotentency.Finally,we wanted to find out the molecular mechanism of human embryonic stem cell differentiation caused by the deletion of the MTA gene by adding small molecular inhibitors of different kinases.The results showed that addition of ID8 could effectively decrease the differentiation degree caused by down-regulation of the expression of MTA2 or MTA3.Alkaline phosphatase staining also showed that the number of positive clones increased in the presence of ID8 compared with the group without ID8 treatment.At the same time,the expression levels of the marker genes of mesendoderm were also inhibited by ID8 after knodowndown of MTA2 or MTA3 transcript.ID8 is a selective inhibitor of the DYRK(Dual-specificity tyrosine phosphorylation-regulated kinase),including DYRK1 A,DYRK1B,DYRK2,DYRK3 and DYRK4 members.Therefore,we used RNA interference to down-regulate each gene expression in MTA2 or MTA3 knockdown human embryonic stem cell lines.The results showed that DYRK2 could inhibit the differentiation of human embryonic stem cells induced by MTA2 shRNA,but could not reverse the differentiated phenotype caused by MTA3 shRNA.Notably,suppression of DYRK4 gene could prevent the differentiation induced by MTA2 or MTA3 deletion,indicating that the signal-regulated by DYRK could mediate the function of MTA2 and MTA3 on the regulation of the fate decision of human embryonic stem cells.In summary,our study revealed the role of MTA in regulating the pluripotentency of human embryonic stem cells and revealed part of the mechanism downstream of MTA2 and MTA3 knockdown.These results enrich the regulation theory of the pluripotency of embryonic stem cells and deepen our understanding of stem cell regulatory network,which will facilitates future application of stem cells.