新基因TRIM67在结肠癌中甲基化的功能研究及其临床应用价值

TRIM67 is a genuine novel gene in tripartite motif(TRIM) gene family. Its role and clinical application value in colon cancer remains to be elucidated. Further study on colon cancer revealed that TRIM67 was silenced or down-regulated in eight colon cancer cell lines by promoter methylation. TRIM67 was down-regulated in colon tumor tissues as compared with colon normal tissues. In concordance with transcriptional down-regulation, DNA methylation was detected in colon tumor samples. Meanwhile,TRIM67 was expressed at high level in normal colon mucosa, without promoter DNA methylation. Importantly, ectopic expression of TRIM67 in silenced cancer cells was found to suppress cell growth and modulated the expression of many genes involved in important biological processes, such as cell proliferation, cell cycle regulation, etc. Based on these findings we aim: 1) to explore the clinical application values of TRIM67 promoter methylation on a large-scale cohort of colon tumor samples; 2) to investigate the functions of TRIM67 on cell proliferation, apoptosis, cell cycle regulation, migration/invasion and tumorigenecity by both in vitro and in vivo studies after ectopic expression in silenced colon cancer cells; 3)to elucidate the molecular mechanism by which TRIM67 functions by comparing the genome-wide mRNA expression profiles of TRIM67-overexpressioned and empty vector-transfected colon cancer cells. The study will provide a comprehensive understanding of the role of TRIM67 in colon cancer and the potential clinical application of its promoter methylation in non-invasive diagnosis and prognosis of colon cancer.

TRIM67是TRIM基因家族中的潜在抑癌基因。研究显示：TRIM67在8个结肠癌细胞系和结肠癌组织中由于启动子DNA甲基化导致其表达沉默，但正常结肠粘膜无甲基化。转染该基因显著抑制癌细胞生长，基因表达芯片揭示：该基因在肿瘤中调控多个重要下游基因。根据此前期研究结果，本课题旨在进一步研究：1）大规模结肠癌组织标本中检测TRIM67的甲基化水平，探索其临床价值；2）观察过表达/敲低该基因对结肠癌细胞增殖、凋亡、周期、侵袭能力和裸鼠致瘤性等的影响，揭示其功能。3）分析并验证TRIM67的分子调控信号通路，阐明其作用的分子机制。该研究将全面揭示新基因TRIM67在结肠癌中的临床意义和功能机制。

三结构域(Tripartite motif, TRIM)家族蛋白参与多种细胞进程。本研究通过DNA甲基化芯片、二代测序、染色质免疫沉淀（ChIP）、实时qPCR、细胞转染、western blot、生物信息学分析等方法对TRIM家族新成员TRIM67在大肠癌（CRC）中的功能及其临床应用价值进行探讨，证明了TRIM67在CRC中通常低表达，其下调与低生存率相关。通过TCGA数据库及DNA甲基化芯片结果分析，证实TRIM67在大肠癌中的低表达与TRIM67 DNA甲基化密切相关。体外研究发现TRIM67的过表达抑制了大肠癌细胞的增殖、集落形成、迁移和侵袭，而基因敲低起相反作用，展示了TRIM67在大肠癌中的抑癌作用。在ApcMin/+小鼠中TRIM67基因敲除增加了大肠肿瘤的发病率和肿瘤负荷。同样，在小鼠的结肠特异性敲除TRIM67显著增加了AOM诱导的大肠癌的肿瘤数目及肿瘤负荷。这些在体实验结果，证实TRIM67在大肠癌中的抑癌基因角色。.为分析TRIM67的作用机制，RNA-seq结果显示TRIM67的抗癌作用是通过激活p53信号通路实现的。免疫共沉淀鉴定TRIM67与p53 C末端直接相互作用，通过泛素连接酶MDM2阻止p53降解。此外，ChIP分析表明TRIM67是p53转录调控的靶基因。在细胞应激时，p53与TRIM67启动子结合，介导TRIM67的显著上调，从而形成TRIM67-p53自增强环路，促进p53诱导的细胞生长抑制和凋亡。因此，肿瘤中p53正调控的缺失严重损害了p53对化疗诱导DNA损伤的应答。在TRIM67敲除小鼠的肿瘤和TRIM67敲除胚胎成纤维细胞中均观察到p53应答的抑制。体外和在体实验均证实TRIM67的再活化恢复了p53的活性。同时RNA测序结果还发现蛋白丝裂原活化蛋白激酶11(MAPK11)是TRIM67调控的下游基因，在大肠癌中TRIM67的表达与MAPK11的表达呈显著负相关，体外实验证实阻断MAPK11的表达显著改善TRIM67敲低对大肠癌细胞的增殖、集落形成、迁移和侵袭的促进作用。故MAPK通路亦是TRIM67发挥抑癌基因作用的潜在机制。研究还发现TRIM67提高了p53野生结直肠癌的化疗敏感性。.综上所述，TRIM67在大肠癌中起着关键的抑癌作用，是阻断大肠癌进展及提高化疗反应性的潜在靶点，研究成果具有较好的转化应用前景。