ESRG Maintains The Self-renewal And Pluripotency Of Human Pluripotent Stem Cells In Collaboration With MCM2

Background and objectives:Human pluripotent stem cells(h PSCs),including human embryonic stem cells(h ESCs)and induced pluripotent stem cells(hi PSCs),are a class of pluripotent cells with the ability of self-renewal,and have the potential to differentiate into a variety of cells and tissues.In recent years,h PSCs have been widely used in basic research,cancer and congenital disease research,cell replacement therapy and other fields.To elucidate the mechanisms of self-renewal and pluripotency maintenance of h PSCs is of great significance for the study of embryo development and tissue engineering as well as the exploration of human health.ESRG(Embryonic stem cell related gene),a novel gene which was first cloned in our previous study,is highly expressed in h ESCs and is significantly decreased after differentiation.These results suggest that ESRG may play an important role in the maintenance of self-renewal and pluripotency of h ESCs and even h PSCs.In addition,online prediction websites and RNA pull-down combined with mass spectrometry were used to screen the proteins interacting with ESRG,and it was found that ESRG may interact with MCM2,a DNA replication licensing factor,suggesting that ESRG is likely to maintain the self-renewal of h PSCs in collaboration with MCM2.This study intends to further clarify the function of ESRG as long non-coding RNA(lnc RNA)in h PSCs and the mechanism of ESRG and MCM2 in maintaining the self-renewal of h PSCs,so as to lay a good foundation for revealing the biological function of ESRG and clarifying the molecular regulatory mechanism in maintaining the self-renewal and pluripotency of h PSCs.Methods:1.Study on the expression of ESRG in cells:(1)The coding ability of ESRG is predicted by bioinformatics softwares or online websites(CPC2,PORTRAIT,Phylo CSF and NONCODE)and verified by experiments.(2)The expression of ESRG in cells and fetal tissues was detected by q RT-PCR and Northern blot.(3)ISH and FISH assays were conducted to investigate the localization of ESRG in cells.Then,the nuclear RNA and cytoplasmic RNA were extracted respectively,and the localization of ESRG was further verified by q RT-PCR.2.Biological functions of ESRG in h PSCs:(1)The expression of ESRG in h ESCs and hi PSCs was knocked down by RNAi method and the cell morphology was observed.Then the self-renewal ability of h ESCs and hi PSCs was detected by flow cytometry for analysis of cell proliferation,cell cycle and apoptosis.(2)The expression of ESRG in h ESCs and hi PSCs was knocked down by RNAi method,and cell pluripotency was detected and analyzed by q RT-PCR,Western blot,teratoma and other methods.3.Study on the binding sites of ESRG and MCM2:(1)The co-location of ESRG and MCM2 in h PSCs was observed under confocal microscope by FISH and immunofluorescence assays.(2)The interation between ESRG and MCM2 was verified by RNA pull down combined with Western blot and RIP in h ESCs and hi PSCs.(3)Vectors containing different ESRG truncations and MCM2 domains were constructed,and the specific binding sites of ESRG and MCM2 were determined by RNA pull-down and other techniques in h PSCs and 293 T cells.4.Study on the regulatory relationship between ESRG and MCM2:(1)Immunofluorescence and confocal microscopy were used to observe the changes in the cellular localization of MCM2 before and after ESRG knockdown;the nuclear and cytoplasmic proteins of the cells were further extracted and the changes in the cellular localization of MCM2 were verified by Western blot.(2)The expression of ESRG in h PSCs was knocked down by RNAi method,and the expression level of MCM2 was detected by Western blot.Then the stability and ubiquitination of MCM2 before and after ESRG knockdown were analyzed by treatment with protein synthesis inhibitor,proteasome inhibitor and Co-IP assay.(3)In order to comprehensively analyze the influence of MCM2 on the function of ESRG in h PSCs,the rescue experiment was performed by knocking down ESRG and overexpressing MCM2 in h PSCs.The cell morphology was observed,and then the pluripotency markers were detected by Western blot.At the same time,the cell proliferation,cell cycle and apoptosis were detected by flow cytometry.5.Investigating the molecular mechanism of ESRG2-MCM2:(1)The common biological functions of ESRG and MCM2 in h PSCs were investigated,and the downstream pathways jointly regulated by ESRG and MCM2 were analyzed combined with RNA-seq.(2)After knocking down the expression of ESRG or MCM2,the key genes of the downstream pathway were inhibited,and the rescue experiment was performed to evaluate the self-renewal ability of h PSCs to verify the downstream pathway co-regulated by ESRG and MCM2.Results:1.According to website prediction and experimental verification,ESRG does not have the ability to encode protein;ESRG was only expressed in h ESCs and hi PSCs,and its expression decreased with the differentiation of h PSCs,which was similar to the changes of OCT4,SOX2 and NANOG.ESRG was mainly localized in the nucleus of h PSCs,and a small part was localized in the cytoplasm.2.Knockdown of ESRG expression in h PSCs results in loose and elongated cells with differentiation.At the same time,cell proliferation slowed down and apoptosis were observed,and cell cycle was arrested in G2/M phase.After knockdown of ESRG expression in h PSCs,the expression of pluripotency marker genes decreased,while the expression of endoderm,mesoderm,ectoderm and trophoblastic ectoderm increased;the volume of teratoma generated by subcutaneous injection of NSG mice was smaller than that of cells without knocking down ESRG.3.ESRG colocalizes and interacts with MCM2 in the nucleus of h PSCs;2001-3153 nt of ESRG can bind to 1-256 amino acids residues of MCM2.4.MCM2 was decreased in the nucleus and increased in the cytoplasm after knockdown of ESRG in h PSCs.After knocking down the expression of ESRG in h PSCs,the protein level of MCM2 was significantly downregulated,and the knocking down of ESRG promoted the binding of MCM2 to TRAIP(a ubiquitin ligase),leading to ubiquitination degradation of MCM2.Knockdown of ESRG and overexpression of MCM2 in h PSCs can partially rescue the reduction of cell pluripotency and self-renewal ability caused by ESRG knockdown.5.Knockdown of ESRG or MCM2 can cause DNA damage in h PSCs,and overexpression of MCM2 can rescue the DNA damage caused by ESRG knockdown to a certain extent;the KEGG enrichment pathway analysis after RNA-Seq showed that ESRG knockdown activates the p53 signaling pathway;inhibition of p53 can partially rescue the DNA damage and reduced cell proliferation caused by knockdown of ESRG or MCM2.Conclusions:1.ESRG is an lnc RNA that is highly and specifically expressed in h PSCs.2.Specific inhibition of ESRG results in the loss of self-renewal and pluripotency maintenance ability in h PSCs.3.The 2001-3153 nt of ESRG can specifically bind to the 1-256 amino acids residues of MCM2.4.ESRG can maintain the expression stability and nuclear localization of MCM2.5.Knocking down the expression of ESRG will lead to abnormal MCM2(ubiquitination and nuclear translocation),which will affect DNA replication,lead to DNA damage,and then activate the p53 signaling pathway,and ultimately lead to the loss of self-renewal and pluripotency maintenance ability in h PSCs.