肿瘤细胞中线粒体基因组胚系突变的异质性转换研究

The mitochondrial DNA (mtDNA) mutations have always generated a mixed population of molecules within the cell, which termed heteroplasmy, and also have emerged as a major cause of cancers and other human diseases. Heteroplasmic mtDNA mutations include inherited germline mutations and acquired somatic mutations, and how to identify two kinds of mutations is of great significance in tumor study. However, traditional strategies for detection of mtDNA mutations are difficult to identify the origin of mtDNA mutations. In our previous work, mtDNA of 20 hepatocellular carcinoma (HCC) patients were detected by next generation sequencing (NGS). We found that the heteroplasmic level of many low frequency mtDNA germline mutations is significantly changed between blood and HCC tissues by heteroplasmy shift. It suggested that many germline mutations may be recognized as somatic mutations in many studies before. Here, we aim to (1) systematically identify the heteroplasmy shift of mtDNA germline mutations and their pattern in HCC, colorectal carcinoma, and renal carcinoma; (2) analyze and elucidate the biological role and mechanism of the heteroplasmy shift of mutations in cancer cells. This study will help to recognize the origins of mtDNA mutation and its functions of dynamic transformation in tumor development, it also lay a solid foundation for the elucidation of molecular mechanisms underlying mtDNA mutation-mediated carcinogenesis.

线粒体基因组（mtDNA）突变常以异质性状态存在，即野生型和突变型mtDNA共存于同一细胞中，与肿瘤及多种疾病密切相关。mtDNA异质性突变包括先天遗传的胚系突变和后天新生的体细胞突变，正确识别突变起源对于认识其在肿瘤中作用机制具有重要意义。然而，传统突变检测方法难以区分mtDNA异质性突变真实来源。本项目组在前期肝癌mtDNA深度测序分析中，发现大量血液中极低（高）频率的胚系突变在癌组织中频率大大提高（降低），发生了显著的异质性频率转换，提示以往研究极有可能将极低频率胚系突变误判成后天新发的体细胞突变。基于此，本项目拟：(1)系统性鉴定肝癌、肠癌及肾癌等多种肿瘤mtDNA胚系突变异质性转换及其模式特征；(2)初步阐明这种异质性转换在肿瘤中的生物学意义及驱动机制。本项目的完成有助于正确认识mtDNA突变起源及其动态转换在肿瘤发生发展中的功能作用，为深入理解肿瘤的分子病理机制奠定理论基础。

线粒体基因组（mtDNA）突变常以异质性状态存在，即野生型和突变型mtDNA共存于同一细胞中，与肿瘤及多种疾病密切相关。以往由于测序精度限制，往往容易造成mtDNA胚系突变和体细胞突变判定错误。我们发现肿瘤细胞中存在一种突变模式，即正常组织（血液）中极低（高）频率的突变在肿瘤组织中突变碱基频率大大提高（降低），我们将其定义为异质性转换（heteroplasmic shift），属于胚系突变，以往研究容易将其判定为体细胞突变。.本项目基于项目组前期建立的mtDNA捕获测序技术，制定了mtDNA异质性转换鉴定流程，在肝癌、肠癌组织样本中验证mtDNA异质性转换客观存在，并进一步研究了肿瘤mtDNA异质性转换突变模式特征与规律，最后初步探讨了mtDNA异质性转换的产生机制及其临床应用潜能。.项目组圆满完成项目目标，并取得如下研究成果：建立了胚系突变异质性转换鉴定标准，在此基础上完成了12例肝癌、13例肠癌多点取材样本和156例肝癌、116例肠癌大样本的异质性转换鉴定，证实了肿瘤组织中存在大量异质性转换位点；进一步分析发现肝癌组织中异质性转换发生频率、剧烈异质性转换位点（switch）的比例均显著高于癌旁组织；发现肝癌特异性异质性转换位点中switch位点比例显著高于炎症特异性异质性转换位点；发现肝癌与肠癌的D-loop区平均每个位点的异质性转换数量均显著高于Complex区和RNA区；通过细胞学实验发现，与未处理的野生型细胞相比，干涉或过表达Drp1的细胞中出现了mtDNA异质性转换位点，并且突变转换发生在干涉、过表达处理时，随着细胞连续培养，转换后的位点频率相对稳定。最后，发现晚期肝癌患者中，mtDNA switch比例显著升高，提示mtDNA异质性转换或能影响肝癌进展。本项目的完成对于正确认识mtDNA突变起源及其动态转换在肿瘤发生发展中的功能作用具有重要意义。