NGFR甲基化活化VEGF-C促进结直肠癌侵袭和转移的机制研究

Our preliminary studies found that the hypermethylation of NGFR are associated with colorectal cancer lymph node and distant metastasis, Tumors with high expression of VEGF-C hadmore remote lymph node involvement ; The promoter hypermethylation of NGFR was observed in some cases which also show a high expression of VEGF-C protein. As a result, we first proposed that a new mechanism that the hypermethylation of NGFR promote colon cancer cell invasion and metastasis through activation of VEGF-C and MAPK signaling pathway. In the next step, we intend to obtain the evidence that NGFR activates the VEGF-C gene in three-dimensional cell culture model by use of laser scanning confocal microscope; Reveal the role of NGFR and VEGF-C in the process of colorectal cancer invasion and metastasis in cell、animal and patient, View the relationship between the expression level of NGFR and the tumor growth, invasion and metastasis in animal in dynamical way. filter the target genes in the downstream MAPK pathway involving in invasion and metastasis, reveal the regulatory mechanisms through a series of in vitro and in vivo functional experiments. This subject will provide a theoretical basis to the targeted therapy.

前期研究显示结直肠癌淋巴结及远处转移与NGFR高甲基化密切相关，VEGF-C高表达可增强肿瘤细胞的侵袭转移；进一步研究发现NGFR高甲基化的病例常同时呈现VEGF-C的高表达。由此，我们首次提出NGFR高甲基化通过活化VEGF-C、关联下游MAPK信号通路促进肠癌细胞侵袭转移的新机制。本课题拟采用激光共聚焦等技术在三维细胞培养模型中获得NGFR活化VEGF-C的可靠证据；通过动物活体实验，动态观察NGFR表达水平对肿瘤生长及侵袭转移的影响，从"细胞-动物-病人"多个层次揭示NGFR和VEGF-C在肠癌侵袭转移过程中的作用；筛选参与侵袭转移的下游MAPK通路靶基因，通过系列体外及体内功能实验揭示其调控机制。本课题将为结直肠癌的靶向治疗提供理论依据。

结直肠癌是一种常见的消化道恶性肿瘤，甲基化突变近来被认为是肿瘤中抑癌基因改变的途径之一。在前期研究的基础上，我们发现NGFR基因在结直肠癌肿瘤组织中存在启动子区域高甲基化及NGFR高甲基化的患者易发生淋巴结及远处脏器转移，相关结果已经发表。此外，我们采用甲基化试剂处理结直肠癌细胞株后，NGFR的甲基化水平、mRNA表达量、细胞生长会出现变化。采用慢病毒介导的shRNA干扰能有效减少结直肠癌裸鼠移植瘤中NGFR的表达,降低NGFR的表达能促进结直肠癌移植瘤的生长。通过免疫组化方法检测110例结直肠癌患者手术切除的癌组织和癌周正常组织中NGFR和VEGF-C的表达情况。研究发现，NGFR与VEGF-C表达水平均与结直肠癌淋巴结转移有关(P＜0.05)。本研究从“细胞-动物-病人”多个层次揭示NGFR和VEGF-C在肠癌发生、发展过程中的作用，将为结直肠癌的靶向治疗提供理论依据。