The Function And Mechanism Of RBPJ In Decidualization Of Stromal Cells Related To Recurrent Pregnancy Loss

Objective This study aims to identify differentially expressed genes(DEGs)in the endometrium of recurrent pregnancy loss(RPL)population;reveal differentially expressed molecules in different endometrial cell subpopulations;study the dynamic expression of recombination signal binding protein for immunoglobulin kappa J region(RBPJ),a transcription factor of the Notch signaling pathway,in the peri-implantation endometrium;explore the function and molecular mechanism of RBPJ in regulating stromal cell decidualization,and provide a new theoretical basis for RPL prevention and treatment.Method 1.Analyze the microarray chip dataset of endometrium in RPL population using bioinformatics methods to obtain DEGs;verify the distribution of DEGs in different endometrial cell subpopulations in RPL endometrium by single-cell transcriptome sequencing technology;detect the expression of Notch signaling pathway transcription factor RBPJ and decidualization-related molecules in peri-implantation human endometrium and human endometrial stromal cell line(h ESCs)induced differentiation process by immunoblotting,QRT-PCR,and immunofluorescence methods;carry out functional studies on RBPJ regulation of decidualization by constructing h ESCs cell lines with inducible RBPJ deletion,and integrate and analyze sequencing results by bioinformatics methods through transcriptome sequencing(RNA-sequencing,RNA-Seq)and chromatin immunoprecipitation(Ch IP)sequencing technology to explore the molecular mechanism of RBPJ regulation of decidualization.Results 1.Analyze the microarray chip data of gene expression in endometrium and find some genes related to endometrial decidualization.For example,IL6 R,DDK2 and COX2 are up-regulated in the PRL group,and IL17 RD,IL16,SOX4,CREBBP and POFUT1 are down-regulated in the RPL group.Among the components of the Notch signaling pathway,downstream core transcription factor RBPJ shows a downward trend in the endometrium of the RPL group.2.The single-cell RNA sequencing results of endometrium in RPL patients show that RBPJ is widely expressed in various types of endometrial cells,among which decidual cells,immune cells and macrophages have higher expression levels.In addition,RBPJ has a downward trend in decidual cells of the RPL group.3.Immunohistochemical localization shows that RBPJ is widely expressed in various types of endometrial cells at different stages.In the early and middle stages of proliferation,RBPJ is mainly expressed in glandular epithelium and is also widely distributed in stromal cells;as time progresses,RBPJ expression in glandular epithelium and stromal cells becomes more obvious;during the process from proliferation to secretion,RBPJ still expresses in stromal and epithelial cells;during the mid-secretory phase and implantation window period,RBPJ is expressed more uniformly in stromal and epithelial cells;when the endometrium is about to shed in the late secretory phase,RBPJ shows a higher level of expression in stromal(decidual)cells.4.Treatment of h ESCs with NOTCH signaling pathway inhibitor DBZ inhibits decidualization,mainly manifested as that the cells do not change into an epithelial-like appearance morphologically,but still maintain a fibroblast-like appearance of stromal cells,showing a flat and cord-like distribution with less cytoplasm.At the same time,m RNA levels of decidualization marker molecules PRL and IGFBP1 are significantly reduced.5.Based on CRISPR-Bac system construction of RBPJ-deficient h ESCs cell lines,consistent with the inhibitor experiment,the cells also show impaired decidualization function after induction of decidualization,specifically manifested as significant reduction of m RNA levels of PRL and IGFBP1 and inhibition of protein expression of FOXO1 and IGFBP1.6.By analyzing the sequencing results of RNA-Seq and Ch IP,it was found that there were 201 genes whose expression changed significantly after RBPJ deletion directly bound to regulatory sequences:among them,FOXO1 showed consistent down-regulation of m RNA and protein after RBPJ deletion;In addition,we also noticed that after RBPJ deletion,SLC40A1,a key molecule regulating intracellular iron homeostasis,was down-regulated and TFRC was up-regulated.This causes an imbalance in cellular iron homeostasis and an increase in divalent iron ions in cells.Conclusions 1.In the population of recurrent miscarriage,RBPJ expression is reduced in the endometrium,and there is also a decrease in decidual cells.2.RBPJ is an important regulatory factor for decidual differentiation of endometrial stromal cells,which can directly regulate the expression of decidualization key transcription factor FOXO1.At the same time,RBPJ can also participate in the regulation of intracellular divalent iron ion homeostasis by directly regulating SLC40A1 and TFRC.