The Mechanism Of Etv5 In The Regulation Of Imprinted Gene H19 And ERK Phosphorylation In Mouse Embryonic Stem Cells

ETS(E-twenty six)family plays an important role in various physiological processes,including cell proliferation,differentiation,migration,apoptosis and tumorigenesis.Etv5(ETS variant 5)is a member of the ETS family.It has a general expression in spermatogonial stem cells,tumor cells,testis and ovary.It is closely related to many development processes and diseases such as animal infertility,prostate cancer,endometrial cancer and so on.However,the role and mechanism of Etv5 plays in mouse embryonic stem cells(m ESCs)is not clear yet.This study was aimed to determine the expression profile of Etv5 and investigate the effect of Etv5 on pluripotency in mESCs,and to provide a theoretical basis for exploring the multifunctional regulatory mechanism of m ESCs.The main results of this study are as follows:1.The expression profile of Etv5 in different cell lines and tissues.By comparing the expression profile of Etv5 in different cell lines and tissues though Bio-GPS database,it showed that Etv5 has specificity high expression in pluripotent stem cells and lung tissues.The transcriptional activity of Etv5 in mESCs and MEFs was analyzed by using Chip-seq database,and the transcriptional activity of Etv5 in mESCs was higher than MEFs.Finally,it was found that Etv5 expressed highly in stem cells through RT-qPCR analysis.2.The effect of Etv5 on mESCs pluripotency.In this study,Etv5 knockdown mESCs lines(Etv5 KD mESCs)was established.The multifunctional differences between the Etv5 knockdown and the control group were compared by Ap staining,immunofluorescence,RT-qPCR,and Western Blot.It was found that there was no significant difference between the experimental group and the control group in terms of cell morphology,AP staining degree,and pluripotent gene expression.3.The effects of Etv5 on the regulation of lncRNA H19.Through high throughput analysis of lncRNAs,we compared the expression profile of lncRNAs between Etv5 KD mESCs and control group.By using the scattered point diagramand heatmap analysis,we found 16 lncRNAs that differentially expressed in Etv5 KD mESCs,including 7 upregulated lncRNAs and 9 downregulated lncRNAs.Through RT-qPCR analysis,we compared the expression of Etv5 and H19 in Etv5 KD mESCs,Etv5 rescue m ESCs and control group,and found there was a negative relationship between Etv5 and H19 in m ESCs.4.The regulatory effects of Etv5 on FGF/ERK signal pathways and Gata6.In this study,the Etv5 knockout mESCs(Etv5 KO mESCs)was established,through RT-qPCR and Western Blot,it was found that Etv5 knockout resulted in the significantly reduced of Fgfr2 and Gata6 expression in m ESCs.At the same time,the ERK phosphorylation(pERK)levels were significantly reduced as well.By using the PD inhibitors(PD0325901)to treat mESCs,it was found that the expression of Etv5 was significantly reduced after pERK was inhibited through time gradient and concentration gradient experiments.Conclusion: this study confirmed the high expression level of Etv5 in mESCs,and found Etv5 knockdown had no significant effect on pluripotency maintenance,demonstrated the negative regulatory relationship between Etv5 and H19,it was proved that Etv5 could regulate the expression of Gata6 by FGF/ERK signaling pathway to mediate the differentiation of the original endoderm layer of mESCs.This study enriched the regulatory function of Etv5 in mESCs and provided a theoretical basis for the study of the regulatory mechanism in early embryonic development.