BRD4调控EZH2转录对膀胱癌细胞生物学功能的影响及机制研究

发布时间：2018-06-01 12:39

本文选题：膀胱癌 + BRD4　；参考：《华中科技大学》2016年博士论文

【摘要】：膀胱癌是泌尿系统的第二大恶性肿瘤,在世界范围内仍然是一个棘手的难题。尽管目前手术治疗及辅助化学治疗等治疗手段对一部分病人有效,但膀胱癌的总体治疗效果仍然远远不能令人满意,因此探寻新的有效的治疗策略仍是有必要的。膀胱癌的复发及转移往往与其复杂的分子调控机制有关,发现异常的基因调控和新的治疗靶点可以帮助我们更好的理解膀胱癌的发病机制及生物学特性。BET蛋白家族能够重塑染色体结构,作为转录调控蛋白发挥重要的作用。BRD4是BET蛋白家族中的重要成员,已发现能够在多种肿瘤中调节肿瘤细胞的生物学活性。BET抑制剂能够阻断BET蛋白与染色体的结合,从而发挥抗肿瘤活性。在许多肿瘤中已经证实BET抑制剂能有效抑制肿瘤的生长,然而BRD4蛋自在膀胱癌中的作用仍然不明确,BET抑制剂对膀胱癌生物学活性可能的影响仍未报道。因此,明确BRD4在膀胱癌中的作用机制有助于我们更好地理解其在肿瘤发生发展中起到的作用。本课题中,我们将探讨BRD4在膀胱癌中的表达和作用,并通过体内外实验评估药物抑制BRD4蛋白对膀胱癌生物学功能的影响,进一步地补充和完善膀胱癌的分子调控机制。本课题分为以下三个部分：第一部分 BRD4基因在人膀胱临床标本和细胞系中的表达研究目的：检测BRD4基因在人正常膀胱组织与膀胱癌组织及细胞中的表达差异,分析其表达水平与膀胱癌临床分期分级可能存在的关系。方法：1.收集我院55例行膀胱癌根治切除术的手术标本,获取癌组织和配对正常膀胱组织,行荧光实时定量PCR(RTPCR)和western blot法分别检测并分析组织标本及细胞系中BRD4基因的mRNA水平和蛋白水平,并分析BRD4的表达水平与膀胱癌临床分期分级的关系。2.随机选取40例配对的膀胱正常组织与癌组织,使用免疫组织化学染色法(IHC)检测BRD4蛋白在正常膀胱与癌组织中的表达,并分析其差异。结果：1.与正常膀胱组织相比,BRD4基因mRNA水平在膀胱癌组织中整体上明显升高(p0.05)：BRD4蛋白表达水平在膀胱癌组织中大部分升高(p0.05)。以SV-HUC-1细胞为对照,膀胱癌细胞系EJ和T24细胞中的BRD4基因的mRNA和蛋白表达水平均显著上调(p0.05)。2.免疫组织化学染色结果表明：BRD4蛋白在膀胱癌组织中大部分表达上调；此外,BRD4蛋白表达主要集中于细胞核。3.55例膀胱癌病例中,38例BRD4呈高表达。19例浅表性癌中9例BRD4呈高表达,39例浸润性癌中29例BRD4呈高表达;29例无淋巴结转移癌中16例BRD4呈高表达,26例伴淋巴结转移癌中22例BRD4呈高表达；48例无远处转移癌中34例BRD4呈高表达,7例有远处转移癌中4例BRD4呈高表达。结论：在膀胱癌组织和细胞系中,BRD4基因显著高表达,其表达水平可能与淋巴结转移和肿瘤分级有关(p0.05)第二部分沉默BRD4及BET抑制剂对膀胱癌细胞生物学功能影响的体内外实验研究目的：研究BRD4基因在膀胱癌中的作用,检测基因干扰BRD4或BET抑制剂对膀胱癌细胞生物学功能的潜在影响。方法：1.构建并合成BRD4 shRNA以沉默BRD4,并转染入EJ和T24细胞。以不同浓度的BET抑制剂JQ1处理膀胱癌细胞,应用MTT来检测细胞生存率,流式细胞术来检测各处理组细胞周期和凋亡的变化,并应用EdU法来检测细胞增殖的变化。2.构建裸鼠膀胱癌异位移植成瘤模型,检测JQ1及沉默BRD4在裸鼠体内能否抑制膀胱癌的生长。结果：1.MTT实验、流式细胞周期检测、凋亡检测及EdU增殖实验结果显示在EJ与T24细胞中,沉默BRD4或JQ1药物处理可使两种膀胱癌细胞的生存率降低,细胞周期阻滞于G0/G1期(G0/G1细胞增加,S期减少),细胞增殖能力显著降低,凋亡率明显增加。2.裸鼠成瘤实验显示BRD4 shRNA及JQ1均可明显抑制膀胱癌的生长。结论：沉默BRD4基因或JQ1药物抑制BRD4均可对膀胱癌细胞的周期、凋亡、增殖及膀胱癌肿瘤模型的生长产生明显影响,BRD4对膀胱癌细胞生物学行为的调控可能起重要作用。第三部分膀胱癌中BRD4通过C-MYC调控EZH2转录的具体机制研究目的：探讨BRD4在膀胱癌中的作用机制,进一步完善和补充上游基因在转录水平对EZH2的调控机制。方法：1.行荧光实时定量PCR(RTPCR)和western blot法分别检测并分析组织标本中EZH2、C-MYC基因的mRNA水平和蛋白水平,使用免疫组织化学染色法(IHC)检测EZH2、C-MYC蛋白在正常膀胱与癌组织中的表达,并分别分析EZH2、C-MYC的表达水平与BRD4表达水平之间的相关性。2.构建EZH2启动子双荧光素酶报告载体,使用BRD4 shRNA沉默BRD,并转染EJ和T24细胞,或JQ1药物处理细胞后,检测EZH2、C-MYC基因mRNA和蛋白的表达变化,以及通过双荧光素酶实验检测EZH2启动子活性。3.设计“逆转”实验,构建EZH2过表达载体,将其和shBRD4共同转入到EJ和T24细胞,或将EZH2过表达载体转入到JQ1处理的细胞,检测EJ和T24的细胞周期、凋亡和增殖能力。4.构建C-MYC shRNA和过表达载体,将C-MYC过表达质粒和shBRD4共同转入到EJ和T24细胞,或将C-MYC过表达质粒转入到JQ1处理的细胞,检测EZH2基因mRNA和蛋白的表达变化,以及EZH2启动子活性的变化。5.CHIP实验检测BRD4与C-MYC启动子区域的结合、C-MYC与EZH2启动子区域的结合作用。结果：1.C-MYC及EZH2在膀胱癌组织中均高表达,他们的表达水平分别与BRD4的表达存在正性相关。2.ShRNA沉默BRD4或JQ1能够降低EZH2和C-MYC的mRNA和蛋白水平的表达,并能够抑制EZH2启动子活性。3.过表达EZH2能够部分逆转抑制BRD4引起的细胞周期阻滞,细胞凋亡和增殖能力的变化。4.过表达C-MYC能够部分逆转抑制BRD4引起的EZH2 mRNA、蛋白水平和启动子活性的变化。5.CHIP实验证实BRD4能够结合于C-MYC启动子区域,C-MYC能够结合于EZH2启动子区域。抑制BRD4能够减弱C-MYC与EZH2启动子区域的结合。结论：BRD4通过调控C-MYC的转录影响C-MYC与EZH2启动子区域的结合,进而促进EZH2转录,促进膀胱癌细胞增殖,抑制细胞凋亡。JQ1能够通过抑制C-MYC-EZH2通路,诱导膀胱癌细胞凋亡,抑制细胞增殖。
[Abstract]:Bladder cancer is the second major malignant tumor of the urinary system, which is still a difficult problem worldwide. Although surgical treatment and adjuvant chemotherapy are effective for some patients, the overall therapeutic effect of bladder cancer is still far from satisfactory. Therefore, it is still necessary to explore new effective treatment strategies. The recurrence and metastasis of bladder cancer is often related to its complex molecular regulation mechanism. It is found that abnormal gene regulation and new therapeutic targets can help us to better understand the pathogenesis and biological characteristics of bladder cancer. The.BET protein family can remould the structure of chromosomes and play an important role in the regulation of transcriptional regulatory proteins,.BRD4 It is an important member of the BET protein family. The biological activity of.BET inhibitors that can regulate tumor cells in a variety of tumors can block the binding of BET proteins to chromosomes and thus play an antitumor activity. In many tumors, BET inhibitors have been proved to effectively inhibit the growth of swelling tumors. However, BRD4 eggs are in the bladder cancer. The effect of BET inhibitors on the biological activity of bladder cancer is still not reported. Therefore, the mechanism of BRD4's role in bladder cancer will help us to better understand its role in the development of cancer. In this subject, we will explore the expression and role of BRD4 in bladder cancer and through the experiment in vivo and in vitro To evaluate the effect of drug inhibition BRD4 protein on biological function of bladder cancer and further complement and improve the molecular mechanism of bladder cancer. This topic is divided into three parts: the first part of the BRD4 gene expression in human bladder clinical specimens and cell lines: detection of BRD4 gene in human normal bladder tissue and bladder cancer The expression difference in tissue and cell was analyzed, and the possible relationship between the expression level and the clinical staging of bladder cancer was analyzed. Methods: 1. a total of 55 cases of radical resection of bladder cancer in our hospital were collected to obtain cancer tissue and matched normal bladder tissue. The fluorescence real-time quantitative PCR (RTPCR) and Western blot were used to detect and analyze the tissue respectively. The mRNA level and protein level of BRD4 gene in the specimens and cell lines, and the relationship between the expression level of BRD4 and the clinical staging of bladder cancer..2. randomly selected 40 matched normal bladder tissues and cancer tissues. The expression of BRD4 protein in normal bladder and cancer tissues was detected by immunohistochemical staining (IHC), and the difference was analyzed. Results: 1. compared with normal bladder tissue, the mRNA level of BRD4 gene increased significantly in bladder cancer tissue (P0.05), the expression level of:BRD4 protein was most elevated in bladder cancer tissue (P0.05). SV-HUC-1 cells were used as control. The mRNA and protein expression levels of BRD4 genes in the bladder cancer cell line EJ and T24 cells were significantly up (P0). .05).2. immunohistochemical staining showed that most of the expression of BRD4 protein in bladder cancer tissues was up-regulated; in addition, the expression of BRD4 protein was mainly concentrated in the.3.55 cases of bladder cancer in the nucleus, 38 cases of BRD4 showed high expression of.19 cases with high expression of BRD4, and 29 cases of BRD4 were highly expressed in 39 cases of impregnated cancer; 29 cases had no lymph nodes. 16 cases of metastatic carcinoma showed high expression of BRD4, 26 cases with lymph node metastasis and 22 cases of high expression of BRD4, 48 cases with no distant metastasis and 34 cases of high expression of BRD4, 7 cases of distant metastatic carcinoma, 4 cases of BRD4 expressed high expression. Conclusion: in bladder cancer tissues and cell lines, the BRD4 gene is highly expressed, its expression level may be associated with lymph node metastasis and tumor. Classification related (P0.05) second partially silencing the effects of BRD4 and BET inhibitors on the biological function of bladder cancer cells in vitro and in vivo: To investigate the role of BRD4 gene in bladder cancer and to detect the potential effect of gene interference on the biological function of bladder cancer cells by BRD4 or BET inhibitors. Methods: 1. construction and synthesis of BRD4 shRNA to sink in the bladder cancer cells. BRD4 and transfected into EJ and T24 cells. Bladder cancer cells were treated with BET inhibitor JQ1 with different concentrations. MTT was used to detect cell survival rate. Flow cytometry was used to detect the changes of cell cycle and apoptosis in each treatment group, and EdU method was used to detect cell proliferation in nude mice to construct a tumor heterotopic tumor model of bladder cancer in nude mice and detect JQ1 and Whether the silence of BRD4 could inhibit the growth of bladder cancer in nude mice. Results: the results of 1.MTT experiment, flow cytometry, apoptosis detection and EdU proliferation test showed that the survival rate of two bladder cancer cells decreased, the cell cycle was blocked at G0/G1 phase (G0/G1 cell increase, S phase decreased) in EJ and T24 cells. The proliferation ability of cells decreased significantly, the apoptosis rate increased obviously in.2. nude mice and showed that BRD4 shRNA and JQ1 could obviously inhibit the growth of bladder cancer. Conclusion: silent BRD4 gene or JQ1 drug inhibition of BRD4 can significantly affect the cycle, apoptosis, proliferation of bladder cancer cells and the growth of bladder cancer tumor model. BRD4 has a fine effect on bladder cancer. The regulation of cellular biological behavior may play an important role. Third the specific mechanism of BRD4 in the regulation of EZH2 transcription by C-MYC in bladder cancer: To explore the mechanism of BRD4 in bladder cancer and to further improve and supplement the regulation mechanism of the upstream gene at the transcription level to EZH2. Method: 1. lines of real-time quantitative quantitative PCR (RTPCR) and Weste RN blot method was used to detect and analyze the mRNA level and protein level of EZH2, C-MYC gene in tissue specimens. The expression of EZH2, C-MYC protein in normal bladder and cancer tissues was detected by immunohistochemical staining (IHC), and the correlation between the expression level of EZH2, C-MYC and the level of BRD4 surface was analyzed, and the dual fluorescence of the promoter was constructed. BRD4 shRNA was used to silence BRD, and EJ and T24 cells were transfected, or JQ1 drug treated cells were used to detect the expression of mRNA and protein in EZH2, C-MYC gene, and the "reverse" experiment was used to detect the activity.3. design of EZH2 promoter by double luciferase experiment. Cells, or EZH2 overexpressed vectors, were transferred into JQ1 treated cells to detect the cell cycle of EJ and T24, and the apoptotic and proliferative capacity.4. constructed C-MYC shRNA and overexpressed vectors to transfer C-MYC overexpressed plasmids and shBRD4 into EJ and T24 cells, or to transfer the C-MYC over expression plasmids to the cells treated, and to detect the genes and proteins. Changes in expression, and changes in the activity of EZH2 promoter.5.CHIP test to detect the binding of BRD4 to the promoter region of C-MYC and the binding of C-MYC to EZH2 promoter region. Results: 1.C-MYC and EZH2 are highly expressed in bladder cancer tissues, and their expression levels are in positive correlation with BRD4 expression in.2.ShRNA silencing BRD4 or JQ1 can be reduced. The expression of mRNA and protein levels of low EZH2 and C-MYC, and the inhibition of EZH2 promoter activity.3. overexpression EZH2 can partly reverse the inhibition of cell cycle arrest, apoptosis and proliferation ability of BRD4,.4. overexpression C-MYC can partly reverse the inhibition of EZH2 mRNA, protein level and promoter activity induced by BRD4 Experiments show that BRD4 can combine in the C-MYC promoter region, C-MYC can be combined with EZH2 promoter region. Inhibition of BRD4 can weaken the combination of C-MYC and EZH2 promoter region. Conclusion: BRD4 can promote the transcription of EZH2, promote the proliferation of bladder cancer cells and inhibit cell withering by regulating the binding of C-MYC transcription to C-MYC and EZH2 promoter region. .JQ1 can induce apoptosis of bladder cancer cells and inhibit cell proliferation by inhibiting C-MYC-EZH2 pathway.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R737.14

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