VMAT1基因拷贝数变异在结直肠癌发生及恶性演变中的作用及机制研究

Our previous findings showed that the odds ratio (OR) of colorectal cancer (CRC) increased to 16.7(P = 6.4×10-5) among the individuals with VMAT1 (vesicular monoamine transporter 1) germline deletions (CRC 1628, control 4321). Analysis of TCGA CRC dataset revealed that the deletions of VMAT1 were associated with decreased mRNA expression levels. The gene expression of VMAT1 was significantly down regulated in CRC tissues than in paired normal tissues. CRC cases with lower expression levels of VMAT1 had shorter overall survival compared with those with higher expression levels of VMAT1. However, the mechanism underlying CNV formation and the function of VMAT1 in colorectal cancer was unclear.We proposed to map the breakpoints , reveal the structure of the CNV and the mechanism of CNV formation by high density array CGH and sequecing. Furthurmore, we will detect the copy number status, mRNA and protein level of VMAT1 in colorectal cancer tissues. The functional effect of VMAT1 will be examined in vitro and in vivo. This study will help to elucidate the role of VMAT1 in the maligant change of colorectal cancer.

本项目前期发现携带VMAT1基因拷贝数缺失的人群结直肠癌患病风险为该基因拷贝数正常或拷贝数重复的人群的16.7倍（结直肠癌患者1628例，对照4321例，P = 6.4×10-5）。TCGA数据显示，VMAT1拷贝数缺失与低表达相关，且VMAT1在结直肠癌组织的转录水平显著低于正常组织，VMAT1低表达预后差。但VMAT1拷贝数变异(copy number variants, CNV)的发生机制和在结直肠癌中的作用还不明确。本项目拟用高密度aCGH芯片及二代测序技术等解析CNV的精细结构，精确定位断裂点及分析断点序列以揭示CNV的发生机制；在结直肠癌组织样本中检测VMAT1 CNV、mRNA水平及蛋白表达水平，分析VMAT1 CNV、表达与结直肠癌临床指标、5年生存率的相关性；从体外细胞实验和动物实验研究VMAT1蛋白对结直肠癌发生和转移的影响；阐明VMAT1在结直肠癌恶性演变中的作用。

本项目通过aCGH芯片实验明确了结直肠癌组织和正常组织存在的拷贝数变异为不同的拷贝数变异，癌组织存在染色体大片段缺失，而正常组织为涉及VMAT1基因的小片段的缺失（chr8:19986563-20025949）。根据拷贝数变异数据显示，这段缺失并不是新发的拷贝数变异。本研究发现VMAT1基因缺失与不利的临床病理特征相关。在TCGA数据中VMAT1基因缺失与患者的浸润深度、淋巴结转移、远处转移、TNM分期均显著相关。VMAT1缺失的患者其总体生存率显著低于VMAT1基因拷贝数正常或重复的患者。在本实验室结直肠癌样本中，我们发现VMAT1缺失与远处转移显著相关。VMAT1低表达与淋巴结转移和远处转移有关。VMAT1低表达与结直肠癌预后不良相关。VMAT1低表达可作为结直肠癌不良预后指标。体外细胞学实验表明VMAT1具有抑制结直肠癌增殖、侵袭、迁移的作用。小鼠实验表明VMAT1具有抑制结直肠癌转移的能力。