| Objective: BAP18,a traditional member of MLL1/MOF complex,is a critical histone reader which had the capacity of recognizing histone H3K4me3 loci to act its function.BAP18 has also been considered as a potential interaction of NURF(Nucleisome remodeling factor).For now,we identified BAP18 as a novel androgen receptor(AR)co-activator in Drosophila system which enhanced AR-mediated transactivation activity,playing an important role in prostate cancer and castration resistant prostate cancer process.However,other functions of BAP18 protein especially acting as a histone reader also need further exploration.Belonging to a nuclear receptor superfamily,ER? experiences conformations changes and translocates from the cytosol to the nucleus for binding to specific estrogen response elements(EREs)to regulate gene transcription upon estrogen treatment.Estrogen-dependent gene expression recruits a series of highly organized complexes,including various transcription factors(TFs),epigenetic enzymes,and chromatin remodeling to be essential for breast cancer process.Estrogen-induced genes,such as MYC,MTA3,and CARM1 play vital roles in cell-cycle regulatory,tumor metastasis and endocrine treatment resistance in ER?-positive breast cancer.Thus,further exploring the molecular mechanism of regulation of ER?-E2 signaling pathway in breast cancer would be extremely essential for identification of novel therapeutic targets of ER? positive breast cancer and endocrine resistance.Female breast cancer is one of the most common malignant tumors in women in cohorts.Clinical diagnosis data had revealed that more than three quarters of female breast cancer patients indicating estrogen receptor positive,and over 80% of them showed high expression of ER?.Since estrogen receptor has a role in promoting the occurrence,development and metastasis of breast cancer mainly through estrogen-mediated gene activation,anti-estrogen therapy is often adopted in patients with positive ER?.However,about 20% of ER? positive patients in clinical treatment showed congenital resistance to or insensitivity to endocrine therapy,and more than half of patients underwent long-term anti-estrogen therapy turned to treatment resistance with malignant transformation.Numerous studies have shown that ER?-mediated gene transcription in anti-estrogen therapy resistance plays an important role,and the abnormal expression of auxiliary adjustment factor is one of the main reasons for abnormal transcription factors mediated gene transcription,suggest we found new ER? auxiliary adjustment factor and to elucidate its effect on transcription factors mediated gene transcription regulation could be revealed ER?-positive endocrine therapy of breast cancer drug resistance or become the new therapeutic targets.In this study,our objectives were :(1)Clarify the role of BAP18 in breast cancer process;(2)Clarify the genome-wide distribution of BAP18 as HR in breast cancer cells,and the characteristics of BAP18 binding sites and the regulation of endogenous genes;(3)Explore whether BAP18 is a new co-regulator of ER? and to regulate gene transcription mediated by ER?;(4)Explore the deeper molecular mechanism by which BAP18 regulates ER? mediated gene transcription;(5)Explore the biological function of BAP18 in the development and anti-endocrine therapy of breast cancer.Methods:(1)The relationship between the protein expression level of BAP18 and the prognosis and survival of tumor patients was analyzed by immunohistochemical(IHC)staining,and the protein expression level of BAP18 in clinical breast cancer patients was detected by western blotting and IHC,so as to clarify the relationship between BAP18 and the disease classification and disease severity of breast cancer;(2)Through high-throughput genome-wide protein chromatin immunoprecipitation sequencing(Ch IP sequence),the characteristics of BAP18 distribution and its correlation with ER? recruitment and H3K4me3 modification level were investigated.And real-time q-PCR and western blotting were used to verify that BAP18 affected ER?-E2 signaling pathway and gene expression with estrogen treatment;(3)Protein co-immunoprecipitation assays(co-IP)were used to determine whether BAP18 interacts with ER?,immunofluorescence assays were used to verify the localization of BAP18 and ER? in breast cancer cells,and then double-luciferase reporter gene assays were used to verify the effect of BAP18 on transcriptional activity of ER? mediated genes;(4)Explore the molecular mechanism of BAP18's influence on ER?-E2 signaling pathway.Detect BAP18,ER? and COMPASS complex interactions between the members of the core complex by co-immunocoprecipitation experiments.And then using chromatin immunocoprecipitation(Ch IP)assays to validate knockdown of BAP18 whether affected core members of COMPASS complex function on ER? Target gene transcription activity.Finally using dual luciferase report gene experiments to detect BAP18 and COMPASS compounds respectively affect ER? mediated gene transcription activity;(5)Biological function experiments were conducted to verify the effect of BAP18 on the proliferation of breast cancer cell lines and subcutaneous tumorigenesis growth in nude mice.The effect of BAP18 on endocrine therapy resistance in vivo was checked by drug feeding treatment.Finally,immunohistochemistry of subcutaneous tumor-forming tissues in mice,real-time q-PCR and western blotting were utilized to verify the relationship between the proliferation,drug resistance and BAP18 in breast cancer.Results:(1)Use tissue microarray assays by immunohistochemical method to analyze the expression of BAP18,ER?,PR and HER2 protein with histological grading.Patients with high expression of BAP18 had higher clinical grade and worse prognosis than those with low expression.Western blotting and immunohistochemical experiments showed that the expression of BAP18 in breast cancer tissues was significantly higher than that in adjacent tissues and had close inter-relationship with ER?;(2)Ch IP sequence of genome-wide BAP18 recruitment in ER? positive breast cancer cells MCF7 with or without E2 stimulation,to explore the characteristics of genome-wide BAP18 recruitment sites.The results showed that most of the recruitment sites of BAP18 were located in the proximal promoter region of the gene in the presence or absence of E2.From the gene clusters with the recruitment sites of BAP18 in the promoter region,we found classical ER? regulatory genes such as CCND1,MYC and KCNK5,and non-classical signaling pathway genes such as EGF and CCK.Sequence analysis of binding sites indicated that BAP18 may interact with transcription factors such as half-ERE,Sp1 and Ap1,or interact with DNA-binding proteins such as CTCF or BORIS to affect gene transcription.Through the analysis of gene recruitment peak,it was found that BAP18 was recruited in the proximal promoter region of these genes,which was closely related to H3K4me3 level;(3)In MCF7 and T47 D cells of HEK-293 and ER? positive breast cancer cell lines,we found that BAP18 could interact with ER? and up-regulate the transcriptional activity of ER?.Through immunofluorescence localization experiments,it was found that BAP18 and ER? could co-locate in the nucleus under E2 stimulation;(4)Use the protein immunoprecipitation experiment,we found that BAP18 could interact with COMPASS complex and ER? without affecting the interaction between COMPASS complex and ER?.Using Ch IP assays experiment,we found that the use of silencing RNA to reduce BAP18 thus affected the recruitment of COMPASS complex in MYC-ERE and PS2-ERE regions and affected the modification level of H3K4me3 and H4 ac.Using luciferase double reporter experiments,it was found that the expression plasmids of co-transfecting BAP18 and COMPASS complex members WDR5,ASH2 L and DPY30 could respectively enhance the up-regulation effect of COMPASS complex on transcriptional activity of era-mediated gene;(5)in ER? positive breast cancer cells MCF7,T47 D and BT474,lentivirus knockdown of BAP18 was injected into immunodeficient mice for double axillary tumorigenesis and Tamoxifen was fed to the experimental group.It was found that knockdown of BAP18 could significantly reduce the Tamoxifen resistance of BT474 cells and reverse the treatment sensitivity of breast cancer cells to Tamoxifen.Through the Cell growth curve experiment(Cell growth curves assays)and Cell clone formation experiment(Cell colony formation)prove BAP18 on low to slow ER? positive cells in the body into a tumor(nude mice experiment)and the growth of the external environment,the treated ectopic tumors for IHC and q-PCR experiments,results show that after knocking at low BAP18 MYC in ectopic tumors,such as CCND1 gene expression.Conclusion:(1)BAP18 is highly expressed in ER?-positive breast cancer with poor prognosis;(2)As a histone reader,BAP18 is recruited in gene promoter region in the majority of the whole genome and is associated with gene transcriptional activation;(3)BAP18 can interact with ER? and up-regulate gene transcription mediated by ER?;(4)As a bridge protein,BAP18 can interact with the COMPASS complex and co-recruit to the hormone response element regulating the downstream target gene of ER? to change the modification level of histone H3K4me3 and H4 ac.Moreover,BAP18 can up-regulate the regulation of era-mediated gene transcription by COMPASS complex members ASH2 L,WDR5 and DPY30,respectively;(5)BAP18 can promote the growth of ER? positive breast cancer cells and enhance the sensitivity of ER? positive breast cancer cells to the treatment of Tamoxifen. |
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