dlk1基因在非小细胞肺癌中异常高表达及其通过Notch信号通路促进肿瘤转移的分子机制研究

Previously microarray based study of our group discovered a number of genes that were differentially expressed between squamous cell lung cancer with or without lymph node metastasis. Annotation with biological database made dlk1 as a candidate for detailed study. Our preliminary work found that dlk1 expressed higher in tumor tissues compared with adjacent normal lung. Without a copy number aberration detected in dlk1 region in the genome, we found that its aberration expression was partially because of abruption in DNA methylation. A couple of transcription factor (TF) binding site were also detected in the promoter region of dlk1 gene. Up-regulation of dlk1 could also activate Notch signaling pathway. Upon these results, we suggest that aberration in epigenomics and TF regulatory could cause up-regulation of dlk1 expression in lung cancer and consequently promote tumor invasion through Notch signaling pathway. In this study, we first confirm the expression of dlk1 in protein level with a cohort of surgical resected tumor tissues from NSCLC patients and explore the relationship between dlk1 expression and clinical characters. We employ bisulfate sequencing method to define the CpG area in which methylation changes could cause aberrant dlk1 expression. ChIP was also employed to confirm the regulation of predicted TF to dlk1. Meanwhile, we investigate dlk1's effects on cell invasion both in vitro and in vivo, and explore the signal transduction pathways involved in dlk1 regulated cell invasion. Together, we explain dlk1's role in human NSCLC in combination of illustrating the mechanism of its aberrant expression and investigating how this aberration function in cell behavior.

申请者前期利用表达谱芯片筛选出一组与人非小细胞肺癌淋巴结转移相关的差异表达基因；生物信息学挖掘筛选出dlk1基因进行深入研究。预实验发现，dlk1在非小细胞肺癌肿瘤组织中表达高于癌旁正常组织，其上游具有CpG岛及多个转录因子结合位点，dlk1过表达可以激活Notch信号通路。据此我们推测，dlk1所在区段的表观遗传学改变与上游调控因子异常引起其在肺癌中异常表达，进而激活Notch信号通路促进肿瘤细胞的转移。本项研究首先在蛋白水平明确dlk1在大样本肺癌组织中的表达状态及其与肺癌临床特征间的关系；继而从表观遗传学与转录调控因子两个方面界定引起dlk1异常表达的机制；同时，从体内及体外两个方面研究dlk1在肺癌转移中的生物学效应，明确其促进肺癌细胞转移的信号传导途径。从上、下游两个角度，揭示dlk1在肺癌发生演进中的作用，并探讨将其作为候选的转移相关分子标志的可能性。

本项目立题始于申请者前期利用基因表达谱芯片筛选得到的与人非小细胞肺癌淋巴结转移相关的差异表达基因，DLK1。通过本项目的研究，我们对该基因与非小细胞肺癌的关联及其分子机理进行了深入的分析。我们首先在一组大样本的非小细胞肺癌肿瘤组织中证明DLK1基因存在异常高表达，且表达强度与肿瘤分期、肿瘤大小显著相关。进一步分析发现DLK1基因上游CpG甲基化岛异常甲基化是其异常高表达的原因之一。对其在非小细胞肺癌中分子机理的研究证明，DLK1基因通过激活Notch信号传导通路，上调细胞中基质金属蛋白酶9（MMP9）的表达及活性形式的分泌，从而促进细胞的侵袭与转移。在此基础上，我们通过免疫共沉淀及蛋白质谱分析的方法，获得了细胞中DLK1的相互作用蛋白，并进行了初步的分析。通过本项目的研究，我们明确了非小细胞肺癌中新的癌基因DLK1，并对其功能进行了研究。该项目的研究结果整理成2篇研究论文，发表于国际及国内专业期刊。