本文選題：膀胱癌 + BRD4��； 參考：《華中科技大學(xué)》2016年博士論文

【摘要】：膀胱癌是泌尿系統(tǒng)的第二大惡性腫瘤,在世界范圍內(nèi)仍然是一個(gè)棘手的難題。盡管目前手術(shù)治療及輔助化學(xué)治療等治療手段對(duì)一部分病人有效,但膀胱癌的總體治療效果仍然遠(yuǎn)遠(yuǎn)不能令人滿意,因此探尋新的有效的治療策略仍是有必要的。膀胱癌的復(fù)發(fā)及轉(zhuǎn)移往往與其復(fù)雜的分子調(diào)控機(jī)制有關(guān),發(fā)現(xiàn)異常的基因調(diào)控和新的治療靶點(diǎn)可以幫助我們更好的理解膀胱癌的發(fā)病機(jī)制及生物學(xué)特性。BET蛋白家族能夠重塑染色體結(jié)構(gòu),作為轉(zhuǎn)錄調(diào)控蛋白發(fā)揮重要的作用。BRD4是BET蛋白家族中的重要成員,已發(fā)現(xiàn)能夠在多種腫瘤中調(diào)節(jié)腫瘤細(xì)胞的生物學(xué)活性。BET抑制劑能夠阻斷BET蛋白與染色體的結(jié)合,從而發(fā)揮抗腫瘤活性。在許多腫瘤中已經(jīng)證實(shí)BET抑制劑能有效抑制腫瘤的生長(zhǎng),然而BRD4蛋自在膀胱癌中的作用仍然不明確,BET抑制劑對(duì)膀胱癌生物學(xué)活性可能的影響仍未報(bào)道。因此,明確BRD4在膀胱癌中的作用機(jī)制有助于我們更好地理解其在腫瘤發(fā)生發(fā)展中起到的作用。本課題中,我們將探討B(tài)RD4在膀胱癌中的表達(dá)和作用,并通過(guò)體內(nèi)外實(shí)驗(yàn)評(píng)估藥物抑制BRD4蛋白對(duì)膀胱癌生物學(xué)功能的影響,進(jìn)一步地補(bǔ)充和完善膀胱癌的分子調(diào)控機(jī)制。本課題分為以下三個(gè)部分：第一部分 BRD4基因在人膀胱臨床標(biāo)本和細(xì)胞系中的表達(dá)研究目的：檢測(cè)BRD4基因在人正常膀胱組織與膀胱癌組織及細(xì)胞中的表達(dá)差異,分析其表達(dá)水平與膀胱癌臨床分期分級(jí)可能存在的關(guān)系。方法：1.收集我院55例行膀胱癌根治切除術(shù)的手術(shù)標(biāo)本,獲取癌組織和配對(duì)正常膀胱組織,行熒光實(shí)時(shí)定量PCR(RTPCR)和western blot法分別檢測(cè)并分析組織標(biāo)本及細(xì)胞系中BRD4基因的mRNA水平和蛋白水平,并分析BRD4的表達(dá)水平與膀胱癌臨床分期分級(jí)的關(guān)系。2.隨機(jī)選取40例配對(duì)的膀胱正常組織與癌組織,使用免疫組織化學(xué)染色法(IHC)檢測(cè)BRD4蛋白在正常膀胱與癌組織中的表達(dá),并分析其差異。結(jié)果：1.與正常膀胱組織相比,BRD4基因mRNA水平在膀胱癌組織中整體上明顯升高(p0.05)：BRD4蛋白表達(dá)水平在膀胱癌組織中大部分升高(p0.05)。以SV-HUC-1細(xì)胞為對(duì)照,膀胱癌細(xì)胞系EJ和T24細(xì)胞中的BRD4基因的mRNA和蛋白表達(dá)水平均顯著上調(diào)(p0.05)。2.免疫組織化學(xué)染色結(jié)果表明：BRD4蛋白在膀胱癌組織中大部分表達(dá)上調(diào)；此外,BRD4蛋白表達(dá)主要集中于細(xì)胞核。3.55例膀胱癌病例中,38例BRD4呈高表達(dá)。19例淺表性癌中9例BRD4呈高表達(dá),39例浸潤(rùn)性癌中29例BRD4呈高表達(dá);29例無(wú)淋巴結(jié)轉(zhuǎn)移癌中16例BRD4呈高表達(dá),26例伴淋巴結(jié)轉(zhuǎn)移癌中22例BRD4呈高表達(dá)；48例無(wú)遠(yuǎn)處轉(zhuǎn)移癌中34例BRD4呈高表達(dá),7例有遠(yuǎn)處轉(zhuǎn)移癌中4例BRD4呈高表達(dá)。結(jié)論：在膀胱癌組織和細(xì)胞系中,BRD4基因顯著高表達(dá),其表達(dá)水平可能與淋巴結(jié)轉(zhuǎn)移和腫瘤分級(jí)有關(guān)(p0.05)第二部分沉默BRD4及BET抑制劑對(duì)膀胱癌細(xì)胞生物學(xué)功能影響的體內(nèi)外實(shí)驗(yàn)研究目的：研究BRD4基因在膀胱癌中的作用,檢測(cè)基因干擾BRD4或BET抑制劑對(duì)膀胱癌細(xì)胞生物學(xué)功能的潛在影響。方法：1.構(gòu)建并合成BRD4 shRNA以沉默BRD4,并轉(zhuǎn)染入EJ和T24細(xì)胞。以不同濃度的BET抑制劑JQ1處理膀胱癌細(xì)胞,應(yīng)用MTT來(lái)檢測(cè)細(xì)胞生存率,流式細(xì)胞術(shù)來(lái)檢測(cè)各處理組細(xì)胞周期和凋亡的變化,并應(yīng)用EdU法來(lái)檢測(cè)細(xì)胞增殖的變化。2.構(gòu)建裸鼠膀胱癌異位移植成瘤模型,檢測(cè)JQ1及沉默BRD4在裸鼠體內(nèi)能否抑制膀胱癌的生長(zhǎng)。結(jié)果：1.MTT實(shí)驗(yàn)、流式細(xì)胞周期檢測(cè)、凋亡檢測(cè)及EdU增殖實(shí)驗(yàn)結(jié)果顯示在EJ與T24細(xì)胞中,沉默BRD4或JQ1藥物處理可使兩種膀胱癌細(xì)胞的生存率降低,細(xì)胞周期阻滯于G0/G1期(G0/G1細(xì)胞增加,S期減少),細(xì)胞增殖能力顯著降低,凋亡率明顯增加。2.裸鼠成瘤實(shí)驗(yàn)顯示BRD4 shRNA及JQ1均可明顯抑制膀胱癌的生長(zhǎng)。結(jié)論：沉默BRD4基因或JQ1藥物抑制BRD4均可對(duì)膀胱癌細(xì)胞的周期、凋亡、增殖及膀胱癌腫瘤模型的生長(zhǎng)產(chǎn)生明顯影響,BRD4對(duì)膀胱癌細(xì)胞生物學(xué)行為的調(diào)控可能起重要作用。第三部分膀胱癌中BRD4通過(guò)C-MYC調(diào)控EZH2轉(zhuǎn)錄的具體機(jī)制研究目的：探討B(tài)RD4在膀胱癌中的作用機(jī)制,進(jìn)一步完善和補(bǔ)充上游基因在轉(zhuǎn)錄水平對(duì)EZH2的調(diào)控機(jī)制。方法：1.行熒光實(shí)時(shí)定量PCR(RTPCR)和western blot法分別檢測(cè)并分析組織標(biāo)本中EZH2、C-MYC基因的mRNA水平和蛋白水平,使用免疫組織化學(xué)染色法(IHC)檢測(cè)EZH2、C-MYC蛋白在正常膀胱與癌組織中的表達(dá),并分別分析EZH2、C-MYC的表達(dá)水平與BRD4表達(dá)水平之間的相關(guān)性。2.構(gòu)建EZH2啟動(dòng)子雙熒光素酶報(bào)告載體,使用BRD4 shRNA沉默BRD,并轉(zhuǎn)染EJ和T24細(xì)胞,或JQ1藥物處理細(xì)胞后,檢測(cè)EZH2、C-MYC基因mRNA和蛋白的表達(dá)變化,以及通過(guò)雙熒光素酶實(shí)驗(yàn)檢測(cè)EZH2啟動(dòng)子活性。3.設(shè)計(jì)“逆轉(zhuǎn)”實(shí)驗(yàn),構(gòu)建EZH2過(guò)表達(dá)載體,將其和shBRD4共同轉(zhuǎn)入到EJ和T24細(xì)胞,或?qū)ZH2過(guò)表達(dá)載體轉(zhuǎn)入到JQ1處理的細(xì)胞,檢測(cè)EJ和T24的細(xì)胞周期、凋亡和增殖能力。4.構(gòu)建C-MYC shRNA和過(guò)表達(dá)載體,將C-MYC過(guò)表達(dá)質(zhì)粒和shBRD4共同轉(zhuǎn)入到EJ和T24細(xì)胞,或?qū)-MYC過(guò)表達(dá)質(zhì)粒轉(zhuǎn)入到JQ1處理的細(xì)胞,檢測(cè)EZH2基因mRNA和蛋白的表達(dá)變化,以及EZH2啟動(dòng)子活性的變化。5.CHIP實(shí)驗(yàn)檢測(cè)BRD4與C-MYC啟動(dòng)子區(qū)域的結(jié)合、C-MYC與EZH2啟動(dòng)子區(qū)域的結(jié)合作用。結(jié)果：1.C-MYC及EZH2在膀胱癌組織中均高表達(dá),他們的表達(dá)水平分別與BRD4的表達(dá)存在正性相關(guān)。2.ShRNA沉默BRD4或JQ1能夠降低EZH2和C-MYC的mRNA和蛋白水平的表達(dá),并能夠抑制EZH2啟動(dòng)子活性。3.過(guò)表達(dá)EZH2能夠部分逆轉(zhuǎn)抑制BRD4引起的細(xì)胞周期阻滯,細(xì)胞凋亡和增殖能力的變化。4.過(guò)表達(dá)C-MYC能夠部分逆轉(zhuǎn)抑制BRD4引起的EZH2 mRNA、蛋白水平和啟動(dòng)子活性的變化。5.CHIP實(shí)驗(yàn)證實(shí)BRD4能夠結(jié)合于C-MYC啟動(dòng)子區(qū)域,C-MYC能夠結(jié)合于EZH2啟動(dòng)子區(qū)域。抑制BRD4能夠減弱C-MYC與EZH2啟動(dòng)子區(qū)域的結(jié)合。結(jié)論：BRD4通過(guò)調(diào)控C-MYC的轉(zhuǎn)錄影響C-MYC與EZH2啟動(dòng)子區(qū)域的結(jié)合,進(jìn)而促進(jìn)EZH2轉(zhuǎn)錄,促進(jìn)膀胱癌細(xì)胞增殖,抑制細(xì)胞凋亡。JQ1能夠通過(guò)抑制C-MYC-EZH2通路,誘導(dǎo)膀胱癌細(xì)胞凋亡,抑制細(xì)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.
【學(xué)位授予單位】：華中科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：R737.14

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