食管癌中NTRK3基因重排的鉴定及其作用机制研究

Chromosomal and gene rearrangements are common features of human malignant tumors. Chromosomal rearrangements may influence gene expression through several ways. First, fusion genes may be resulted from chromosomal translocations, inversions, and insertions, and thus expressing new fusion proteins with abnormal functions. Second, the regulatory elements are fused to the vicinity of normal genes, and dysregulate the gene expression. Third, gene truncations may be resulted from the gene breakage, and thus express the truncated proteins. All of the situations above might lead to the malignant transformations..The implications and the clinical values of gene rearrangements have been emphasized since years ago. Many fusion genes have been reported to play important roles in tumor development and progression, and several have been used for the assistant diagnosis as well as the targets for therapies. For a long period, the majority of gene rearrangements were discovered and in hematological malignancies. The findings for epithelial cancers were main reasons were the technical limitations and the complex karyotypes. Until recently, more and more fusion genes have been identified in multiple types of epithelial cancers, including prostate, lung, breast, colorectal, gastric, hepatoma carcinoma and so on. .Esophageal squamous cell carcinoma (ESCC) is one of the common cancers with high incidence and death rate in China. Until now, the studies and findings about the gene rearrangements in ESCC are still limited. In the previous study, we detected the breakage of NTRK3 gene in the ESCC cell line KYSE450 and KYSE510, and identified a fusion transcript in KYSE450. Then we carried out FISH experiments on the primary ESCC tumors, and found that 16.7% of the cases exhibited NTRK3 breakages, suggesting that rearrangements involving NTRK3 may play important roles in ESCC. .Based on the findings, the aim of our study is to indentify the rearrangement forms of NTRK3 in ESCC tumors as well as their roles and the molecular mechanisms in ESCC cells. We will use the NRTK3-breakage-positive ESCC tissue samples which have previously been verified previously. A series of molecular biological and cell biological methods, such as RACE and genome walking strategies, will be adopted to amplify the abnormal transcripts, breakpoints and junctions of NTRK3 rearrangements. The products will be cloned and sequenced, followed by FISH or PCR validations in the positive cases. The protein expression of the rearrangement forms will be analyzed by western blot or immunohistochemistry. Then validation will be performed in the large sample. Using the cell and node mice model system, the impact on the malignant phenotype and the tumorigenic capacity in nude mice will be analyzed in vitro and in vivo, respectively. Further, the signaling pathways and the downstream molecules will be investigated, in order to reveal the molecular mechanisms of NTRK3 rearrangements in ESCC.

基因重排与肿瘤发生发展的关系愈来愈受到重视，我们在前期工作中发现食管癌细胞系KYSE450和KYSE510中存在NTRK3基因的断裂，并在KYSE450中鉴定出NTRK3基因形成了融合转录本。对临床食管癌组织标本进行FISH检测，发现16.7%的病例中存在NTRK3基因的断裂，提示NTRK3涉及的基因重排可能在食管癌中起重要作用。本研究拟在前期发现的基础上，选取存在NTRK3断裂的肿瘤组织样本，利用分子生物学和细胞生物学技术，针对NTRK3的断裂连接点进行序列分析，证实基因在断裂阳性病例中NTRK3基因的重排方式和蛋白表达情况，并扩大样本进行验证；继而利用细胞和动物模型，研究NTRK3基因重排对食管癌细胞恶性表型和裸鼠致瘤能力的影响；进一步分析NTRK3基因重排在食管癌中参与的信号通路及其影响的下游基因，揭示其在食管癌中发挥作用的分子机制。

食管癌是我国发病率和死亡率较高的恶性肿瘤之一，其发生发展的分子机制尚未阐明。基因重排与肿瘤发生发展的关系越来越受到重视。前期工作中，我们发现食管癌细胞系KYSE450和KYSE510中存在NTRK3基因的断裂，并在KYSE450中鉴定出NTRK3基因的融合转录本NTRK3-Chr5。在临床食管癌肿瘤组织标本中发现16.7%的病例中存在NTRK3基因断裂。本课题在此基础上，针对KYSE450细胞中NTRK3的基因组断裂点进行基因组步移分析，缩小了基因组DNA断裂点范围，同时鉴定出食管癌细胞中融合转录本NTRK3-Chr5的蛋白表达产物大小。进一步选取NTRK3基因断裂的肿瘤组织，利用荧光原位杂交技术和比较基因组杂交微阵列分析，发现食管癌肿瘤组织中NTRK3基因存在NTRK3-Chr5和ETV6-NTRK3两种融合形式。针对我们近期发表的食管癌外显子组测序结果、以及另外两组食管癌高通量测序结果进行分析，发现食管癌肿瘤组织中还存在NTRK3基因突变，包括热点突变V21F和R130H、以及在蛋白酪氨酸激酶催化结构域中的突变K746T和T749R。克隆了NTRK3-Chr5融合转录本，在食管癌细胞中转染NTRK3-Chr5，能够显著增强食管癌细胞的增殖能力，而不影响细胞的侵袭迁移和凋亡；初步机制研究提示该融合转录本能够激活AKT信号通路。这些发现为进一步深入研究NTRK3基因在食管癌细胞增殖中的作用机制，以及阐明食管癌发生发展的分子基础提供理论依据。