受甲基化调控的抑癌基因TLX2在食管癌早期诊断中的应用及作用机制研究

Esophageal cancer is one of malignant tumor with poor prognosis and low survival rate. Early diagnosis can effectively improve the survival rate of patients with esophageal cancer. We find the methylation level of TLX2 promoter region is significantly higher than that of paracancerous tissues and the expression of TLX2 is down-regulated in cancer tissues through data analysis of genomic DNA methylation chip, Methyl-target and q-PCR test validation. This suggests that TLX2 may be used as a biomarker of early diagnosis of esophageal cancer. Several experiments, such as cell proliferation and nude mouse tumor formation, initially confirm that TLX2 is a methylation-regulated tumor suppressor gene. At present, the specific mechanism of TLX2 in the diagnosis and development of esophageal cancer is not yet clear, including its diagnosis efficacy in esophageal cancer, the regulation of methylation and the downstream target genes. This project intends to find TLX2’s co-regulator factors and downstream target genes and explore the mechanism of signal pathway of TLX2 regulating esophageal development and provide reliable experimental data for revealing the mechanism of esophageal carcinogenesis through a series of molecular cell experiments. Furthermore, this project also intends to detect and analysis the methylation level of circulating DNA in plasma from patients with esophageal cancer and normal control subjects and establish the relationship with the main risk factors of esophageal cancer and the early diagnosis of esophageal cancer model, in order to obtain a diagnostic strategy with independent intellectual property rights.

食管癌预后差，生存率低，早期诊断可以有效地提高食管癌病人的生存率。通过全基因组DNA甲基化芯片数据分析、Methyl-target及q-PCR检测验证，我们发现食管癌组织中TLX2基因启动子区域异常高甲基化，同时伴随表达量的显著下降，提示TLX2可能作为食管癌早期诊断的标志物。细胞增殖和裸鼠成瘤等多个实验初步证实TLX2是受甲基化调控的抑癌基因。目前TLX2对食管癌诊断效能，甲基化修饰调控，下游靶基因等一系列参与调控食管癌发生发展的具体机制尚不清楚。本项目拟通过一系列分子细胞实验寻找与TLX2基因互作的蛋白及下游靶基因，探究以TLX2为节点的信号网络调控食管癌发生发展的作用机制，为揭示食管癌发生发展机制提供可靠的实验数据；同时检测其在正常对照及食管癌人群血浆游离DNA中TLX2甲基化程度，建立其与食管癌主要风险因素的关系及食管癌早期诊断模型，以期获得一项具有自主知识产权的诊断策略。

食管鳞癌（ESCC）恶性程度高，患者五年生存率较低，严重危害国民健康。前期研究发现，TLX2在ESCC组织呈高甲基化低表达，其在癌组织与癌旁组织间的甲基化差异具有潜在的临床诊断价值。基于此，本项目筛选并鉴定了在ESCC中TLX2的互作蛋白p53及下游调控基因p21，初步表明TLX2可与p53相互作用来促进p21的表达，进而发挥其抑癌作用。.为进一步探究TLX2是否可作为转录因子对下游靶基因进行调控，本项目利用RNA-Seq结合实时荧光定量PCR实验、Western Blot实验证实TLX2可上调IFIT2的表达水平。通过双荧光素酶报告系统实验、ChIP-PCR证明，TLX2作为转录因子可与IFIT2启动子区结合，激活了IFIT2的转录活性。并明确了靶基因IFIT2在ESCC中的抑癌作用。构建TLX2同源盒结构域缺失型变体发现，TLX2通过同源盒结构域发挥转录调控作用及肿瘤抑制作用。进一步阐明了TLX2-IFIT2信号轴调控ESCC发生的分子机制，为ESCC的临床诊治提供新的理论依据和策略。.受该基金资助，在食管癌中研究了另一个受甲基化调控的抑癌基因ZNF132，探明了该基因在食管癌中低表达的详细分子机制，为深入理解食管癌的发病机理提供了新思路。还研究了甲基转移酶样3（METTL3）在食管鳞癌中的生物学意义，为进一步阐明METTL3在食管鳞癌发生发展中的分子机制奠定基础。.总结已有的研究成果，部分结果已经整理成科研论文，其中在SCI期刊上已经发表5篇。累积影响因子约26分，部分结果还在继续撰写将投稿至相关专业SCI期刊上。

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