CPEB3通过调控ATG5介导的自噬影响结直肠癌细胞对5-FU敏感性的机制研究

The frequent emergence of clinical drug resistance continued to be an important factor that impeded the successful treatment of colorectal cancer (CRC) cancer with chemotherapeutics. As a result, it is urgent to search for the more effective therapeutic molecular targets which will decrease the metastasis rate and improve the prognosis of patients with CRC. Recent research has showed that autophagy played a protective role in the induction and maintenance of drug resistance in tumors. In our preliminary study, we showed that autophagy related gene 5 (ATG5) probably was a downstream target of CPEB3 by using bioinformatics analysis and RIP-sequence which was further confirmed in CRC cells and tissues. Meanwhile, CPEB3 could suppress the 5-FU induced autophagy and potentiated 5-FU induced cell apoptosis in CRC cells. Based on the previous data, we hypothesized that CPEB3 regulated the chemotherapy resistance of CRC cells through ATG5 mediated autophagy pathway. In this study, Flow cytometry, transmission electron microscope, RIP-qPCR, EMSA, luciferase reporter gene assays will be employed to test the influence and molecular mechanism of the CPEB3-ATG5-autophagy signal pathway on the chemotherapy resistance in CRC cell lines. Finally, the results will be further confirmed using nude mouse tumorigenesis test in vivo as well as the clinical analysis using CRC tissues and clinical data. With good design and strong research basis, this study will provide novel strategy for the autophagy-inhibited therapy combined with chemotherapy in CRC treatment.

化疗耐药是导致结直肠癌治疗失败的重要因素之一，寻找有效的治疗靶点提高化疗药物疗效，对减少术后复发转移、改善结肠癌患者预后具有重要意义。研究表明，自噬是调控结直肠癌化疗耐药的重要机制。前期研究中，通过生物信息学分析、RIP-sequence测序等发现自噬相关基因ATG5可能是抑癌基因CPEB3新的下游靶基因，且通过组织学及细胞学对二者的关系进行了初步验证；同时体外预实验也提示CPEB3可能参与了5-FU诱导的结直肠癌细胞自噬。由此，我们提出CPEB3通过ATG5调控细胞自噬、影响结直肠癌5-FU化疗敏感性的科学假说。在本研究中，拟通过流式细胞术、透射电镜、RIP-qPCR、EMSA、荧光素酶报告基因等技术，探索CPEB3-ATG5-自噬轴对结直肠癌5FU化疗敏感性的影响及调控机制，并在动物体内及临床组织标本中进行进一步验证，最终为结直肠患者术后化疗抵抗提供新的预警标记和治疗靶点。

结直肠癌是最常见的恶性肿瘤之一，死亡率高，已成为威胁国民健康的重大问题。因此，寻找有效的治疗靶点，对改善结肠癌患者预后具有重要意义。本研究中，我们首先证实了CPEB3在结直肠癌中是一个抑癌基因，能够增加结直肠癌细胞对5-FU的敏感性，并探索了该分子通过调控自噬相关分子发挥作用的分子机制。随后，我们构建了5-FU耐药的结直肠癌细胞株，以进一步验证 CPEB3对结直肠癌细胞5-FU敏感性的影响，结果发现，CPEB3在5-FU耐药株中同样能够抑制5-FU诱导的自噬相关分子的表达，但在两株5-FU耐药株中改变CPEB3表达，其增殖及凋亡水平与其相应对照相比均无明显差异，该结果与我们前期在结直肠癌裸株中的结果不一致，考虑可能为在结直肠癌耐药株的构建过程中出现了部分基因突变或表达变化所致。因此，为明确CPEB3的抑癌机制，我们重新对CPEB3过表达结直肠癌细胞进行了基因组学检测。结果发现，CPEB3显著抑制JAK/STAT通路活性。众所周知，JAK/STAT通路是IL-6的重要下游应答信号通路，由此，我们进一步通过CCK-8及Transwell实验证实：CPEB3可通过抑制JAK/STAT抑制IL-6对结直肠癌细胞增殖、转移的促进作用。IL-6是肿瘤相关巨噬细胞（TAMs）分泌的重要细胞因子，通过共培养体系我们进一步证实CPEB3能够通过抑制IL-6通路影响TAMs对结直肠癌细胞的作用。同时，我们还发现CPEB3能够通过抑制结直肠癌细胞上清中CCL-2的分泌调控TAMs的分化，而JAK/STAT是CCL-2的重要上游调控通路。综上，CPEB3通过抑制结直肠癌细胞中JAK/STAT的活性调控结直肠癌细胞与TAMs的互相作用，通过抑制该通路：一方面抑制TAMs分泌的IL-6对结直肠癌细胞的促进作用，另一方面通过抑制结直肠癌中CCL-2的分泌调控TMAs的分化。为结直肠癌治疗提供了新的靶点。