线粒体DNA 10398位点突变异质性对非小细胞肺癌放射敏感性的影响

In our previous study, in patients with lung cancer who received comprehensive treatments (including radiotherapy), we found that those who carried mtDNA 10398A and lower mtDNA content have poor prognosis. This finding indicated that missense mutation in mtDNA 10398 locus may be associated with the radiosensitivity. However, the relationship between prognosis and mtDNA 10398G/A mutation had been reported in several studies with controversial conclusions. The reason why there were controversial may be related to heteroplasmic mutation. Herein, in our further study, the mtDNA 10398G/A heteroplasmic mutation in tumor tissue of non-small lung cancer(NSCLC) who have received radiotherapy will be detected to estimate the range of this heteroplasmic mutation. And then, the model of heteroplasmic mutation in NSCLC cells will be constructed in vivo and in vitro for studying the influence of these cells on radiosensitivity and determine the ratio of the heteroplasmic mutation. After that, the content and activity of respiratory chain complex I , the changes of morphology and metabolism in mitochondrial (ROS, lactate, apoptosis)，and the mitochondrial retrograde signaling(HIF-1α signal pathway, nuclear DNA damage and repair) will be detected to investigate the mechanism that how these changes affects the radiosenstivity of NSCLC cells. The results of this research will provide an effective approach for a prediction of radiosensitivity , a evaluation of prognosis, and a potential molecular target of radiosensitization. This work will help for developing the theory that mitochondrial regulates radiosensitivity.

我们前期研究发现临床接受综合治疗（含放疗）的肺癌患者组织标本mtDNA含量低并伴有mtDNA 10398A的患者预后差，提示10398位点突变可能与放射敏感性有关。然而多项该位点突变影响肿瘤治疗敏感性和预后的研究结论并不一致，分析其原因可能与突变异质性有关。本研究拟检测接受放疗的非小细胞肺癌患者组织标本mtDNA 10398G/A突变比例，估计该位点突变异质性范围；在此基础上建立体外和体内非小细胞肺癌mtDNA 10398G/A突变异质性模型，研究其对放射敏感性的影响，并从呼吸链复合酶I的活性和含量变化、线粒体形态和功能代谢改变（ROS、ATP、乳酸代谢、凋亡等）及线粒体对细胞核逆向调控（HIF-1α通路、核DNA损伤修复等）等方面探讨其可能机制，为非小细胞肺癌放射敏感性的预测及预后评估提供有效手段，也为肺癌放疗增敏提供潜在的分子靶标，并进一步丰富放射敏感性的理论研究。

首先，课题组收集了129例非小细胞肺癌患者病理组织标本，检测其中mtDNA 10398 异质性突变比例在0.31% 到97.04%之间，且异质性突变比例高的非小细胞肺癌患者的生存时间显著长于异质性突变比例低的患者，提示mtDNA 10398 突变异质性可以作为一种新的预后分子靶标。且在122例宫颈癌患者中也发现高mtDNA 含量加上10398 A可以作为宫颈癌预后不良的一个标志。其次，通过低剂量EB诱导培养构建mtDNA稳定缺失的肺癌细胞模型（H1299ρ0），实验发现mtDNA缺失能减少ATP及ROS产生、延长G2/M期阻滞、抑制细胞凋亡、加快DNA损伤的修复，导致H1299ρ0细胞放射敏感性降低。进一步采用表达谱基因芯片分析H1299 ρ0及正常H1299细胞之间线粒体逆向调控信号通路的显著性差异基因以及细胞实验进行验证，表明mtDNA缺失通过转录因子NF-KB调节的逆向信号通路调节核基因PI3K/AKT/mTOR通路的表达，引起凋亡抗拒及周期阻滞，导致肿瘤细胞放射抗拒。最后，通过长期微量EB诱导培养建立mtDNA部分缺失的结肠癌SW480细胞模型；mtDNA部分缺失使SW480细胞发生了有氧糖酵解的代谢表型转换，并表现出对化疗药物5-氟尿嘧啶和奥沙利铂可逆的凋亡抵抗；mtDNA部分缺失激活了SW480中PI3K/AKT/mTOR通路，抑制PI3K或恢复mtDNA可以降低细胞内葡萄糖含量，恢复其对化疗药物的敏感性。本研究项目揭示了mtDNA 10398异质性突变比例对非小细胞肺癌患者预后判断的价值，并证明mtDNA缺失所引起的线粒体-核逆向信号通路（NF-KB /HIF-1α /PI3K/AKT/mTOR）表达增强与肺癌细胞放射抗拒密切相关，为肺癌放疗增敏提供潜在的分子靶标；此外，还证明了mtDNA部分缺失通过激活PI3K/AKT/mTOR通路，诱导结肠癌代谢表型转换以及对化疗药物抵抗，丰富了结肠癌的化疗增敏的理论研究。