miR-19a对结肠癌化疗药物敏感性的影响

Colon cancer(CC) is one of the most common types of malignant tumors in the western world, so is in China. Oxaliplatin-based chemotherapy is a major protocol for CC therapy in both adjuvant and metastatic setting. However, resistance to chemotherapeutic reagents is the most challenging issue in the treatment of CC, leading to relapse and poor patient prognosis. Therefore, discovering novel biomarkers for CC treatment is crucial to realize the aim of personalized medicine.The development of drug resistance is often a multifactor process, which involves multiple genetic and cellular mechanisms. microRNAs (miRNAs) are endogenous small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level. miRNAs regulate a large number of genes by interacting with complementary sites in the 3'untranslated region (3'UTR) of target genes. Currently, extensive studies have indicated that the acquisition of drug resistance by cancer cells may also be modulated via the changes in miRNA levels. In various types of cancers, many miRNAs are differently expressed and can serve as either oncogenes or tumor suppressors. Our previous studies have demonstrated that miR-19a was highly expressed in colon cancer tissue; miR-19a could significantly reduce the sensitivity of colon cancer cells to oxaliplatin; the target gene of miR-19a was TP53INP1; in the oxaliplatin-resistant cell line miR-19a was highly expressed and TP53INP1 expression was reduced. The hypothesis of our study is that miR-19a may reduce the sensitivity to oxaliplatin for colon cancer by targeting TP53INP1. We will investigate the impact of miR-19a on the sensitivity to oxaliplatin via lentiviral vectors; explore the mechanism of oxaliplatin resistance in the level of miRNA and proteins by the suspension array system and 2-D gel chromatography; construct an animal model to observe the influence of oxaliplatin on the implanted tumor; and investigate the impact of miR-19a and TP53INP1 on colon cancer patients after surgery. We aim to provide a new clue for the treatment of oxaliplatin resistance by serial investigations of the impacts and mechanisms of miR-19a on oxaliplatin sensitivity.

耐药是导致结肠癌化疗失败最主要的原因，严重阻碍化疗方案的实施，寻找新的生物学标记从而实现个体化治疗结肠癌刻不容缓。我们前期工作发现：miR-19a在结肠癌组织中高表达；miR-19a显著降低结肠癌细胞对奥沙利铂的药物敏感性；miR-19a的靶基因是TP53INP1；在奥沙利铂耐药细胞株中miR-19a表达显著增加，TP53INP1蛋白表达明显下降。我们推测miR-19a通过调节TP53INP1影响结肠癌对奥沙利铂药物敏感性。本研究通过慢病毒介导载体研究miR-19a对药物敏感性的影响；利用悬液芯片及2-D凝胶电泳技术，从miRNA及蛋白层面阐明肿瘤细胞的耐药机制；建立移植瘤动物模型观察奥沙利铂对移植瘤的作用；临床观察miR-19a及TP53INP1对术后化疗的影响。本项目旨在通过对miR-19a系统研究，深入探讨miR-19a对奥沙利铂药物敏感性的影响，为临床结肠癌治疗提供新的思路。

结直肠癌的发病率和死亡率居高不下，以手术为主的术后辅助化疗是目前治疗结肠癌的最主要方法。耐药是目前结肠癌治疗中最常见的问题，严重阻碍化疗方案的实施，寻找新的影响药物敏感性的生物学分子已成为结肠癌治疗中的热点问题。针对这个研究背景，我们设计了两方面的研究课题：第一，利用qRT-PCR检测5-Fu耐药细胞株HCT8/Fu及敏感细胞株HCT8中miR-19a的表达差异;MTT法检测miR-19a对细胞5-Fu药物敏感性的影响;双荧光素酶报告基因系统对miR-19a与TP53INP1的靶基因关系进行鉴定;Western blot检miR-19a对TP53INP1表达的影响，最后miR-19a在HCT8/Fu中的表达量是HCT8细胞的7.12倍,显著高于敏感HCT8细胞株的表达量(P<0.05)；miR-19a通过作用于TP53INP1的3’-UTR负调控其表达，进而促进耐药，这为下一步建立miR-19a/TP53INP1通路在结直肠癌中的治疗提供新策略。同时，我们也进一步探索，发现TRIM11在结直肠癌细胞中表达量明显高于癌旁组织，且高表达TRIM11的结直肠癌患者临床预后较差。在研究TRIM11为什么表达上调方面，我们通过生物信息、双荧光素酶报告实验、Western blot、real-time PCR等方法明确了miR-24-3p可以直接作用于TRIM11的3’UTR区域，并且负调控TRIM11的表达，这个发现也为靶向TRIM11提供了新的策略。通过过表达TRIM11以及利用Cas9技术敲低TRIM11，并通过CCK8、克隆形成、凋亡实验以及体内动物实验我们发现，TRIM11具有促进结直肠癌细胞增殖、克隆形成和促进体内成瘤能力，和具有抑制结直肠癌细胞凋亡的作用，这些数据充分说明TRIM11在结直肠癌细胞中可能发挥促癌作用已经可以作为潜在的治疗靶点。本研究课题发表相关研究论文2篇。