基于eIF3a基因多态、microRNA及其启动子DNA甲基化的铂类耐药分子机制研究

Lung cancer is a severe harmful tumor with common occurrence for human life and health all over the world. The chemotherapy of patients with lung cancer is mainly treated with platinum-based drugs, but a great deal of the individual diffrences and resistances of drugs response exist commonly in the treatment course of lung cancer patients. Recently, the role of epigenetics involved in the DNA methylation modification and miRNA modulation in drug response differences is gradually concerned and emphasized by the people. In the our previous studies, we found that the genetic polymorphisms of eukaryotic translation initiation factors-3 (eIF3a) can modulate the chemotherpy sensitivity in lung cancer patients treated with platinum-based medicine by regulating the protein expression of DNA repair pathway. On the basis of these results, this project is going to clarify the effects of two novel eIF3a mutants (eIF3a C1458T and G2554A, found by our groups) on eIF3a biological funtion and molecular mechanism, screen the microRNAs and promoter methylation sites related to eIF3a function by the use of the Luciferase reporter gene assay and methylation PCR technology as well as elucidate the molecular mechanisms of microRNAs and promoter methylation level regulating the eIF3a function. Finally, we are going to investigate the relationship between the genetic polymorphism of eIF3a, microRNAs, and promoter methylation sites and the efficacy of chemotherapy in large samples of lung cancer patients treated with platinum-based drugs. The aim of this projects will can provide some new mechanism explanations with the resistance occurrence in the treatment of platinum-based drugs in patients with lung cancer from epigenetics analysis and theoretical basis and experimental evidence with formulation of individualized chemotherapy program to gene-guidance platinum-based treatment.

肺癌化疗主要以铂类药物为主，但个体反应差异性大，耐药现象十分严重。近年来表观遗传学在药物反应差异中的作用逐渐受到极大关注，主要包括DNA甲基化修饰以及microRNA调控。我们前期工作发现，eIF3a基因及多态可以通过调控DNA修复途径蛋白表达来调节肺癌铂类药物化疗敏感性。在此基础上，本课题首先使用激光共聚焦和Gel shift等方法进一步阐明我们发现的eIF3a C1458T和G2554A新突变对eIF3a功能的影响，然后使用荧光素酶报告基因和甲基化PCR方法筛选影响eIF3a功能的microRNA和启动子甲基化位点，阐明其影响eIF3a功能的分子机制；最后查明调控eIF3a基因的多态、microRNA和DNA甲基化位点与肺癌铂类药物化疗敏感性的关系。本课题旨在从表观遗传学的角度为肺癌铂类药物耐药的发生提供新的解释机制，对以基因为导向的铂类药物化疗提供理论依据和实验依据。

背景：肺癌化疗主要以铂类药物为主，但个体反应差异性大，耐药现象十分严重。近年来表观遗传学在药物反应差异中的作用逐渐受到极大关注，主要包括DNA甲基化修饰以及microRNA调控。我们前期工作发现，eIF3a基因及多态可以通过调控DNA修复途径蛋白表达来调节肺癌铂类药物化疗敏感性。.研究内容：本课题旨在阐明调控eIF3a基因的多态、eIF3a相关microRNA和DNA甲基化位点与肺癌铂类药物化疗敏感性的关系，从基因多态性和表观遗传学的角度为肺癌铂类药物耐药的发生提供新的解释机制，对以基因为导向的铂类药物化疗提供理论和实验依据。.重要结果、关键数据：.1.eIF3a基因多态性与肺癌铂类药物的化疗疗效及毒性的关系已完成，发表相关文章2篇：.1.1通过HapMap软件筛选了eIF3a的127个SNP位点，并在325例非小细胞肺癌患者的血液样本中提取DNA进行了基因分型发现，eIF3a的5个SNPs (rs1409314, rs4752219, rs4752220, rs7091672 和 rs10510050) 与非小细胞肺癌患者铂类化疗毒性相关。.1.2通过对185个基因的416个SNPs进行基因分型，使用logistics回归方法建立了进展期非小细胞肺癌患者铂类化疗疗效和毒性的预测模型。.2.eIF3a相关microRNA调控肺癌铂类化疗敏感性已完成，发表相关文章1篇：.通过生物信息学预测和双荧光素酶报告基因实验验证microRNA-488能通过作用于eIF3a的3’UTR来抑制eIF3a的表达，并通过一系列实验发现MiR-488通过活化eIF3a介导的NER信号通路来抑制非小细胞肺癌细胞的增殖和铂类化疗敏感性。.3.DNA甲基化与肺癌的关系已完成，发表相关文章1篇：.通过DNA甲基化检测分析发现肺鳞癌的5214个差异甲基化探针、449个异常甲基化基因，以及潜在诊断标志物CLDN1, TP63, TBX5, TCF21, ADHFE1和HNF1B。.4.发明了一种检测与肺癌易感性及化疗敏感性相关基因的多态性的试剂盒。.科学意义：本课题率先证明了eIF3a基因多态、eIF3a相关microRNA和DNA甲基化位点与肺癌铂类化疗疗效及毒副反应的相关性，为寻找肺癌的诊断和治疗新靶点，以及提高肺癌早期诊断率和化疗疗效、减少化疗毒性提供理论和实验依据。