DHX32在结肠癌5-FU耐药中的作用及机制

5-Fluorouracil (5-FU)-based adjuvant chemotherapy is routinely given to the great majority of patients with colorectal cancer. However, chemoresistance is a major obstacle for colorectal cancer treatment which highly impact on the outcome of treatment, and as high as 50% of metastatic colorectal cancer patients are resistant to 5-FU-based chemotherapy.Although many different mechanisms for 5-FU induced apoptosis as well as the development of 5-FU resistance have been proposed, the underlying molecular details remains obscure. Our lab identified DHX32 by DD-PCR from colorectal cancer tissues and found that the level of DHX32 gene expression was significantly associated with malignant degree of colorectal cancer. Further analysis showed that knockdown of DHX32 in colorectal cancer cell was able to inhibit cell proliferation and promote 5-FU induced cell apoptosis. We also investigated that the kinase activity of AKT was suppressed after the interferance of DHX32 expression. Therefore, we propose the possible mechanisms of 5-FU resistance : DHX32 regulates colorectal cancer cell apoptosis via PI3K/AKT pathway. This project intends to confirm our guess at the level of cells, tissues and mice model throuth follow-up experiments.This subject is expected to explore the roles of DHX32 in the development of colorectal cancer and chemosensitivity to 5-FU and provide a new theoretical basis for DHX32 as a target of anti-tumor therapy.

5-氟尿嘧啶（5-FU）是目前肠道肿瘤的首选化疗药物，但是许多患者对以5-FU为主的化疗产生耐药，严重影响了结肠癌的治疗和预后。DHX32是一种新型的RNA螺旋酶，该蛋白在结肠癌中的功能尚未有文章报道。前期工作表明在结肠癌细胞中干扰DHX32表达能够明显地抑制细胞增殖并促进5-FU诱导的细胞凋亡，提高结肠癌细胞对5-FU的敏感性；信号通路分析显示DHX32对AKT的磷酸化起正调控作用。因此，我们提出DHX32通过PI3K/AKT信号通路影响结肠癌对5-FU耐药的假设。本项目拟进一步研究DHX32对PI3K/AKT通路的调控方式以及介导5-FU耐药的下游凋亡信号，并从组织水平和动物模型对上述机制进行验证。对DHX32调控结肠癌细胞增殖和凋亡的研究有助于探索结肠癌发生发展的分子机理，探讨DHX32与5-FU疗效的相关性及抵抗耐药的机制将有望为结肠癌的临床治疗提供新的靶点和思路。

结肠癌是常见的恶性肿瘤之一，近年来结肠癌的发病率在我国呈现逐年上升的趋势。化疗是结肠癌临床治疗最主要的手段之一，其中5-FU是目前肠道恶性肿瘤的首选化疗药物。但是许多患者对以5-FU为主的化疗产生耐药，严重影响了结肠癌的治疗和预后。结肠癌患者对5-FU耐药的机制较为复杂，是一个多基因参与的过程。对这些基因的研究是目前癌症研究的热点，对于探讨结肠癌的发病机制和治疗抵抗具有重要的意义。DHX32是近年来新发现的一种RNA螺旋酶，本项目组在前期筛选结肠癌的差异表达基因时发现DHX32基因在结肠癌组织中表达上调。本项目中我们也发现了DHX32的蛋白水平在结肠癌病人组织中出现明显上调，并且与结肠癌的分化程度相关。细胞水平的功能实验结果表明，DHX32能促进结肠癌细胞增殖、迁移和侵袭，抑制5-FU诱导的细胞凋亡。qPCR芯片的筛选结果显示，敲低DHX32抑制了血管生成因子VEGFA的mRNA水平，预示着DHX32可能参与了肿瘤血管生成的调控。本项目研究发现，在结肠癌细胞中DHX32能激活Akt，并且通过与Akt下游beta-catenin结合稳定其蛋白水平从而上调beta-catenin下游靶基因VEGFA的转录；由此提高了肿瘤微环境中VEGFA的蛋白水平，促进了血管内皮细胞的募集和肿瘤细胞的血管生成，由此导致了对5-FU的耐药。裸鼠移植瘤实验结果表明，敲低DHX32能显著降低结肠癌细胞裸鼠移植瘤的血管密度并抑制裸鼠移植瘤成瘤；在结肠癌裸鼠移植瘤中注射5-FU能减小移植瘤大小，而同时注射靶向DHX32的siRNA核酸药物能进一步提高5-FU的疗效。结肠癌病人组织水平的研究也验证了结肠癌组织中DHX32与beta-catenin的蛋白表达水平及肿瘤血管密度呈正相关，并且癌组织中DHX32的高表达提示了结肠癌病人的不良预后。本项目的研究明确了DHX32促进结肠癌发生发展的功能，探索了DHX32在结肠癌中发挥调控作用的分子机制，并在动物模型中初步探讨了以DHX32作为靶点的RNA核酸干扰药物的疗效。为临床研究结肠癌的发病机理及发现相关的诊断和治疗靶点提供了潜在的生物标志物，为以DHX32为靶点的药物开发提供了理论和实验基础，具有重要的科学研究意义及潜在的临床应用价值。