利用全基因组敲除筛选鉴定奥沙利铂耐药的相关基因

Oxaliplatin (L-OHP) in combination with other chemotherapeutic drugs is the standard first-line therapy for colon cancer patients, however, as the therapy progresses almost all patients will develop resistance eventually leading to a failure of chemotherapy and a relapse of tumors. At current stage, the clinical problem with respect to resistance to platinum drugs developed by tumor cells remains and there are no sophisticated and comprehensive studies that clearly reveal the underlying molecular mechanisms. This study is aimed to identify L-OHP resistance-related genes at genome-wide level by using CRISPR-cas9 knockout technology. A library of retroviruses called GeCKO library has been created to perform a genome-wide in HCT116 cell line under the selective pressure of L-OHP treatment. Through deep-sequencing, the depleted genes will be analyzed to identify candidate genes that render HCT116 colon cancer cells resistant to L-OHP. Subsequently, the ability of candidate genes to control sensitivity to L-OHP will be validated both in vitro and in vivo by individual knockout and re-expression of those genes. Using public resources of databases biological functions and processes that the candidate genes may execute and participate will be dissected to reveal the molecular mechanisms of L-OHP acquired resistance. Results obtained from this study would help elucidate molecular basis on acquired resistance to L-OHP from new perspectives, and provide a scientific fundamental for the rational use of drugs in clinics and find ways or strategies of preventing the development of resistance during treatment or reversing it once established.

奥沙利铂(L-OHP)联合用药是结肠癌患者的一线化疗方案，然而随着治疗的进行，几乎所有患者都将产生耐药并最终导致化疗失败、肿瘤复发。目前，针对肿瘤细胞发生铂类药物耐药的临床治疗难题，并没有全面的研究清楚揭示其分子机制。本研究拟通过CRISPR-cas9基因敲除技术，从全基因组层面发现介导奥沙利铂耐药的相关基因。利用GeCKO病毒敲除文库，在L-OHP筛选压力下，对结肠癌细胞HCT116实施全基因组敲除。通过深度测序，分析细胞中发生敲除的基因，获得L-OHP耐药的相关候选基因系列。随后在体外体内采用单基因敲除、回补表达等方法，验证候选基因的有效性。利用公共数据库分析其生物学功能及参与的生物学行为，揭示候选基因介导L-OHP耐药的分子机制。本研究结果将有助于从全新的角度揭示L-OHP获得性耐药的分子基础，为临床合理用药和寻找防止耐药产生、逆转已出现的耐药的方法和策略提供科学依据。