The Cooperative Regulation Mechanism Of ERAP2 And ER? And The Function Of ERAP2 In Breast Cancer

Objective: At present,breast cancer is a high incident malignant tumor that threatens women's physical and mental health.Its morbidity and mortality are increasing rapidly all over the world,and the age of onset is getting younger.A large number of in-depth studies have been conducted on breast cancer.In particular,ER positive breast cancer,which accounts for over 70% of all breast cancers,has attracted the most attention.ER can accept estrogen stimulation,promote the development and progression of breast cancer.ER is a member of the nuclear receptor superfamily,including ER? and ER?.There are three subtypes of ER?,namely ER? 66,ER? 46 and ER? 36.ER? 66 is the full length expression of ER?,and it is also the most studied ER subtype in breast cancer.Generally,ER? of an article refers to ER? 66.The structure of ER? includes: non-ligand independent activation function1;DNA binding domain(DBD);Ligand binding domain(LBD);ligand-dependent activation function 2.AF-2 can combine with different ligands to form different configurations,so as to determine the co-activators and co-repressors that need to be combined for the transcription of target genes,thus promoting or inhibiting the transcription of target genes.Co-regulators play an core role in the regulation of ER target genes.Instead of altering gene sequences,the co-regulators induce structural changes by chromatin remodelling or histone modification,which keep the chromatin in an active or non-active state and promote or inhibit the gene transcription.Markers of chromatin activation include high levels of H3K4 me,H4K16Ac,and so on.They can change chromatin from a folded state to an open state and facilitate transcription.ERAP2 is a component of a variety of methyltransferase complexes.It forms a complex named WRAD with WDR5,ASH2 L and RbBP5,which is the conservative subunit in SET1/MLL complex family.The SET1/MLL complex family plays the important role of H3K4 methylation,and it is believed that it catalyzes the majority of H3K4 methylation in cells.In the absence of WRAD complex,SET1/MLL subunits only have a weak function of H3K4 monomethylation.When it combines with WRAD complex,it have a strong effect on promoting H3K4 dimethylation and trimethylation.It might have something to do with their spatial conformation.WRAD complex and SET1/MLL form a channel-like structure,while H3K4 is bound and fixed in this channel.The catalytic domain of SET1/MLL binds to H3K4,which results in the methylation of H3K4.Especially in the case of increased ERAP2,H3K4 trimethylation level significantly increased and became more stable.The downstream target genes regulated by ERAP2 were more coincident with ER?.We hypothesized that ERAP2 may act as a co-activator of ER? to regulate the transcription of ER? target genes.We conducted relevant studies around this hypothesis,confirmed the conjecture,and analyzed the biological function of ERAP2 in breast cancer cells and tissues,providing new ideas and possible targets for the treatment of breast cancer.Methods:1.Differentially expressed genes between the breast cancer tissues and the adjacent normal tissues were screened out in the GEO database by data mining.Then combined with the differentially expressed genes of early recurrence and late recurrence of ER positive breast cancer for analysis.The top 50 differentially expressed genes in common of the three databases were selected.Finally,the 36 th ERAP2 was selected as the main factor of the study.2.Dual-luciferase reporter assay is performed to examine the regulation of ER? transcription by ERAP2 with or without estrogen treatment in HEK-293,MCF-7 and T47 d cells.3.To examine the regulation of ER? target genes transcription and protein by ERAP2 with Realtime PCR and western blot.4.The interaction between ERAP2 and ER? was determined by co-immunoprecipitation assay.5.The intracellular localization and co-localization of ERAP2 and ER? were examined by immunofluorescence assay with confocal laser scanning microscope.6.Chromatin immunoprecipitation assay was performed to detect whetherERAP2 can bind to the ERE region of ER? target genes as ER? coactivator.7.To construct a T47 d and MCF-7 cell line with ShERAP2 lentivirus.To clarify the biological function fo ERAP2 in breast cells,Clony formation assay,MTS assay and flow cytometry were performed.8.Western blot was performed to detect the ERAP2 expression in breast cancer tissues and adjacent normal tissues.The expression of ERAP2 in ER positive breast cancer tissues was detected by immunohistochemistry assay.The role of ERAP2 in breast cancer was confirmed by the analysis of ERAP 2 expression with clinicopathological and prognostic data.Results:1.The dual-luciferase reporter assay confirmed that ERAP2 upregulate ER?transcriptional activity.2.The knockdown of ERAP2 reduced the transcription level of some ER?target genes,such as c-MYC,CyclinD1,CDK2 and so on.3.Co-immunoprecipitation assay was performed to determine the interaction of ERAP2 and ER? in vivo.4.Confocal laser scanning was performed to detect the localization of ERAP2 and ER?.The localization of ERAP2 is in the nuclear.The localization of ER? is mainly in the cell plasma without E2 and in the nuclear with E2.5.Chromatin immunoprecipitation assay was performed to detect that ERAP2 can bind to the promotor of ER? target genes as ER? coactivator.ERAP2 also increased the level of H3K4me3.6.ERAP2 can promote the Clony formation,proliferation and cell cycle of breast cancer cells.7.Compared with the adjacent normal tissues,the expression of ERAP2 increases in breast cancer tissues.Immunohistochemistry assay was performed to examine the expression level of ERAP2 in ER? positive breast cancer section.The expression level is corelated with tumor size,TNM stage(P<0.05).Conclusions:1.ERAP2 as a coactivator of ER? promotes ER? induced gene transcription by binding with ER? and improving the level of H3K4me3.2.ERAP2 coordinately regulates the expression of ER? target genes: CyclinD1,c-MYC,CyclinE,CDK2,TFF1 and VEGF in breast cancer cells.3.ERAP2 promotes the proliferation of ER? positive breast cancer cells.