甲基化介导沉默MicroRNA-124调控SNAI2蛋白在结直肠癌肝转移上皮间质转化中的机制研究

The epithelial-mesenchymal transition (EMT) is one of the significant mechanisms of colorectal liver metastases. MicroRNA-124 (miR-124) has been known as the EMT related specific MicroRNA. However, its function in colorectal carcinogenesis has not been clarified. Our previous research firstly found: ①the miR-124 expression was significantly downregulated in colorectal cancer. MiR-124 was an independent prognostic factor and involved in regulation of differentiation, tumor invasion and metastasis. ②The methylation level of CpG islands in miR-124 promoter regions was high. The expression of miR-124 increased in colorectal cancer cell after demethylation. ③ As a potential target gene of miR-124, thebexpression of EMT regulator SNAI2 was negatively correlated with miR-124 expression. Therefore, we hypothesize that methylation-mediated silencing of miR-124 targeting SNAI2, is functionally involved in regulating EMT and colorectal liver metastases. To test the hypothesis, we will construct lentiviral vector and mutant vectors containing 4 mutated bases on the separately predicted binding sites. Applying dual-luciferase reporter system, qMSP and mouse model of colorectal liver metastasis, we will explore the function of miR-124 and target gene SNAI2 in colorectal carcinogenesis to deeply understand the process of EMT and colorectal liver metastases, and provide new evidence to uncover the mechanism of colorectal cancer invasion and metastasis.

上皮间质转化（EMT）是结直肠癌肝转移发生的重要机制之一。作为EMT相关特异性MicroRNA，MicroRNA-124（miR-124）在结直肠癌中的功能尚未阐明。我们前期研究首次发现：在结直肠癌中①miR-124下调表达，与预后相关，参与调控分化及侵润转移；②miR-124高甲基化，去甲基化处理使miR-124表达增高；③EMT调控因子SNAI2是miR-124潜在靶基因。我们提出假设：miR-124启动子区CpG岛DNA甲基化介导miR-124基因沉默，通过直接靶基因SNAI2，参与调控结直肠癌肝转移EMT。为验证上述假设，本课题继续构建慢病毒包装载体及融合靶基因3’UTR重组载体，采用双荧光素酶报告基因分析、qMSP及结肠癌肝转移小鼠模型。对miR-124及其靶基因SNAI2在结直肠癌中功能进行探索，旨在深入认识结直肠癌肝转移EMT发展过程，为揭示结直肠癌侵润转移机理提供新的证据。

结直肠癌在全球范围内发病率和死亡率均位于第3位。而我国30年来，结直肠癌发病率发病率猛增3倍，甚至超过部分发达国家的水平，而病死率也随之升，也已成为破坏人民健康及经济发展的重大疾病。其中，大约50%~60%的结直肠癌患者在确诊时已发生远处转移，其中肝转移占一半以上。另有25％患者在接受结直肠癌根治术后仍出现肝转移，严重影响患者术后生存率。临床数据表明结直肠癌的侵袭转移，特别是肝转移是导致结直肠癌病死率逐年递增的关键因素。.本课题首次揭示表观遗传miR-124启动子区CpG岛DNA甲基化介导miR-124基因沉默，miR-124调节结直肠癌肝转移EMT；通过结构鉴定、表达验证，证明在结直肠癌中SNAI2是miR-124直接靶基因；运用单/双基因沉默、共表达、结肠癌肝转移裸小鼠模型，揭示miR-124调控SNAI2参与结直肠癌肝转移EMT的作用机制。采用临床-基础-临床的转化医学理念，从细胞和分子水平对miR-124及其靶基因SNAI2在结直肠癌中的功能提出新的认识，其结果有助于深入认识结直肠癌肝转移EMT发展过程，为揭示结直肠癌侵润转移机理提供新的证据。