Effects Of EIF3d On Proliferation And Differentiation Of Mouse Embryonic Stem Cells

Mouse embryonic stem cells are derived from the inner cell mass of preimplantation blastocysts,which can be cultured in vitro under appropriate conditions,providing an effective in vitro model for exploring early embryonic development in vivo.Mouse embryonic stem cells have the ability of self-renewal and differentiation,can keep their undifferentiated state and proliferate indefinitely during the culture process,and have the potential to differentiate into any somatic cell.At present,the self-renewal and differentiation of mouse embryonic stem cells are mainly studied through four directions:transcript network,non-coding RNA,proteomics and epigenetic regulation.The previous work of our research group has discovered some RNA binding proteins that are highly expressed in mouse spermatogonial stem cells.Because mouse embryonic stem cells and spermatogonial stem cells have many similarities in self-renewal and pluripotency,we want to study these the role of RNA binding protein in mouse embryonic stem cells.Through mutation screening in mouse embryonic stem cells,we discovered the translation initiation factor e IF3d.The current research on e IF3d mainly focuses on translation regulation and cancer cell proliferation regulation.The research on e IF3d in pluripotent stem cells is still There are many unknowns,and this is what we want to study in this article.In order to study the role of e IF3d in mouse embryonic stem cells,we used CRISPR-Cas9technology to construct e IF3d mutant mouse embryonic stem cells Through the identification and analysis of cell genotypes,we found that the e IF3d^+/-mouse embryonic stem cell model was constructed.Through cell counting and cell cycle detection,it was found that the number of e IF3d^+/-cells was reduced and blocked in the G2/M phase,indicating that the proliferation ability of e IF3d^+/-mouse embryonic stem cells had declined;by detecting clone formation and clone size,it was found that e IF3d^+/-The number of cell clones is reduced,and the clone area is significantly smaller,indicating that the cloning ability of e IF3d^+/-mouse embryonic stem cells has decreased;by detecting cell apoptosis,it is found that the number of e IF3d^+/-cell apoptosis has increased.In order to study the effect of e IF3d on the pluripotency and differentiation of mouse embryonic stem cells,we tested the changes of pluripotency marker genes and differentiation marker genes in e IF3d^+/-mouse embryonic stem cells at the molecular level.Under LIF culture conditions with feeder cells,no difference in gene expression was observed.Interestingly,under2i culture conditions with the addition of PD0325901 and CHIR99021 small molecule inhibitors,Otx2 expression increased.Through research on the differentiation ability of mouse embryonic stem cells,it is found that after removing LIF,e IF3d^+/-mouse embryonic stem cells can inhibit cell differentiation,and embryoid body differentiation experiments have found that e IF3d^+/-embryoid body can successfully express external Marker genes of germ layer and endoderm,but it has a strong inhibitory effect on the expression of mesoderm marker genes.To further verify the above conclusions,we overexpress e IF3d in mouse embryonic stem cells.It is found that after over-expression of e IF3d,the state of mouse embryonic stem cells is more prone to differentiation,the proliferation ability has been strengthened,and the expression of Otx2 has recovered to a certain extent.These data more fully illustrate the validity of our conclusions.In order to study the effect of e IF3d on the transcriptome of mouse embryonic stem cells,we performed transcriptome sequencing on wild-type and e IF3d^+/-mouse embryonic stem cells.It was found that there were 592 down-regulated genes in e IF3d^+/-mouse embryonic stem cells,and 896 up-regulated genes.Differentially expressed genes are mainly related to the development process and the regulation of stress response,and are mainly enriched in cancer cell pathways,cell membrane-related signaling pathways,and immunological-related signaling pathways.In summary,by studying e IF3d^+/-mouse embryonic stem cells and e IF3d overexpressing mouse embryonic stem cells,we found that:(1)e IF3d can regulate the proliferation of mouse embryonic stem cells;(2)when embryonic stem cells cultured in 2i media,e IF3d can inhibit the expression of Otx2;(3)e IF3d can affect the differentiation of mouse embryonic stem cells.Studying the role of e IF3d in mouse embryonic stem cells provides a new direction for in-depth exploration of pluripotent gene regulatory networks,and also provides theoretical support for understanding the role of translation initiation factors in pluripotent stem cells.