NTRK3调控c-Myc在结直肠癌浸润转移中的作用及相关机制研究

c-Myc is one of the most importance classic oncogenes. Methylation-induced downregulation of NTRK3 expression in colorectal cancer (CRC) has been identified and validated in our previous studies. Next, we found that NTRK3 can suppress c-Myc expression as being a transcriptional suppressor factor. In the current study, we apply the methylation-specific PCR and Pyrosequencing to NTRK3 gene. qRT-PCR and tissue microarrays will be used to detect the mRNA and protein expression levels of NTRK3 and c-Myc genes. The correlation between NTRK3 and c-Myc expression will be studied comprehensively. In addition, the functions of NTRK3 gene, including the suppression of c-Myc, tumor invasivenss and metastasis, will be investigated on colon cell lines with altered NTRK3 expression by using NTRK3-specific short-hairpin RNA (shRNA) knockdown or Lenti-virus-infected NTRK3-transfection with/without c-Myc in vitro, and assessed by using a in vivo SCID mice model intensively. The mechanisms of downregultion of c-Myc induced by NTRK3 will be explored by using the single-allel-mutated c-Myc construct in the promotoer region and by luciferease assays. These findings will provide the foundamental evidences for the machnisms of NTRK3 in the setting of the epigenectic target therapry in CRC.

c-Myc是最重要的癌基因之一。我们已经证明在结直肠癌中由于NTRK3启动子区域甲基化造成表达缺失；进而发现NTRK3具有转录抑制功能，可特异性下调c-Myc表达。因此提出"NTRK3调控c-Myc参与结直肠癌浸润转移"的科学假设。本项目拟采用组织芯片探讨结直肠腺瘤、局限性及转移性结肠癌中NTRK3和c-Myc各自表达规律及它们之间相关性；改变结肠癌细胞中NTRK3表达，经体外细胞试验和动物活体成像模型，动态观察干预NTRK3基因对c-Myc表达及肿瘤生长及转移的影响，从 "细胞-动物-病人"多个层次揭示NTRK3和c-Myc在结直肠癌浸润转移中的作用。并通过启动子定点突变、荧光素酶分析、EMSA和CHIP近一步探讨NTRK3下调c-Myc的具体机制。该研究的完成将对阐明结直肠癌浸润转移机制和发现新的治疗靶标提供依据。

我们先前的研究表明在结直肠癌中由于NTRK3启动子区域甲基化造成表达缺失；进一步发现NTRK3具有转录抑制功能，可特异性下调癌基因c-Myc的表达。因此提出"NTRK3调控c-Myc参与结直肠癌浸润转移"的科学假设。本研究项目通过甲基化芯片、QMSP、焦磷酸测序等方法发现和验证了结直肠癌组织中抑癌基因NTRK3启动子区高甲基化，并在mRNA和蛋白质层面上证实了NTRK3启动子区高甲基化会抑制NTRK3的表达，阻碍NTRK3抑癌作用的发挥，进而促进肿瘤的发生发展。通过大样本临床组织中验证了NTRK3启动子区高甲基化提示预后不良，以及NTRK3甲基化程度联合CA19-9水平可提供更多更准确的预后信息。通过构建单碱基分辨率下检测基因组甲基化水平的新技术（QASM）及其定量系统，为NTKR3基因单个碱基的甲基化水平检测提供了便利。创新性地提出了一种合作临床研究模式，为甲基化分子标志物或多因素模型的临床队列验证开拓了广阔前景。在研究中还发现了一些其他相关的甲基化预后标志物，为完善NTRK3甲基化调控网络进行了重要补充。该研究的完成对阐明结直肠癌浸润转移的机制和发现新的预后标记物提供了重要依据。