候选抑癌基因Nkx2.5在结直肠癌中的作用及机制研究

It has been reported that homeobox transcriptional factor Nkx2.5 was hypermethylated in primary colorectal cancer tissues, as well as in colon cancer cell lines. Nkx2.5 hypermethylation results in significantly attenuated expression or even silencing of the gene in colorectal cancer. However, further studies or reports of the significance of this phenomenon have not been carried out so far to date. The reported phenomenon has been verified in our preliminary studies using colorectal cancer tissues and cell lines. For further study of the roles of Nkx2.5 in colorectal cancer, Nkx2.5-overexpression colon cancer cell line has been established, and inhibited proliferation, promoted apoptosis and cell cycle arrest have been observed in the Nkx2.5-overexpression cancer cells. Therefore, we hypothesized that Nkx2.5 might function as a tumor suppressor gene in colorectal cancer. Furthermore, results from related database supported this hypothesis. This project is designed to prove this hypothesis and further explore its regulatory mechanisms in vitro and in vivo using cell models, mouse models, and colorectal cancer tissue specimens. After Nkx2.5 expression restored in methylated CRC cell lines by transfecting with lentivirus, and Nkx2.5 expression suppressed in FHC cells by siRNA, cell and mouse models will be used to investigate changes in cell/tumor growth and the related signaling pathways. Furthermore, downstream target genes will be screened using Microarray Technology. The expression of the identified target genes will be artificially managed to observe their influence on the biological behavior of colon cancer cells. Then, the interactions between Nkx2.5 and the related signaling pathways will be verified in colorectal cancer specimens. The association of alterations of Nkx2.5 and its related signaling pathways with clinical characteristics and prognosis of CRC patients will be analyzed. The prognostic evaluation value of methylated Nkx2.5 as a biomarker will also be evaluated. The results of this study will provide novel scientific evidences to improve the prognostic evaluation and targeted therapy for CRC.

文献报道同源盒转录因子Nkx2.5在结直肠癌组织标本及细胞系中发生高甲基化，且表达下降甚至不表达，但对其意义并未作进一步的研究和报道。申请人在前期研究中验证了该报道中的现象，而后为了初步探讨Nkx2.5在结肠癌细胞中的作用，申请人在结肠癌细胞中过表达Nkx2.5，发现其能抑制癌细胞增殖，促进凋亡，并导致细胞周期停滞。因此我们提出了Nkx2.5在结直肠癌中发挥潜在抑癌作用的假设。相关数据库的检索结果也支持这一假设。本课题将在细胞-动物-组织标本三个层次求证这一假设并探索其机制。在细胞和小鼠模型中研究Nkx2.5表达量改变对癌细胞生物学特性及相关信号通路的影响；应用基因芯片技术筛选靶基因并研究其机制;而后在临床组织标本中分析并验证Nkx2.5及其相关信号通路与肿瘤的临床病理特征及患者预后之间的关系，评估Nkx2.5作为预后判断标志物的可能性。本课题将为结直肠癌的预后判断和靶向治疗提供新的理论依据。

文献报道同源盒转录因子Nkx2.5在结直肠癌组织标本及细胞系中发生高甲基化，并认为Nkx2.5在结肠癌中是可能性极大的候选抑癌基因，但并未作进一步的深入研究。与此同时，同源盒转录因子NK2家族的其他基因均陆续被报道与癌症的发生发展相关。在我们的前期研究中，我们在结肠癌细胞HCT116中过表达Nkx2.5，发现其能抑制癌细胞增殖，促进凋亡，并导致细胞周期停滞。因此提出了Nkx2.5在结直肠癌中发挥抑癌作用的假设。我们在5例结肠癌组织标本中验证了Nkx2.5基因启动子在癌组织标本中甲基化程度高于正常结肠粘膜组织，但在后续14例结肠癌组织及正常结肠粘膜组织标本的检测中发现Nkx2.5的mRNA表达在两者之间无显著性差异，而Nkx2.5的蛋白水平在癌组织中较高。对GEO及TCGA中数据进行分析发现结肠癌组织与正常结肠粘膜组织中Nkx2.5的mRNA表达无显著差异，但癌组织中mRNA表达量离散度更大。而在数据库中进行生存分析后发现，Nkx2.5在肿瘤中表达更高的结肠癌患者预后越差，这个现象与Nkx2.5为抑癌基因的假设相悖。而后为了进一步探讨Nkx2.5在结肠癌细胞中的作用，我们对常见的8种结肠癌细胞进行检测发现Nkx2.5的蛋白水平与癌细胞的恶性程度呈现正相关性。我们在另一种结肠癌细胞SW480中过表达Nkx2.5，但是Nkx2.5的过表达对SW480的增殖、凋亡及细胞周期没有任何影响。经查询数据库并对细胞进行测序后我们发现结肠癌细胞中Nkx2.5蛋白水平高低与TP53基因是否发生突变具有显著相关性，其中HCT116中TP53基因为野生型，Nkx2.5蛋白水平低；SW480中TP53基因为突变型，Nkx2.5蛋白水平高。因此我们提出了Nkx2.5是依赖p53蛋白发挥其抑癌作用的假设。后续研究中我们发现Nkx2.5能结合p53蛋白，并增加野生型p53蛋白的转录活性，特别是能增加抑癌蛋白p21的表达，并对下游一系列细胞周期相关蛋白的表达具有显著影响；但对于突变型p53蛋白则没有增加转录活性作用，也不影响抑癌蛋白p21的表达。本研究揭示了Nkx2.5在结肠癌中的抑癌作用及其作用机制，将为结直肠癌的预后判断和靶向治疗提供新的理论依据。