Preliminary Study On The Effect And Mechanism Of ATF6 On Endometrial Physiological Repair

In the non-pregnant state,the endometrium is jointly regulated by estrogen and progesterone,and undergoes periodic proliferation,differentiation,breakdown and shedding,and regular changes in regeneration and repair.This periodic disintegration,shedding,regeneration and repair,namely menstruation,constantly renew the endometrium,which is of great significance to women's reproductive health.In this thesis,a menstrual-like model of wild type(WT)and Activating Transcription Factor 6(ATF6)knock out(KO)pseudopregnant mice was established to explore the effects and mechanism of ATF6 on the physiological repair of the endometrium.It mainly includes three aspects:the expression level of ATF6 in the human menstrual period endometrium and the mouse menstrual-like model uterus,the effect of ATF6 knockout on the physiological process of mouse menstrual-like model endometrium,and the screening and verification of downstream genes of ATF6 during endometrial repair.1.The expression level of ATF6 in the endometrium of human menstrual cycle and the uterus of mouse menstrual-like model.Immunohistochemical staining revealed that human endometrial ATF6 has a higher expression level during menstruation and early proliferation.Subsequently,a mouse pseudopregnancy menstrual-like model was established,and the uterine tissues of Day 6,Day 7,and progesterone(P4)withdrawal for 24 h,32 h,40 h,and 48 h were collected.Gross and HE staining showed that the endometrium on Day 6 and Day 7 was in a state of decidualization,the endometrium after progesterone withdrawal for 24 h completely disintegrated,and the endometrium from 24 h to 48 h was in the proceeding of physiological repair.Immunohistochemical staining results,qRT-PCR results and Western blot results of nuclear and cytoplasmic protein showed that the expression level of ATF6 in the uterus of menstrual-like model significantly increased during 40?48 h.It suggests that the expression level of ATF6 increases during the physiological repair of the endometrium.2.The effect of ATF6 on the physiological repair process of the endometrium in pseudopregnant menstrual-like mice.Firstly,the genotype of KO mice was identified by RT-PCR,DNA electrophoresis,DNA sequencing and Western blot,and the reproductive ability of transgenic mice was preliminarily evaluated.Secondly,establish pseudopregnancy menstrual-like models on WT and KO mice,and extract Day 6 and Day 7 uterine tissues respectively,and use HE staining,immunohistochemical staining of PRL,qRT-PCR and western blot to determine the effect of ATF6 on decidualization.Uterine tissues were then collected at 24 h,32 h,40 h and 48 h after progesterone withdrawal.Comparing the gross and HE staining of the uterus of the two groups,as well as the immunohistochemical staining of Ki67 and CK17,it was found that knocking out ATF6 had no effects on the decidualization and disintegration of the endometrium,while it significantly delayed the physiological repair process of the endometrium.The phenotypic difference of the delayed physiological repair at 40 h was the most obvious.3.Screening and verification of downstream genes of ATF6.Frozen samples of the uterus at 40 h were selected for the expression profile of gene chip detection,and downstream genes were screened and verified through GO enrichment analysis of differentially down-regulated genes,KEGG enrichment analysis,ab initio prediction of Atf6 target genes,and PPI network analysis.The results of qRT-PCR and Western blot results of uterine tissues from 24 h to 48 h showed that the levels of mRNA and protein expression level of CXCR2 and MMP9 in the uterus of the two groups increased significantly at 40 h,which was consistent with the change trend of ATF6;and knocking out ATF6 decreased the mRNA and protein expression level of CXCR2 and MMP9.It suggested that ATF6 may activate the inflammatory factors CXCR2 and MMP9 to promote the physiological repair of the endometrium.Finally,the regulatory effects of ATF6 and its effects were verified at cell level of HESCs.The ATF6 of HESCs was knocked down/overexpressed by siRNA-lipo3000/plasmid-lipo3000,and the effect of knockdown/overexpression was verified by qRT-PCR and Western blot technology.The results of qRT-PCR and Western blot showed that the high/low mRNA and protein expression levels of CXCR2 and MMP9 were related to the increase/decrease of ATF6.The results of cell proliferation and migration experiments showed that knocking down ATF6 inhibited the proliferation and migration of HESCs,while overexpression failed to affect cell proliferation and migration.In summary,ATF6 may positively regulate inflammation-related genes CXCR2 and MMP9,promote cell proliferation and migration,and promote endometrial regeneration and repair.