Study On The Molecular Mechanism Of Phenolic Environmental Estrogens Promoting The Proliferation Of Uterine Leiomyoma Cells Based On High Throughput Omics

Part 1:The Influence of Phenolic Environmental Estrogen on the Transcriptome of Uterine Leiomyoma Cells:A Whole Transcriptome Profiling-Based AnalysisBackground:Uterine leiomyomas are the most common benign tumors in the female reproductive system.As a type of environmental endocrine disruptors,environmental estrogen enters the human body and accumulates through digestion and absorption,which increases the actual load of estrogen in the body and exerts its role as endocrine disruptors.Uterine fibroids are also targets of environmental estrogen.Humans are more exposed to environmental estrogen,such as Bisphenol A(BPA),Nonylphenol(NP)and other phenolic environmental estrogens,which are an important source of in vitro intake of estrogen.Existing studies have shown that phenolic environmental estrogens are related to the occurrence and development of uterine leiomyomas,while the effect and mechanism of action of phenolic environmental estrogens on the occurrence and development of uterine leiomyomas are still not very clear.Objective:The goal of this study is to deeply analyze the effect of BPA on the expression of key genes through epigenetic regulation,identify the key transcription factors in the proliferation of human leiomyoma cells induced by BPA,and verify their functions,so as to provide theoretical basis and new insights for the pathogenesis of endocrine disruptors exposed to BPA and other similar effects.Methods:After successfully constructing of primary uterine leiomyoma cells,CCK-8 was used to detect the effects of phenolic environmental estrogens BPA and NP on the proliferation of uterine leiomyoma cells,and flow cytometry was used to detect the effects of BPA and NP on the cell cycle of uterine leiomyoma cells.After the optimal intervention concentration and time were used to intervene uterine leiomyoma cells,the samples of BPA group,NP group and control group were collected for transcriptome high-throughput sequencing(RNA-seq)and data analysis.Those genes affected by BPA and NP were identified.Then,Gene Ontology(GO)enrichment,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment,and Protein-protein Interaction(PPI)analysis were performed.Quantitative real-time polymerase chain reaction(q-PCR)and western blot were used to verify the differentially expressed gene and protein.Results:Phenolic environmental estrogens BPA and NP promote the cell cycle progression and cell proliferation of human uterine leiomyoma cells.The sequencing results showed that the number of differentially expressed genes in BPA group was 1288 compared with the control group,and the number of differentially expressed genes in NP group was 3735 compared with the control group.The number of differential genes regulated by both NP and BPA is 739,including 255 up-regulated genes and 484 down-regulated genes.The GO enrichment analysis of 739 differentially expressed genes co-regulated by BPA and NP showed that the most enriched GO items were connective tissue development and G1/S transition of mitotic cell cycle,and extracellular matrix.The results of KEGG enrichment analysis showed that differentially expressed mRNA were enriched mainly in three primary pathways,including environmental information processing,human diseases,and cellular processes.The cell cycle,PI3K-AKT signaling pathway are significantly enriched.The qPCR and western blot verified the cell cycle associated genes and proteins were upregurated in both BPA group and NP group.Both BPA and NP activated the PI3K-AKT signaling pathway.Conclusion:After exposure to BPA and NP,the expression of genes E2F1,CCND1,ccne2,MCM2,MCM3,MCM4,MCM5 and MCM6 related to cell proliferation and cell cycle in uterine leiomyoma cells is up-regulated,and the activation of the PI3K-AKT signaling pathway may also play a key role.In addition,as an external stimulant,phenolic estrogen promotes the upregulation of inflammatory factors IL-6 and IL-8 in uterine leiomyomas.Part 2:Integrated RNA-seq and ChIP-seq analysis reveals XBP1 as a regulator in Bisphenol A induced human uterine leiomyoma cell proliferationBackground:BPA is the most researched representative phenolic environmental estrogen.The research results of the first part of this project show that BPA promotes the proliferation of uterine leiomyoma cells,and the high-throughput transcriptome sequencing results show that BPA causes changes in the gene expression levels of human uterine leiomyoma cells.After BPA intervention,human uterine leiomyoma cells need to undergo a series of complex gene regulatory networks to adapt to BPA interference quickly,accurately and harmoniously.This complex biological process is inseparable from the regulation of transcription and chromatin modification.Transcription factors and epigenetic modification play an extremely key role in this process.Objective:Our goal was to identify and characterize a new molecular target of BPA in uterine leiomyoma cells to better understand how this compound may affect uterine leiomyomas growth and development.Methods:Primary cultured cell lines of uterine leiomyoma were treated with 0.1%DMSO or 10.0 μmol/L BPA for 48 h before RNA-seq and histone 3 lysine 27 acetylation(H3K27ac)ChIP-seq were performed.Integrative analysis of ChIP-seq and RNA-seq data identifies the key transcription factor(TF),target gene and signaling pathway.To confirm the expression level of TF and target gene in uterine leiomyomas tissues,we analyzed 10 human uterine leiomyoma tissues and the matched adjacent uterine smooth muscle tissues using western blotting,and 96 paired paraffin-embedded human uterine samples by immunohistochemistry(IHC).The ChIP assay used to confirm the combination between the TF and target gene.In order to verify the functions of key TF,lentivirus was used to knock down the expression level and detect the effects on cell proliferation,cell cycle,and cell tumorigenicity.We further conducted Western blot analysis to confirm the whether the downstream signaling pathway is activated.Results:In the first part of the thesis,the differentially expressed genes after BPA intervention in uterine leiomyoma cells were found through RNA-seq.In this part,the genes with changes in acetylation modification level during BPA intervention in uterine leiomyoma cells were found through H3K27ac ChIP-seq.Integrative Analysis of ChIP-seq and RNA-seq data identifies the differentially expressed genes induced by H3K27ac modification and the key transcription factor XBP1 and its target gene ITGA2.XBP1 and ITGA2 is highly expressed in human uterine leiomyomas.In addition,knocking down the expression of XBP1 can inhibit the proliferation of uterine leiomyoma cells in vivo and in vitro and inhibit the proliferation-promoting effect of BPA on uterine leiomyoma cells.IHC results confirmed that ITGA2 was significantly related to the expression of XBP1(R=0.6708,P<0.001),and ChIP assay showed that XBP1 binds to the predicted promoter region of ITGA2.The enrichment analysis of KEGG signaling pathway show that the PI3K/AKT signaling pathway is significantly enriched,and ITGA2 is a gene enriched in the PI3K/AKT signaling pathway,so further experiments show that the downstream PI3K/AKT pathway is activated.Conclusion:In conclusion,our current work reveals a novel mechanism by which BPA promotes the proliferation in uterine leiomyoma cells.The XBP1 transcription factor regulates and activates the downstream ITGA2/PI3K/AKT pathway.By defining XBP1 as an important regulatory role of BPA in uterine leiomyoma cell proliferation,providing new insights into the pathogenesis related to the exposure to BPA and other endocrine disruptors acting similarly,and XBP1 may serve as a candidate molecular target for intervention and treatment of uterine leiomyomas.