构建结肠癌L-OHP耐药细胞模型并结合临床疗效鉴定新型耐药基因

FOLFOX regimen(combinations of oxaliplatin with 5-fluorouracil and leucovorin) is currently one of the first-line treatments for advanced colorectal cancer(ACC) patients. However,about 50% patients are resistant to the regimen.We have found 24 genes consistently showing differential expression between responders and non-responders of three independent cohorts of ACC patients treated with FOLFOX regimen.Then from these 24 genes, we select four essential L-OHP drug-resistant genes by analysing the expression profile of one L-OHP drug-resistant cell line.This project will verify the L-OHP drug resistance of these four genes（CES2, TUBB3, POLD2,CLDN7） by constructing a L-OHP drug-resistant cell line model HT-29/L-OHP. Through silencing these genes seprately, we will study the impacts of down-regulated target genes on the drug-resistance of cell line model and confirm that these four genes are new drug-resistant ones.By the way of detecting various cell lines gene expression profile through gene chip technics, the differentially expressed genes and enrichment of drug-resistant related signaling pathways will be analyzed to explore their resistance mechanism. Finally, we attempt to further verify the expression pattern in of these drug resistant genes in non-responders by collecting clinical samples, providing a basis for building classifier to distinguish the ACC patients who can benefit from L-OHP chemotherapy.

含奥沙利铂（L-OHP）的FOLFOX方案常用于晚期结直肠癌一线化疗，但仍有约一半患者无效。本课题组前期通过分析3组独立的接受FOLFOX化疗患者组织样本的基因表达谱，发现24个在药物响应组和无响应组间稳定差异表达的疗效相关基因；再分析L-OHP耐药细胞系的基因表达谱，进一步从中筛选CES2、TUBB3、POLD2、和CLDN7基因作为L-OHP耐药相关基因。本项目拟在此基础上构建人结肠癌获得性L-OHP耐药细胞系模型HT-29/L-OHP，以RNAi沉默上述靶基因，检测靶基因下调对细胞模型耐药性的影响，鉴定上述基因是L-OHP的新型耐药基因；以基因芯片检测各类细胞系的基因表达谱，分析其差异表达基因及富集的耐药相关信号通路，探索耐药机制；并采集临床样本，研究上述耐药基因的表达与FOLFOX化疗疗效的相关性，验证耐药基因的疗效预测能力，指导临床化疗方案选择，为后续构建药效预测模型提供依据。

含奥沙利铂（L-OHP）的FOLFOX方案常用于晚期结直肠癌一线化疗，但仍有约一半患者无效。本课题组通过分析前期数据，从中筛选CES2、TUBB3、POLD2、和CLDN7 基因作为L-OHP耐药相关基因。在此基础上，成功构建了结直肠癌奥沙利铂耐药细胞模型，并通过免疫组化分析验证接受了FOLFOX 化疗方案得晚期结直肠癌患者上述基因表达情况，筛选出耐药标记最明显的CES2基因作为核心靶基因进行深入机制探讨，通过RNA干扰技术敲减CES2基因表达用于验证探讨CES2基因与化疗敏感性的关联性，结果提示CES2基因通过调控PI3K/AKT通路的表达从而影响细胞凋亡、增殖等功能学的改变从而导致耐药。同时对前期构建的奥沙利铂耐药模型予以高通量测序，结果提示奥沙利铂耐药细胞株主要与细胞增殖、免疫反应等功能通路相关。为进一步从上游寻找与奥沙利铂耐药相关基因通路，我们搜集了11例治疗前患者的肠镜标本，并予以高通量基因芯片检测其环状RNA、LncRNA以及mRNA表达情况，据此筛选出了效果更为明显的新基因FBXW4，并通过多角度验证该基因与奥沙利铂耐药的关联性，与本项目前期结果相吻合。本课题最终筛选获得了多个与奥沙利铂耐药相关的核心基因，并通过患者标本免疫组化结果得到验证。

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