Epigenetic Mechanism Of KDM3A Upregulation Of AR-mediated Gene Transcriptional Regulation And Its Role In Triple-negative Breast Cancer

Objective: Breast cancer is the most common tumor among women,and in 2020,female breast cancer has become the leading cause of cancer incidence worldwide with2.3 million new cases,accounting for 11.7% of all cancer cases.It is the fifth leading cause of cancer deaths worldwide with 685,000 deaths,posing a serious threat to women’s health.Triple negative breast cancer(TNBC)is a subtype with unique clinical and biological features characterized by the absence of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER2),and accounts for approximately 15-20% of all breast cancers,and its prognosis is poor.AR is a member of the steroid hormone receptor superfamily along with estrogen receptor,progesterone receptor and glucocorticoid receptor and is usually activated by binding to its ligands such as androgens.It may play a dual role in breast cancer,being thought to be involved in tumor suppression in ERα-positive(+)breast cancer and potentially pro-cancer in ERα-negative(-)breast cancer.Therefore,an in-depth study of the regulatory role and molecular mechanism of AR-mediated gene transcription will provide new ideas for the treatment and prognosis determination of TNBC patients.Lysine demethylase 3A(KDM3A),a member of the histone demethylase family,regulates gene transcription mainly through specific demethylation of histone H3K9me1/2.Current studies have confirmed that KDM3 A plays a pro-oncogenic role in a variety of tumors,but its specific mechanism of action is mainly through dehistone modification of the transcription factor binding region,altering the level of histone H3K9me1/2 modification and thus promoting downstream pro-oncogene transcription.In recent years,there have also been reports on the oncogenic role of KDM3 A in the development of gastric and nasopharyngeal cancers.However,the role of KDM3 A in TNBC and its mechanism are not clear.The aims of this study were to(1)clarify the expression of KDM3 A in TNBC;(2)elucidate the interaction between KDM3 A and AR;(3)investigate whether KDM3 A regulates AR-mediated gene transcription;(4)resolve the molecular mechanism of KDM3 A regulation of AR-mediated gene transcription;and(5)elucidate the role of KDM3 A in the developmental process of TNBC.Methods:(1)To detect the expression of KDM3 A in TNBC tissues by Western Blot and immunohistochemistry assay;(2)To clarify whether KDM3 A interacts with AR by protein immunoprecipitation assay(co-immunoprecipitation,co-IP),and to detect the co-localization of KDM3 A and AR in TNBC cells by immunofluorescence confocalization assay;(3)To analyze the regulatory effect of KDM3 A on AR-mediated gene transcriptional activity by dual luciferase assays;(4)To analyze the regulatory effect of KDM3 A on m RNA expression at the genome-wide level by RNA-seq assay,and to further clarify the regulatory effect of KDM3 A on AR downstream genes by Real-time q PCR and Western Blot;(5)Chromatin immunoprecipitation(Ch IP assay)was used To resolve the recruitment of KDM3A/AR to the promoter region of genes downstream of AR and the regulatory effect of KDM3 A on the level of histone modifications in this region;(6)MTT assay,cell cloning assay,scratch assay and Transwell assay to detect the effects of KDM3 A on biological functions such as growth,proliferation and migration of TNBC cells;(7)To detect the effects of KDM3 A on tumorigenesis of TNBC cells in mice by nude mouse tumor loading assay.Results:(1)Western Blot and immunohistochemistry results showed that KDM3 A was significantly low expressed in TNBC tissues;(2)Co-IP results showed that KDM3 A interacted with AR,and immunofluorescence confocal experiments confirmed the colocalization of KDM3 A and AR in TNBC cells;(3)Luciferase assays showed that KDM3 A up-regulates AR-mediated gene transcription through its demethylase activity;(4)RNA-seq assays showed that KDM3 A regulates AR downstream gene transcription at the genome-wide level,and Real-time q PCR and Western Blot verified the regulation of AR downstream gene expression by KDM3A;(5)Ch IP assay confirmed that KDM3 A or AR could be recruited to the promoter region of AR downstream genes,and knockdown of KDM3 A increased the modification level of histone H3K9me1 and H3K9me2 in the promoter region of AR downstream genes;(6)MTT assay,cell cloning assay,scratch assay and Transwell assay showed that knockdown of KDM3 A promoted the growth,proliferation,migration and other biological functions of TNBC cells;(7)nude mice tumor experiment confirmed that knockdown of KDM3 A promoted the growth of TNBC cells in mice.Conclusions:(1)KDM3A is significantly low expressed in TNBC,and low expression of KDM3 A is associated with poor prognosis in TNBC patients;(2)KDM3A can interact with AR,KDM3 A binds to AR through 489-765 aa or 1010-1321 aa regions,and KDM3 A binds directly to the 254-676 aa region of AR;(3)KDM3A upregulates ARmediated gene transcription through its demethylase activity;(4)KDM3A or AR can be recruited to the ARE of the promoter region of AR downstream genes(ATF4,AHNAK,WWC1),and knockdown of KDM3 A increases the level of histone H3K9me1/2 modification;(5)KDM3A inhibits the growth,proliferation and migration ability of TNBC cells.