新候选抑癌基因NLK在肠癌中的功能鉴定及其表观遗传学调控机制的研究

NLK gene is a new tumor suppressor gene in colorectal cancers ,and our previous study had demonstrated that oncogenic miR-199a-3p could targetly down-regulate NLK gene expression in colorectal cancer. NLK expression in colorectal cancer tissues was obviously lower than that of adjacent nontumor tissues, and gene promoter of NLK in normal intestinal epithelial tissues showed low methylation, while hypermethylated in colorectal cancer cells. To further certify the tumor suppresive effects in the initiation and progression of colorectal cancer and its potential mechanism of epigenetic regulation, our study is planed to detect the expression of NLK in colorectal cancer, and certify the correlation between the NLK expression and its clinicopathological features. We will also build the eukaryotic expression vector, siRNA interference vector and cancer model in nude mouse to clarify the suppressive effect of NLK gene in colorectal cancer. Besides that, in order to discuss the molecular mechanism of NLK down-regulation, we will focus on the altered DNA methylation in promoter region and histone acetylation of NLK gene in colorectal cancer treated with 5-Aza or trichostatin A, we also discuss the correlation between low methylation of NLK gene promoter and its clinicopathological features to certify the significance of NLK low methylation for the prognosis of colorectal cancers. Our study will enrich the mechanism of the initiation and progression of colorectal cancer and make for finding a promising molecular target for colorectal cancer diagnosis and therapy.

NLK基因是本课题组新近提出的肠癌候选抑癌基因，前期研究发现在肠癌中具有促癌作用的miR-199a-3p能靶向下调NLK的表达，人肠癌组织中NLK的表达显著低于癌旁正常组织，NLK基因启动子在正常肠上皮组织中呈低甲基化而在肠癌细胞中呈高甲基化状态。为进一步明确NLK在肠癌发生发展的抑癌作用及其表达调控机制，本项目拟首先通过研究NLK基因在肠癌组织的表达分析其与临床病理特征的关系；并通过构建NLK基因真核表达载体、siRNA干扰载体和裸鼠肠癌模型等方法阐明NLK基因表达在肠癌发生、发展中的作用；通过5-Aza及曲古抑菌素A处理对NLK表达影响等实验获得NLK基因表达调控的可靠证据，并在肠癌标本中研究NLK甲基化与肠癌临床病理特征的关系，明确NLK基因启动子低甲基化与肠癌病人预后的关系。通过本项目的实施，将进一步丰富肠癌发生、发展的机制，为肠癌的临床诊治预防、寻找治疗肠癌新靶点提重要的理论依据

我们对miR-199a-3p与肠癌的关系研究，发现miR-199a-3p在肠癌中表达升高，miR-199a-3p可以作为一种促癌的miR影响肠癌病人的预后，并在体外影响肠癌细胞的生长、周期及凋亡变化。部分研究结果被Medical Oncology杂志收录发表（Med Oncol.2013;30:378）。在此基础上，我们就miR-199a-3p调控的靶基因又进行了初步的研究，发现miR-199a-3p能靶向调控NLK基因。并且我们初步检测了NLK基因在肠癌组织中的表达，发现NLK基因在肠癌中较癌旁正常组织表达明显下调，肠癌细胞株中的NLK基因启动子区域甲基化水平较高，5-Aza处理后肠癌细胞中的NLK基因启动子区域的甲基化水平得到不同程度的恢复。这些研究结果提示我们：NLK基因很可能作为一种抑癌基因在肠癌的发生、发展过程中发挥重要作用，并且其启动子区域的甲基化修饰可能是导致NLK基因失活的机制之一。这些研究工作进一步丰富了肠癌发生发展的分子机制，也是本项目重要的立项依据。