Research On The Mechanism Of BRD4 Regulates EZH2 Transcriptionto Influence Biologicalfunction In Bladder Cancer

Bladder cancer, the second most common malignancy of the urinary tract, remains a virtually intractable disease worldwide. Although current targeted strategies range from transurethral resection to systemic chemotherapy are effective in a subset of patients, the overall therapeutic efficacy is still far from satisfactory, indicating the need for innovative therapeutic strategies. Inefficiency and relapse to targeted therapies in bladder cancer has been attributed to the complicated underlying molecularmechanisms that result in functional redundancy among survival pathways. Therefore, exploration of new gene dysregulations and novel therapeutic targets in bladder cancer would facilitate our understanding of the potential etiology and biology. As well-known conserved epigenome readers, the BET proteins link to chromatin remodeling, and function as transcription regulators in a context-dependent manner. The important BET family member BRD4 has been demonstrated to possess significant effect on cell biological activity. Recently, several selective small-molecule chemical compounds that target the acetyl-lysine binding of BET proteins have been developed to exhibit the anti-tumor effects in a variety of malignancies. The specific BET inhibitors have demonstrated apparent efficacy in blocking tumor progression in a range of cancer models. However, a role for BRD4 protein has yet to be reported in bladder cancer, and exploration of new regulatory pathways for BRD4 would broaden our understanding of its contribution in tumor growth.Therefore, in our study, we seek to explore the expression levels and potential role of BRD4 in bladder cancer and evaluate the underlying effect of pharmacologically inhibiting BRED4 protein in bladder cancer cells in vitro and in vivo, and try to extend the knowledge about the molecular mechanism underlying bladder cancer progression.Our study is comprised of the following three parts:Part I Study on expression of BRD4 in clinical specimens and cell linesof bladder cancerPurpose:We aimed to exam the difference of expression of BRD4 among human normal bladder tissues and bladder cancer tissues, normal SV-HUC-1 cell line and bladder cancer cell lines, and analysis the relationship between BRD4 and clinical characteristics in bladder cancer.Methods:1. Fifty-five pairs of fresh bladder cancer tissues and surrounding normal adjacent bladder tissues were selected from patients who underwent partial or radical cystectomy for urothelial carcinomas of bladder at our department. Using real time PCR and western blot, we explored the expression levels of BRD4 mRNA and protein among 55 paired tissue specimens and bladder cancer cell lines, and then we analyzed the relationship between BRD4 and clinical characteristics in bladder cancer.2. Subsequently, we randomly chose40 matched cases of bladder cancer tissues and examined the protein expression of BRD4 using IHC.Results:1. As compared with the normal bladder tissues, the expression levels of BRD4 mRNA was obviously up-regulated in bladder cancer tissues (p<0.05), and the expression of BRD4 protein was dramatically elevated in bladder cancer tissues (p<0.05). As compared with theSV-HUC-1, the expression of BRD4 mRNA and protein levels were both significantly up-regulated in EJ and T24 cells (p<0.05).2. Immunohistochemistry indicated that BRD4 was mainly accumulated in the cell nucleus of malignant cells, and the expression of BRD4 in urothelial carcinoma of bladder was strikingly higher than in normal tissues.3. BRD4 was up-regulated in 38 of 55 samples. BRD4 expression levels were up-regulated in 9 of 19 facial tumors, 29 of 39 invasive tumors,16 of 29 samples absent with lymph node metastasis,22 of 26 samples present with lymph node metastasis,34 of 48 samples absent with distant metastasis,4 of 7 samples present with distant metastasis.Conclusion:BRD4 is up-regulated in bladder cancer specimens and cell lines, and its expression is correlated with lymph node metastasis and tumor grade. These results revealed a potential role of BRD4 protein in promoting bladder cancer progression.Part Ⅱ Study on biological effects of BRD4 knockdown or BET inhibitor on bladder cancer cells both in vitro and in vivoPurpose:We aimed to explore the potential role of BRD4 in bladder cancer and evaluate the underlying effect of pharmacologically inhibiting BRD4 protein in bladder cancer cells in vitro and in vivo.Methods:1. The shRNAs targeting BRD4 were designed and then transfected into EJ and T24 cells. Cells were treated with different concentrations of JQ1. After the transfection or drug treatment, MTT assay was carried out to evaluate the cell viability, flow cytometry was performed to assess the cell cycle and cell apoptosis of bladder cancer cells, and EdU assay was used to evaluate the status of cell proliferation.2. Stable EJ cells transfected with BRD4 shRNA or negative control screened by puromycin were injected into mice to achieve tumor growth.For JQ1 treatment, EJ cells were used and mice were then injected with JQ1 or vehicle.Results:1. The MTT colorimetric assay exhibited that knockdown of BRD4 or JQ1 treatment decreased cell viability in bladder cancer cells.Inhibition of BRD4 using shRNAs or JQ1 treatmentinduced cell cycle arrest at G0/G1 phase in EJ and T24 cells. Meanwhile, JQ1 treatment also induced considerable cell apoptosis in EJ and T24 cells. Consistently, the EdU assay demonstrated that suppression of BRD4 inhibited proliferation of EJ and T24 cells.2. ShBRD4 injected mice showed a reduction in tumor weight and tumor volume at the end compared with their control groups. JQ1-treated mice displayed a considerable reduction in tumor weight and tumor volume at the end of the experiment.Conclusion:Inhibition of BRD4 attenuates proliferation and induces apoptosis of bladder cancer cells in vitro. Suppression of BRD4 impairs bladder cancer tumor growth in vivo. These observations demonstrated that inhibition of BRD4 using shRNAs or treatment with JQ1 could suppress cell biological activity in EJ and T24 cells both in vitro and in vivo, indicating the pro-tumor function for BRD4 in bladder cancer.Part III BRD4 regulates EZH2 transcription through up-regulation of C-MYC in bladder cancerPurpose:We sought to investigate the molecular mechanism of BRD4 on bladder cancer and explore the transcriptional regulation of EZH2.Methods:1. Using real time PCR and western blot, we verified the expression levels of EZH2 and C-MYC mRNA and protein among 55 paired tissue specimens and bladder cancer cell lines, and examined the protein expression of EZH2 and C-MYC using IHC.Then we analyzed the correlations between BRD4 and EZH2, BRD4 and C-MYC in bladder cancer, respectively.2. The EZH2 promoter reporter vector was designed and synthesized. After transfection with shBRD4 or on treatment with JQ1, the mRNA expression levels of EZH2 and C-MYC were analyzed, the EZH2 promoter activity was detected.3. EZH2 expression vector was designed and co-transfected with shBRD4 into EJ and T24 cells, or EZH2 expression vector was transfected into JQ1-treated EJ and T24 cells, then detected the cell cycle, cell apoptosis and proliferation.4. C-MYC expression vector was constructed and co-transfected with shBRD4 into EJ and T24 cells, or C-MYC expression vector was transfected into JQ1-treated EJ and T24 cells, then detected the expression levels of EZH2 mRNA and protein, and EZH2 promoter activity as well. 5. Chromatin immunoprecipitation (CHIP) was carried out subsequently to evaluate the specific bind between BRD4 and C-MYC promoter, C-MYC and EZH2 promoter, respectively.Results:1. We found that C-MYCand EZH2 were up-regulated in bladder cancer tissues, C-MYCand EZH2 expression were positively correlated with BRD4, respectively.2. The expression levels of C-MYCand EZH2 mRNA and protein were decreased in EJ and T24 cells upon transfection of shBRD4 or treatment with JQl, as evaluated by RT-PCR and western blot. Transfection of shBRD4 or JQl treatment resulted in decreased promoter activity of EZH2 evaluated by luciferase reporter system in EJ and T24 cells.3. Ectopic expression of EZH2 reversed cell-cycle arrest and cell apoptosis induced by BRD4 inhibition.4. Ectopic expression of C-MYC efficiently reversed the suppression of EZH2 protein and mRNA levels induced by JQ1 and BRD4 shRNA. Ectopic expression of C-MYC also reversed the attenuation of EZH2 promoter activity upon BRD4 inhibition.5. We found that BRD4 was enriched in the C- MYC promoter region, and there was no obvious recruitment of BRD4 to detected EZH2 promoter region in bladder cancer cells. ChIP experiments also showed that transfection of BRD4 shRNA or JQ1 treatment resulted in considerable loss of C-MYC recruitment to EZH2 promoter region.Conclusion:These data suggested that BRD4 could probably interact with C-MYC promoter to promote its expression, and subsequently enhance the direct binding of C-MYC to EZH2 promoter, which result in the up-regulation of EZH2 transcription.