【作者】 吴丹；

【导师】 胡坚；

【作者基本信息】 浙江大学 ， 外科学（胸心外科）， 2016， 硕士

【摘要】 [目的]溴结构域蛋白4(Bromodomain Containing 4,BRD4)是溴结构域和超末端结构(bromodomain and extraterminal domain,BET)家族成员,其含有两个溴结构域和一个超末端结构,在整个细胞周期中通过溴结构域结合乙酰化的组蛋白及非组蛋白,以及招募不同的转录调节因子,如Mediator、正性转录延伸因子b(positive transcription elongation factor b,P-TEFb)来调节靶基因的表达,在基因的转录调控、细胞周期进展、炎症、肿瘤的恶性进展等生物过程中发挥重要作用。近来研究表明,BRD4的表达水平失调或功能紊乱与黑色素瘤、乳腺癌、结直肠癌、急性髓系白血病等肿瘤的发生发展有关,BRD4shRNA或BET抑制剂具有诱导上述肿瘤发生细胞周期阻滞、凋亡、分化及抑制侵袭转移的作用效应,显示出强大的抗肿瘤活性。上述研究提示,BRD4有望成为上述肿瘤甚至其他肿瘤潜在的治疗靶点。本研究观察乙酰化修饰阅读BRD4在食管鳞癌组织中的表达特征,探讨BRD4的表达与食管鱗癌临床病理特征和预后的关系。以体外培养的人食管鱗癌细胞为疾病模型,采用小分子抑制剂JQ-1靶向抑制BRD4的生物活性,观察细胞生长、增殖的变化情况,进一步分析细胞凋亡和凋亡相关蛋白表达的变化,探讨BRD4是否可以作为食管鱗癌潜在的治疗靶点。[方法](1)乙酰化修饰阅读器BRD4在食管鳞癌组织中的表达特征及临床意义免疫组织化学染色法检测80例食管鱗癌患者手术切除后石蜡包埋标本和配对癌旁正常食管组织中BRD4的表达情况。所有病例均选自2008～2010年在慈溪市人民医院胸外科食管鱗癌住院病人的手术切除标本。根据染色强度(即阴性、弱阳性、中等阳性和强阳性,得分为0-3)和阳性染色细胞比例(即<10%、11%-30%、31%-50%、>50%,得分为0-3),当两者得分之和≥3时判定为BRD4高表达(BRD4High),当两者得分之和<3时判定为BRD4低表达(BRD4Low)。采用卡方检验分析BRD4蛋白的表达情况与临床病理特征的关系,Kaplan-Meier单因素生存分析BRD4蛋白的表达对食管鱗癌术后患者预后的影响。(2)BRD4作为食管鳞癌潜在治疗靶点的研究体外培养人食管鱗癌细胞EC109、TE-1和TE-13,MTT实验观察不同浓度BRD4抑制剂JQ-1作用细胞24、48和72h对其生长的影响;JQ-1作用2w,集落形成实验分析JQ-1是否抑制食管鱗癌细胞EC109、TE-1和TE-13的增殖;JQ-1作用24h,Annexin V-FITC/PI双染法分析TE-1和TE-13细胞凋亡率的变化,Westeen blot实验检测细胞凋亡相关蛋白cleaved caspase3和cleaved PARP表达的水平;研究BRD4是否可以作为食管鱗癌潜在治疗靶点。[结果]BRD4高表达表现为肿瘤细胞的胞浆及细胞核出现棕黄色或棕褐色的颗粒。食管鳞癌组织BRD4高表达率为42.5%(34例/80例),而癌旁正常食管组织高表达率为20.0%(16例/80例),两者相比BRD4的表达具有显著差异(p=0.002)。进一步分析BRD4的表达水平与食管鱗癌临床病理因素的关系,发现与食管鳞癌的分化程度、N分期和病理分期相关(p分别为0.009,0.008和0.003)。34例BRD4高表达的患者1年累积生存率为73.5%,3年累积生存率为11.8%,5年累积生存率为8.8%;46例BRD4低表达的患者1年累积生存率为78.3%,3年累积生存率为50.0%,5年累积生存率为34.8%;BRD4蛋白低表达组的5年生存率明显高于(?)高表达组,差异有统计学意义(P=0.001)。Kaplan-Meier单因素分析发现,BRD4的表达水平、淋巴结是否转移、T分期和病理分期是食管鱗癌患者预后的影响因素。BRD4抑制剂JQ-1呈剂量依赖关系抑制食管鳞癌细胞EC109、TE-1和TE-13的增殖,不同浓度JQ-1(20、40、80和120μM)作用24h,EC109细胞的存活率分别为 101.5%±6.4%、87.2%±5.2%、42.3%±4.3%和 5.6%±2.2%;TE-1 细胞的存活率分别为 96.2%±5.8%、83.7%±4.4%、50.8%±3.2%和 12.4%±2.6%;TE-13 细胞的存活率分别为 105.2%±7.3%、74.8%±5.4%、37.2%±4.2%和 7.2%±2.3%。集落形成实验发现,JQ-1作用EC109细胞2w,空白对照组的集落形成数为148.7±35.2,JQ-1(40、80 和 120μM)组分别为 22.5±15.9、28.7±12.4 和 19.6±8.7,显著抑制 EC109细胞集落克隆的形成(p<0.05);JQ-1作用TE-13细胞2w,空白对照组的集落形成数为 203.4±52.6,JQ-1(40、80 和 120 μM)组分别为 27.5±16.8、28.6±23.0 和19.3±12.9,显著抑制 TE-13 细胞集落克隆的形成(p<0.05)。Annexin V-FITC/PI双染实验结果显示,JQ-1(40、80和120 μM)作用24h,EC019细胞的凋亡率分别为 8.1%±5.2%、49.7%±6.3%和 99.7%±1.0%,与空白对照组(2.6%±4.5%)相比差异有统计学意义(p<0.05);TE-13细胞的凋亡率分别为12.8%±7.3%、52.0%±4.9%和97.3%±1.8%,与空白对照组(7.2%±3.7%)相比差异有统计学意义(p<0.05)。JQ-1(40、80和120 μM)作用EC109和TE-13细胞24h,细胞表达凋亡相关蛋白cleavedcaspase3和cleavedPARP随药物浓度增加而显著上调。[结论]BRD的高表达与食管鳞癌的恶性进展相关,是潜在的预后评估标记物;抑制内源性BRD的生物活性通过诱导细胞凋亡抑制食管鱗癌细胞的增殖,因此BRD4是食管鳞癌潜在的治疗靶点。更多还原

【Abstract】 Background and objectiveBromodomain Containing 4(BRD4)is a member of bromodomain and extraterminal domain(BET)family,and contains two bromodomains and one extraterminal domain.In cell cycle,it combines with acetylated histones and non-histones via bromodomain,and recruits transcriptional regulatory factors such as Mediator,positive transcription elongation factor b(P-TEFb),thus playing a key role in gene transcription regulation,cell cycle progression,inflammation and tumor progression.Recent reports have indicated the disorder and dysfunction of BRD4 bear some relation to tumor genesis and progress in melanoma,breast cancer,colorectal cancer and acute myeloid leukemia,and BRD4 shRNA or BET inhibitor may induce tumor cell arrest,apoptosis,differentiation and inhibit metastasis.It seems BRD4 may be an underlying therapeutic target in tumors mentioned above or not.We observed the expression profiles of BRD4 esophageal squamous cell carcinoma issues to explore the relation between BRD4 expression and clinicopathologic features and prognosis in esophageal squamous cell carcinoma.In vitro cultured human esophageal squamous cell carcinoma cells,we applied BRD4 inhibitor JQ-1,then observed cell proliferation,analyzed cell apoptosis and the expression of related proteins.Experiment methodThe expression profiles of BRD4 esophageal squamous cell carcinoma issues and its clinical meaning.We detected BRD4 expression in 80 samples of esophageal squamous cell carcinoma tissue and the paired para-carcinoma tissue by immunohistochemical staining.Samples were from the thoracic department of Cixi city people’s hospital,gained in operations from 2008 to 2010.We scored the results of immunohistochemical staining by intensity(negative,low,medium,and strong positive,0-3)and the percentage of positive staining cells(≤10%,11%-30%,31%-50%and>50%,0-3),then added the two parts together,if total score>3,BRD4 was considered as high expression,score<3 as low expression.We analyzed the relation between BRD4 expression and clinicopathologic features by chi-square test,and investigate its effect on patients’prognosis by Kaplan-Meier univariate analysis.Whether BRD4 could be an underlying therapeutic target in esophageal squamous cell carcinoma.Human esophageal squamous cell carcinoma cell lines EC 109,TE-1 and TE-13 were cultured in vitro.The effect of cell proliferation by JQ-1 in different concentrations for 24,48,72 hours was analyzed by MTT assay.And in colony forming assay,we investigated the proliferation capacity of EC 109,TE-1 and TE-13 cells after JQ-1 treated for 2 weeks.Treated with JQ-1 for 24 hours,the apoptosis rates of TE-1 and TE-13 cells were analyzed by Annexin V-FITC/PI double staining,and the expression of apoptosis-related proteins,cleaved caspase 3 and cleaved PARP,was explored by Western blot,to further investigate whether BRD4 could be an underlying therapeutic target in esophageal squamous cell carcinoma.ResultIn BRD4 high expression group,we observed brown and dark brown granules in cellular cytoplasm and nucleus.Among cancer tissues,the percentage of BRD4 high expression was 42.5%(34/80)and it was 20.0%among para-carcinoma tissues(16/80),with significant difference(p=0.002).And BRD4 expression showed significant correlation with the differentiation,lymphatic metastasis and pathological stage of esophageal squamous cancer(p=0.009,p=0.008,p=0.003).In BRD4 high expression group,patients’ 1 year cumulative survival rate was 73.5%,3 years cumulative survival rate was 11.8%,and 8.8%in 5 years.While in BRD4 low expression group cumulative survival rates were 78.3%,50.0%and 34.8%respectively,with five-year survival rate significantly higher than that of BRD4 high expression group(P=0.001).In Kaplan-Meier univariate analysis,we found that the level of BRD4 expression,lymphatic metastasis,T stage and pathological stage were prognostic factors of esophageal squamous cell carcinoma.The cell proliferation of esophageal squamous cell carcinoma cells EC 109,TE-1 and TE-13 was inhibited by BIX-01294 in a dose-dependent manner.JQ-1 treated for 24 hours in different concentrations(20,40,80 and 120 μM),the survival rates in EC 109 were 101.5%± 6.4%,87.2%± 5.2%,42.3%±4.3%,5.6%± 2.2%;96.2%±5.8%,83.7%±4.4%,50.8%±3.2%,12.4%±2.6%in TE-1;105.2%±7.3%,74.8%±5.4%,37.2%±4.2%,7.2%±2.3%in TE-13.In colony forming assay,after JQ-1 treated for 2 weeks in EC109,there were 148.7±35.2 colony forming units in control group,22.5± 15.9,28.7± 12.4 and 19.6±8.7 units in 40,80 and 120 μM concentration JQ-1 groups,the colony forming in EC109 was significantly inhibited(p<0.05),and similar results seen in TE-13,203.41±52.6 units in control group,27.5± 16.8,28.6±23.0 and 19.3±12.9 units in 40,80 and 120 μM groups(p<0.05).Annexin V-FITC/PI double staining showed that,after JQ-1(40,80 and 120 μM)treated for 24 hours,EC019 cell apoptosis rates respectively 8.1%±5.2%,49.7%±6,3%and 99.7%± 1.0%,higher than control group(2.6%±4,5%,p<0.05).And in TE-13,apoptosis rates respectively were 12.8%±7.3%,52.8%±4.9%and 97.3%± 1.8%,higher than control group(5.8%±3.7%,p<0.05).After JQ-1 24 hours treatment(40,80 and 120 μM),the expression of apoptosis related proteins,cleaved caspase3 and cleaved PARP significantly upregulated with drug concentration increased.ConclusionBRD4 expression was obviously related to the disease progression of esophageal squamous cell carcinoma,and might be employed as an underlying prognostic marker.Endogenous BRD4 inhibition suppressed the cell proliferation of esophageal squamous cell carcinoma by inducing apoptosis,thus,BRD4 could be a therapeutic target for esophageal squamous cell carcinoma.更多还原