ISOC1/miR-4633与TP53相互作用调控肺癌放化疗抵抗的机制研究

Lung cancer is the leading cause of morbidity and mortality among malignant tumor worldwide. Lung cancer resistance to radiotherapy and chemotherapy is a major problem that affects treatment outcomes. Thus, to elucidate the mechanism of the cancer cell resistance is critical for improving the therapeutic outcomes. Our previous studies using profile microarray and real-time PCR show that the expression of ISOC1 is significantly downregulated in radioresistance cells. We further found that miRNA4633 is harbored in the intron 1. In addition, we detected novel isoforms of ISOC1 in the radioresistance cells, and many CpG islands were found located in the promoter of ISOC1. The preliminary experimental results showed that ISOC1 and miR-4633 also play important roles in chemotherapy based on platinum. ISOC1 interacts with TP53 to mediate HNRNPs, and these genes are also the targets of miR-4633. Base on these findings, we think that ISOC1 play important roles in the mechanism of lung cancer resistance to radiotherapy and chemotherapy. To test this hypothesis, we will firstly use cellular biology approaches to investigate the roles of ISOC1 and miRNA4633 in cancer cell; We will further perform clinical studies to determine methylation status of ISOC1 promoter in human lung cancer cells collected from the patients who received adjuvant radiotherapy and/or chemotherapy after surgical resection and prognosis. To our knowledge, this study is the first attempt to evaluate the role of ISOC1 in the mechanism of lung cancer resistance to radiotherapy and chemotherapy. By defining the mechanism of the therapy resistance, we may be able to elucidate novel therapeutic targets that allow us to treat lung cancer more effectively.

肺癌放化疗抵抗是影响肺癌疗效的主要因素，因此，研究肺癌放化疗抵抗的分子机制对提高肺癌的疗效具有十分重要的意义。课题组用芯片和实时定量PCR技术分析放疗抵抗肺癌细胞株的表达谱数据发现①ISOC1表达量显著下调，②ISOC1第1内含子编码miR-4633，③在经放疗处理的细胞中发现新转录本，④ISOC1启动子区域存在有大量CpG岛。前期实验发现ISOC1与miR-4633在顺铂化疗中也发挥作用，并且ISOC1与TP53相互作用调控HNRNPs多个成员，而这些基因也是miR-4633的靶基因。本研究旨在借助细胞学实验揭示ISOC1和miR-4633在肺癌放化疗抵抗中的作用，并结合临床实验，通过比较分析肺癌术后辅助放化疗患者肿瘤组织、癌旁组织以及外周血中ISOC1启动子甲基化状态及表达量与预后间的关系，进而阐明ISOC1和miR-4633在肺癌放化疗抵抗中的作用机制，为肺癌的个性化治疗提供指导。

放疗和含铂化疗是肺癌治疗的重要策略，近年来，放化疗联合免疫治疗也可以更好的改善患者的预后，因此放化疗在肿瘤治疗中具有重要的作用。但是放疗和含铂化疗抵抗是肿瘤治疗中亟待解决的重要问题。为了全面研究放化疗抵抗的分子机制，我们分别筛选出放疗和含铂化疗抵抗的细胞株，多组学检测和分析发现，ISOC1在所有抵抗的细胞株中显著下调。通过实时荧光定量PCR技术验证了结果的正确性。细胞实验发现过表达ISOC1可以显著增加肿瘤细胞放化疗的敏感性，增加细胞死亡；抑制ISOC1表达显著增加放化疗的抵抗，增加肿瘤细胞的存活。临床样本检测发现ISOC1表达与放化疗疗效显著相关，并且通过数据分析发现，ISOC1低表达与肺癌患者的不良预后显著相关。为了探索ISOC1抵抗放化疗的分子机制，我们进行了免疫共沉淀联合质谱检测实验，结果发现ISOC1互作蛋白包含DNA双链断裂损伤的非同源末端连接（NHEJ）修复的关键蛋白（TP53、PARP1、Ku70、Ku80、NONO、SFPQ），这些蛋白分别形成DNA-PK和Paraspeckle复合体参与NHEJ修复。我们首先通过CO-IP实验验证了ISOC1确实与这些分子存在直接相互作用，随后我们分别检测了ISOC1不同表达状态下DNA双链断裂损伤程度，结果显示ISOC1敲除的肺癌细胞株中γH2AX位点的数目显著少于ISOC1野生型组，而在敲除的肺癌细胞株重新过表达ISOC1的挽救实验中，γH2A位点的数目又显著增多，说明ISOC1的低表达能够显著提高DSB的修复能力，抵抗放化疗造成的DNA损伤，NHEJ报告系统的结果同样表明低表达ISOC1可以显著的增加NHEJ修复效率，说明ISOC1调控NHEJ修复参与肺癌放化疗抵抗。顺铂处理后相关的结果更加显著。我们的实验结果也发现，下调ISOC1的表达可以显著的增加乳酸和IFN-γ的表达，从而参与肿瘤微环境的调控，而微环境的改变也是肿瘤放化疗抵抗以及免疫治疗疗效的一个重要的因素，并且我们的研究结果发现，ISOC1可能通过与Paraspeckle复合体的结合和解离有关。我们还进行了RNA-sequencing检测了ISOC1调控的下游通路，发现多个炎症因子和趋化因子出现显著差异，表明ISOC1确实参与肿瘤微环境的调控，这也是我们需要进一步进行探索的重要问题。