【作者】 彭聪；

【导师】 李桂源； 李小玲；

【作者基本信息】 中南大学 ， 病理与病理生理学， 2003， 硕士

【摘要】 鼻咽癌(nasopharyngeal carcinoma，NPC)是东南亚地区及我国南方各省常见的一种恶性肿瘤，其发生不仅有明显的地域性和家族集聚现象，而且存在基因组不稳定的特性。细胞和分子遗传学研究发现，鼻咽癌基因组存在高频率3p、9p、11q、13q和14q染色体的等位基因的杂合性丢失。我室采用cDNA代表性差异分析的方法克隆了一个新基因BRD7，Genbank登录号为AF152604。 我室近期研究显示，该基因在鼻咽癌细胞和组织中表达明显下调，在BRD7基因的编码区发现了3个cSNP。采用酵母双杂交系统(Two-hybrid system)筛选出与6种BRD7蛋白相互作用的蛋白，包括：溴区包含蛋白3(Bromodomain-containing 3 protein，BRD3)、溴区包含蛋白2(Bromodomain-containing 2 protein，BRD2)、β-IkB激酶(IkB kinase-beta)、KIAA1375蛋白(KIAA1375 protein)、白介素7(interleukin 7，IL-7)和接头相关蛋白复合物3 δ-1亚单位(adaptor-related protein complex 3，delta 1 subunit)。这些都说明BRD7可能是一个与鼻咽癌相关的肿瘤抑制候选基因。本研究拟进一步研究BRD7的功能，阐述其在鼻咽癌发生发展中的作用。 通过生物信息学分析，我们发现BRD-7的溴区蛋白与五个结构已知的溴区蛋白具有高度同源性，这些已知的溴区蛋白具有如下特征：含有四个α螺旋(Z，A，B，C)，螺旋与螺旋之间通过环连接(ZA loop，BC loop)，并且含有一个由疏水氨基酸形成的“口袋”，通过这个结构，彭聪 硕士学位论文9能特异性识别乙酚化的组蛋白。这表明BRD7 可能具有与以上bromodomain蛋白相似的结构。另外，我们采用数字化杂交显示该基因在脑、肺、肝、肾、前列腺、骨骼肌、胎盘等正常组织表达较高。这与我们采用实验室方法得出的结果具有较高的一致性。 我们在构建BRD7的真核表达载体BRD7小CDNA3．1卜）基础上，通过脂质体转染方法，将 BRD7基因导入 NPC细胞株 HNE细胞中，并以HN’EI细胞为对照，通过 RTPCR和 Northern Blot方法检测 BRD7基因的表达水平，挑选出BRD7基因高表达的细胞克隆为研究对象，探讨BRD7基因在鼻咽癌发生、发展中的可能作用。 为了证实 BRD7基因对 HNE恶性表型的影响，我们采用重表达BRD7基因的HNEI细胞株，运用细胞生长曲线、软琼脂糖集落形成实验、裸鼠体内接种实验进行生物学行为的研究。结果显示，pCDNA3．l卜＊BRD7／HNEI细胞的生长速度明显减慢，倍增时间延长，软琼脂中集落形成率较对照组显著下降b狈．05入 裸鼠体内成瘤的时间较对照组明显延长，瘤体的大小较HNE和p cDNA3．1卜）空载体转染组明显减小，将3组肿瘤进行病理切片，均为中、低分化鳞癌，无组间差异，显示BRD7基因对裸鼠移植瘤生长具有抑制作用。以上表明，BRD7基因重表达有助于HNEI细胞恶性表型的部分逆转。 为了研究BRD7基因对NPC细胞的作用机制，我们采用流式细胞仪结合细胞周期素的分析方法。结果显示重表达BRD7基因后，能更多的将细胞阻滞于G厂G；期，降低细胞周期素eyel inDI、B表达。以上结果说明BRD7可能通过下调cyclinDI，B的表达，阻滞细胞于G厂Gl期，从而抑制NPC细胞的过度增殖。 综上所述，BRD7基因作为一个重要的鼻咽癌抑瘤基因侯选者，在鼻咽癌细胞中的过表达后，可导致鼻咽癌细胞 HNE蛋白质表达谱发生改变，逆转其恶性表型，其作用机理可能是：BRD7基因通过其功能域彭聪 硕士学位论文10Bromodomain与乙酚化的组蛋白特异性结合，参于染色体的乙酚化，染色质的组装，从而影响基因转录的调控，最终影响细胞内的信号传导通路并实现对细胞周期的调控，从而发挥抑制鼻咽癌细胞生长的作用。更多还原

【Abstract】 Nasopharyngeal carcinoma (NPC) is a common cancer in South China and Southeast Asia , but is rare in other parts of the world. The tumorigenesis of NPC is characteristic of obvious family history and genomic allelic imbalance. Significant loss of heterozygosity at 3p, 9p,llq, 13q and 14q in nasopharyngeal carcinoma (NPC) had been detected by comparative genomic hybridization (CGH) and genome-wide analysis of genetic alteration with microsatellite allelotyping. A novel NPC related gene, BRD7, was isolated through cDNA representational difference analysis (RDA). GenBank accession number is AF 152604.Our previous studies showed that the BRD7 gene was down-regulated in Nasopharyngeal carcinoma (NPC) cells and NPC biopsy. Three coding-region single nucleotide polymorphisms (cSNPs) were detected in BRD7 cDNA sequence. In addition, six BRD7-interacting proteins (EIPs) were identified by yeast two-hybrid system. These proteins included Bromodomain-containing 3 protein (BRD3), Bromodomain-containing 2 protein (BRD2), IkB kinase-beta , KIAA1375 protein, interleukin 7 ( IL-7) and the delta 1 subunit of adaptor-related protein complex 3. In this study , we further investigated the function of BRD7 in pathogenesis ofNPC and its possible molecular mechanism.Five kinds of proteins homologous to BRD7 of bromodomain were selected through Homology-based sequence analyses. These proteins have the similar structure containing four a helices (Z, A, B and C), and "a hydrophobic pocket" which has the ability to recognize acetylated histone peptide. So, we predicted that the bromodomain of BRD-7 may have the similarity structure. In addition, BRD7 gene expression was detected in brain, lung, liver, muscle, kidney, prostate and in other normal tissues by virtual Northern. The virtual Northern resulted in mainly similar results with laboratory assay.To elucidate the mechanism of BRD7 effect on carcinogenesis in NPC, BRD7 gene was transfected into BRD7 down-expressed HNE1 cell by liposome and a stable cell line overexpressing BRD7 was established. RT-PCR and Northern blot were performed to detect the overexpression of BRD7 in transfected cell.To confirm the effect of BRD7 on the malignant phenotype of HNE1 cell line, three assays, including growth curves 、colony formation and tumor xenografts in nude mice were performed. The results showed that compared with HNE1 and HNE1 cells transfected with pcDNA3.1(+) empty vector, BRD7 transfected cells resulted in declined growth curve, fewer colonies formation, and development of a smaller tumor size. Pathological analysis showed that xenograft were low differentiatesquamous cell carcinoma. These results indicated that the BRD7 can play negative role in the progression of NPC and may partly reverse malignant phenotype of NPC cell line.To determine whether BRD7 gene have effect on cell cycle and apoptosis, the flow cytometry (FCM) was performed to detect the distribution of cell cycle change and expression of cyclins in BRD7 transfected HNE1 cells. The results indicated that the overexpression of BRD7 could delay the progression of GO/G1 to S phase in cell cycle, and the expression cyclinDl and B were significantly decreased, while it had no effect on the apoptosis of cells.In summary, the BRD7 gene acted as a candidate of tumor suppressor gene with NPC. The overexpression of BRD7 can partly reverse malignant phenotype of NPC cell line. The suppression effect of BRD7 on NPC tumorigenesis my be achieved by recognizing acetylated histone peptide through their motif-bromodomain, then modulating gene transcription by taking part in histone acetylating and chromosome remodeling, finally influencing signal-transduction pathways.更多还原