组蛋白去乙酰化酶抑制剂联合自噬增加肺癌放疗敏感性的实验研究

Repairing of DNA double-strand break (DSBs) in the nucleus of tumour cells is main reason for resistance in radiotherapy. Recent studies and our research showed that autophagy could degrade the repairing protein of DSBs and improved the sensitivity of radiotherapy in tumour cells, but the effects were limited. Recent studies also demonstrated that histone deacetylase inhibitor (HDACi) could make the repairing protein of DSBs acetylased, relocated and degraded by autophagy, and subsequently inhibit the repairing of DNA DSBs in yeast cells. However, these effects have never been reported in the tumour cells. We also confirmed that HDACi combined with autophagy decreased the repairing protein of DSBs in the nucleus and increased the sensitivity of radiotherapy in A549 cells. We speculated that HDACi could make the repairing protein of DSBs acetylased, relocated and degraded by autophagy, and subsequently increase the sensitivity of radiotherapy in lung cancer . Therefore, in this project we will observe the sensitivity of radiotherapy in the lung cancer transplanted naked mice using HDACi combined with autophagy, investigate the acetylasation of repairing protein of DSBs, migration and co-location of autophagosome in A549 cells, and explore the mechanism of sensitivity of radiotherapy in lung cancer. We also analyse the correlation between the levels of autophagy and acetylasation, of the samples in lung cancers and the sensitivity and outcome, and provide the evidence for radiotherapy of lung cancer.

肿瘤细胞核内DNA双链断裂（DSBs）修复是放疗抵抗的重要原因。最近研究及我们的工作发现，自噬可降解胞质中DSBs修复蛋白增加肿瘤放疗敏感性，但作用有限。最新研究发现，组蛋白去乙酰化酶抑制剂（HDACi）可促使酵母细胞核内DSBs修复蛋白乙酰化，移位至胞质中被自噬降解，抑制DNA DSBs修复，但在肿瘤细胞中未见报道。我们前期工作证实HDACi联合上调自噬可减少胞核中DSBs修复蛋白，增加A549细胞放疗敏感性。故推测HDACi可使核内DSBs修复蛋白乙酰化并移位到胞质，被自噬降解，增加肺癌放疗敏感性。因此，本项目拟将HDACi联合自噬，观察其增加裸鼠肺癌皮下移植瘤模型放疗敏感性的作用，同时观察A549细胞质中乙酰化DSBs修复蛋白的移位及与自噬小体的共定位，探讨其增加肺癌放疗敏感性的机制；并通过临床肺癌组织标本分析自噬及乙酰化水平与放疗敏感性及预后的关系，为肺癌的放射治疗提供理论依据。

放射治疗是目前肺癌临床综合治疗的重要手段之一，但放射性抵抗极大影响了肺癌放射治疗的效果。组蛋白去乙酰化酶抑制剂（HDACi）联合自噬对NSCLC放疗敏感性具有重要影响。本课题通过体内外观察RAPA联合SAHA对NSCLC细胞增殖、放疗敏感性及周期的影响，结合免疫荧光共聚焦、基因沉默等方法与技术对其放疗增敏的具体机制进行了相关研究，并通过临床研究分析自噬及乙酰化水平与放疗敏感性及预后的关系。首先通过CCK-8实验检测到RAPA联合SAHA对NSCLC细胞增殖具有抑制作用，呈剂量和时间依赖性；细胞克隆实验发现两药联合显著抑制放疗后NSCLC细胞的增殖；流式细胞术检测A549细胞周期变化，结果提示对放疗增敏作用可能是通过抑制细胞周期G2期阻滞造成。通过Western blot、RT-qPCR检测DNA损伤蛋白、DNA双链断裂（Double strand breaks，DSBs）修复蛋白及mRNA水平变化，结果显示两药联合后放疗γ-H2AX表达水平延长，DNA修复蛋白表达下调；经透射电镜、激光共聚焦、Western blot检测自噬及乙酰化，结果显示自噬及乙酰化水平增加，提示乙酰化作用联合自噬诱导剂可抑制A549细胞放疗后DNA DSBs修复蛋白的表达水平；通过沉默Atg5基因下调自噬水平，发现其放疗增敏作用减低，进一步验证了自噬在增加NSCLC放疗敏感性的作用。经动物实验，提示两药联合无明显毒副作用，且增加放疗对NSCLC的杀伤作用。临床研究中发现，与放疗抵抗组相比，放疗敏感组的NSCLC患者自噬及乙酰化水平相对高表达，生存分析显示自噬相关蛋白Beclin1、LC3II高表达组相比低表达组PFS及OS时间明显延长，这提示放疗前NSCLC患者自噬水平高，放疗越敏感，预后可能较好。综合上述结果，本研究为探讨HDACi联合自噬对NSCLC放疗敏感性影响的机制奠定了坚实的工作基础，为NSCLC的放射治疗提供重要证据。