NFYB/E2F1/Chk1通路在结直肠癌对奥沙利铂耐药过程中的作用机制研究

During the chemotherapy treatment for patients with colorectal cancer, the cancer cells usually acquire resistance to chemotherapeutic drugs. As a third-generation platinum drug, Oxaliplatin resistance used to be accompanied with cancer recurrence, metastasis and poor prognosis. However, the involved molecular mechanisms have not been well demonstrated in previous studies. In our preliminary study, the increased protein levels of NFYB and E2F1 were observed in oxaliplatin-resistant colorectal cancer cells. We also revealed that NFYB promoted the expression of E2F1 and resistance to Oxaliplatin in colorectal cancer cells, while Chk1 played an important role in the process. On this basis, we intend to reveal the potential downstream effector proteins of Chk1 and their relationship with Oxaliplatin resistance by means of Chk1 inhibitor and LC–MS/MS in the current project. RNA interference will be adopted to investigate the regulatory mechanisms of NFYB, E2F1, Chk1 and other signaling molecules. Thereafter, the effect of NFYB/E2F1/Chk1 pathway related molecules on Oxaliplatin resistance will be further validated in animal experiments and clinical colorectal cancer samples, thus provide scientific evidences for exploring novel drug-resistant diagnostic or therapeutic targets and improving the clinical chemotherapeutic outcomes of the malignant disease.

在结直肠癌患者的化学治疗过程中，经常发生癌细胞对化疗药物产生耐受的现象。其中，第三代铂类抗癌药物奥沙利铂的耐药性常伴随肿瘤的复发、转移及不良预后，然而相关的耐药分子机制尚不完全明确。我们前期研究发现在奥沙利铂耐药的结直肠癌细胞株中NFYB和E2F1分子发生明显的上调，并且NFYB是通过激活E2F1表达来促进结直肠癌细胞对奥沙利铂产生耐药性，而Chk1在该过程中起到重要作用。本项目拟在此基础上，采用Chk1抑制剂和质谱分析等手段探索潜在的Chk1下游效应蛋白及其与奥沙利铂耐药的关系，采用RNA干扰等方法分别研究NFYB、E2F1、Chk1等信号分子间的调节机制，并在动物实验和临床组织样本中进一步验证NFYB/E2F1/Chk1通路相关分子在奥沙利铂耐药中的作用，争取为探索结直肠癌耐药诊治靶标以及提高临床化疗效果提供新的理论依据。

奥沙利铂作为第三代铂类化学药物，已被广泛用于结直肠癌的治疗。然而，结直肠癌细胞对奥沙利铂的耐药性已成为该治疗方法的主要障碍。在研究中，我们发现DLD1和RKO 结直肠癌细胞奥沙利铂耐药株中核转录因子Y-beta亚基（NFYB）和E2F转录因子1（E2F1）的表达水平均显着高于非耐药细胞。我们还发现高表达的NFYB会激活E2F1基因，这对于维持结直肠癌细胞的奥沙利​​铂耐药性非常关键。 同时，Sirt1的脱乙酰基作用抑制了奥沙利​​铂耐药的结直肠癌细胞中E2F1的促凋亡活性。通过分析敲除E2F1之后的奥沙利​​铂耐药的结直肠癌细胞的转录组，可以确定CHK1是E2F1的靶标。 去除CHK1可以使得耐药结直肠癌细胞对奥沙利铂重新变得敏感。体外和体内表型实验证实，需要完整的NFYB-E2F1-CHK1轴来抑制奥沙利铂诱导的细胞凋亡并维持耐药结直肠癌细胞的致瘤性。敲除奥沙利​​铂耐药的结直肠癌细胞中的E2F1也会降低Polκ的表达，这对于CHK1的激活至关重要。同时，高表达的NFYB，E2F1或CHK1常伴随着结直肠癌患者的较差生存率，这在奥沙利铂治疗组更为明显。总的来说，NFYB-E2F1途径通过诱导CHK1的表达和激活，在结直肠癌细胞奥沙利​​铂化学耐药性中发挥关键作用，为结直肠癌细胞中的奥沙利​​铂耐药提供了可能的治疗靶标。