| Background:Prostate cancer(PCa)remains an important public health concern.As the global population continues to grow and ageing,the incidence of prostate cancer continues to grow.With an estimated 174650 new cases diagnosed and 31620 death cases in America.PCa has become the second most common carcinoma in males worldwide and the most common malignancy in developed countries,only lower than that of lung and bronchial diseases.In China,the incidence of prostate cancer is lower than that of developed countries such as the United States,but it has also shown a significant upward trend in recent years.Established risk factors for this condition include age,race,family history,genetics,and obesity.Currently,radical prostatectomy and radiotherapy are the gold standard treatments for men with early-stage prostate cancer.However,some patients managed with these approaches will subsequently exhibit disease recurrence or metastasis.Once the disease progresses,the first line of therapy becomes androgen deprivation,not a curative treatment for patients with advanced tumors.Hence,exploring signaling pathways in detail and formulating novel targeted therapies is of vital importance in enhancing the survival of patients with advanced PCa.BRD4,the most-studied member of the BET family,binds to acetylated lysine residues(Kac)on histones via its bromodomains.It recruits chromatin remodeling and transcription factors,as well as other related proteins,to specific transcriptional sites.This leads to transcription elongation through the modulation of RNA polymerase Ⅱ.It has been reported to play vital roles in cell proliferation,cycle and inflammation.Carcinogenesis induced by the BRD4 protein was first confirmed in NUT midline cancer.BRD4 binds to testicular nucleoprotein to form an oncogenic protein,which leads to the occurrence of malignant testicular nucleoprotein.Studies have subsequently revealed that BET inhibitors exhibit significant anti-tumor effects in various malignancies.Therefore,to clarify the role of BRD4 in prostate cancer and the biological effects of BET inhibitors on prostate cancer cells will help us better understand its development in prostate cancer.Objectives:1.The expression levels of BRD4 in prostate cancer cell lines and tumor specimens2.The biological effect of BET inhibitors and BRD4 knockdown on prostate cancer cell lines3.The mechanism for the biological effects of BRD4 inhibition in prostate cancer.Methods:1.The Cancer Genome Atlas(TCGA)and Starbase version 2.0 was used to investigate the expression of BRD4 in numerous types of cancer.2.A total of 46 pairs of fresh cancerous prostate tissue and adjacent normal tissue specimens were obtained from patients undergoing surgical treatment in the Department of Urology,Renmin Hospital of Wuhan University.RT-PCR and Immunohistochemistry assays were used to determine the protein expression of BRD4.The clinical characteristics of patients with PCa,the clinical data(Age,Preoperative PSA,Gleason score,Tumor stage,Metastasis)obtained from 46 patients with PCa were also analyzed.3.RT-PCR and western blot assays were used to detect the mRNA and protein expression of BRD4 in RWPE-1,PC3,DU145 and LNCAP cell lines.4.BRD4-targeted shRNA was expressed in lentiviral vector vectors,lentiviral shRNA-containing plasmids were transfected into DU145 and LNCAP cell lines.The cell proliferation was determined by CCK-8 assay,the colony formation capacity of DU145 and LNCAP cells was evaluated,cell cycle and apoptosis were detected by flow cytometry assay,wound healing and transwell assays were used to evaluate cell migration and invasion.5.RT-PCR and western blot assays were used to detect the mRNA and protein expression levels of MYC,Bcl-2,P21 and Cyclin D1 in DU145 and LNCAP cells after transfected with sh-BRD4 or treated with JQ1;RT-PCR and western blot assays were employed to evaluated protein expression levels of MYC and P21 in DU145 and LNCAP cells after transfected with si-p21,then treated with JQ1 or transfected with sh-BRD4.6.RT-PCR and western blot assays were used to detect the mRNA and protein expression levels of p53 and FOXO1 in DU145 and LNCAP cells after transfected with sh-BRD4 or treated with JQ1;DU145 and LNCAP cells were transfected with si-p21,then treated with JQ1 or transfected with sh-BRD4,then cell cycle and apoptosis were determined by flow cytometry assay;the expression levels of FOXO1 and P21 were evaluated by RT-PCR and western blot assays.7.A nude mice model of prostate cancer was constructed,then tumor volume and weight were determined,western blot assay was employed to detect FOXO1,p21 and MYC expression,immunohistochemistry assay was used to determine FOXO1 and p21 expression.Results:1.Data from the Pan-Cancer Analysis Platform indicated that BRD4 was upregulated in numerous types of cancer.2.BRD4 protein expression levels were markedly upregulated in prostate cancer tissues.The clinical data obtained from 46 patients with PCa were analyzed.Among 46 patients with prostate cancer,26 patients with prostate cancer had high expression of BRD4 and 20 patients with low expression of BRD4.Among the 26 cases of T3/T4 cases,18 cases had high expression of BRD4,and 8 cases of 20 cases of T1/T2 stage were highly expressed.Of the 19 patients with cancer metastasis,15 had high expression of BRD4 and 11 of 27 patients with non-cancerous metastasis had high expression.3.when compared with RWPE1 cells,the mRNA and protein expression levels of BRD4 were significantly increased in PCa cell lines(DU145,PC3 and LNCAP).4.The findings form CCK-8 assay suggested that inhibition of BRD4 via shRNA or treatment with JQ1 significantly attenuated cell proliferation of DU145 and LNCAP cells.The results form colony formation assay revealed that BRD4 inhibition significantly suppressed colony formation compared with in the negative control.The results of cell cycle analysis revealed that the majority of shBRD4-treated or JQ1 treated cells were in the G0/G1 phase;The results of cell apoptosis analysis showed that transfected with sh-BRD4 or treated with JQ1 significantly increased apoptosis compared with in the control;Transwell assay and wound healing revealed a significant decrease the ability of cell invasion and migration treated with JQ1 or transfected with shBRD4.5.RT-PCR and western blot assays revealed that BRD4 inhibition via JQ1 treatment or shBRD4 transduction significantly decreased c-Myc and Bcl-2 expression levels while increased p21 and cyclin D1 expression.Reduction of c-Myc expression via BRD4 inhibition may involve p21,and that p21 is an upstream regulator of c-Myc,which may be directly affected by BRD4 inhibition.6.The results indicated that the induction of p21 by BRD4 inhibition may not be mediated by p53,but by FOXO1 and FOXO1 may be a transcription factor that induces p21 when BRD4 is suppressed.7.JQ1-treated or sh-BRD4 transfected mice exhibited a significant reduction in tumor volumes and weights compared with in the control;The data form western blot assay showed that FOXO1 and P21 expression were upregulated,MYC expression was downregulated;Immunohistochemistry assay showed that the expression of FOXO1 and p21 were increased.Conclusion:1.Overexpression of BRD4 in PCa cell lines and tumor specimens,BRD4 expression exhibited a significant association with the tumor clinical stages and metastasis of PCa.2.Inhibition of BRD4 via short hairpin RNA or JQ1 induces cell cycle arrest and apoptosis,and mitigates the invasion of PCa cell lines.3.The anti-tumor effects of BRD4 inhibition in PCa may be mediated via the FOXO1-p21-Myc signaling axis.4.Inhibition of BRD4 delays tumor growth in PCa mouse models via the upregulation of FOXO1 and P21 and downregulation of MYC. |
PDF Full Text Request