MUTYH变异与特发性肺纤维化发生中细胞增殖/凋亡调控的线粒体机制研究

Idiopathic pulmonary fibrosis (IPF) is a progressive, and often fatal form of interstitial lung disease. The exact mechanisms underlying the development of IPF still remain unknown although the increasing evidence indicates that age-related accumulation of oxidative DNA damage has a cause role in IPF pathogenesis. The human mutY homologue (MUTYH) gene is involved in the base excision repair (BER) system, which is critical for repairing oxidized DNA bases and maintaining the fidelity of DNA replication. Our previous work described the novel variation AluYb8MUTYH, a AluYb8 element insertion in MUTYH gene. This variation altered the gene expression patterns by greatly reducing the type 1 MUTYH that was subcellular localized in mitochondria. And then it had effects on mitochondrial DNA (mtDNA) maintenance and decrease the functional mitochondrial mass. On the other hand, The P53 tumor suppressor protein, an important regulator for G1-arrest and apoptosis, naturally occurs in humans in two variants with single nucleotide polymorphisms resulting in Arg or Pro at residue 72 (P53 Arg72Pro). The Arg72 variant shows a selective subcellular localization in the mitochondria and is more efficient in apoptosis induction, whereas the Pro72 variant localizes in nucleus and induces more G1 arrest. Notably, our case-control analysis shows that both of AluYb8MUTYH and the P53 Arg72 were the risk factors for IPF. These results strongly suggest that mitochondrial dysfunction may play a pivotal role in IPF development. Therefore, we propose the study on MUTYH gene variation and mitochrodrial regulation of cell proliferation and apoptosis in IPF pathogenesis. We will use the bleomycin injury model in the MUTYH gene knockout mice to test whether the MUTYH disfunction affects susceptibility to lung fibrosis. To address the mitochondrial mechanism of IPF pathogenesis, we will focus on the genotypes of MUTYH and P53, with the primary cultured fibroblast-like cells, and clinical and molecular epidemiology, to investigate the relationship among oxidative stress, mtDNA damage, expression of signaling molecules, mitochondrial function and cell phenotype characteristics. It is reasonable to believe that the implementation of this study could clarify, in-depth, the mitochondrial role in IPF pathogenesis.

特发性肺纤维化(IPF)是一种渐进发展、死亡率高的间质性肺疾病，发生机制远未阐明，但衰老及DNA氧化损伤累积的病因学作用已引起高度重视。我们前期工作发现，DNA氧化损伤修复关键基因MUTYH存在AluYb8插入变异（AluYb8MUTYH），导致该基因单一亚型（线粒体定位）产物表达缺如，线粒体DNA氧化损伤累积增速、功能降低，并与人群中可改变P53基因亚细胞定位的常见变异体P53 Arg72（产物定位于线粒体）共同构成IPF风险因素。强烈提示线粒体功能异常在IPF发生中的枢纽作用。基于此，我们从MUTYH基因功能缺陷与IPF发病风险，AluYb8MUTYH、P53 Arg72Pro与细胞衰老、增殖/凋亡调控的分子机制两方面入手，结合动物模型、体外细胞调控和IPF患者遗传与靶细胞分子背景，探讨IPF发生的线粒体机制。项目实施可能取得对IPF认识的突破，进而为发展IPF的临床干预措施打下基础。

项目组前期研究发现，人类DNA糖基化酶基因MUTYH存在AluYb8插入变异，介导该基因线粒体定位亚型蛋白产物MUTYH1表达受抑，加速线粒体DNA（mtDNA）损伤，线粒体功能降低，与衰老相关疾病风险相关联。基于此，本项目结合MUTYH基因敲除小鼠模型、体外细胞与分子调控，及特发性肺纤维化（Idiopathic pulmonary fibrosis, IPF）患者遗传易感基因分析与分子流行病学的队列研究，系统观察了IPF发生过程中细胞mtDNA改变、线粒体功能、分子调控状态等，探讨MUTYH变异与IPF发生中线粒体功能障碍的关系及其表型意义。项目实施取得了突破性进展，主要包括：①MUTYH基因敲除对博来霉素（Bleomycin, BLM）诱导小鼠肺纤维化的影响表现为双面性，即可减轻模型小鼠早期的肺部炎症反应，但可加重肺纤维化的表型；②MUTYH AluYb8插入与TP53 Arg72Pro多态性变异在细胞增殖/凋亡调控及提高年龄相关疾病风险方面具有协同作用；③小鼠模型和衰老相关疾病IPF患者临床样本分析均显示，MUTYH功能缺陷可导致mtDNA失稳，在衰老及相关疾病发展中，细胞mtDNA含量出现特征性变化过程，其机制可能涉及mtDNA损伤修复和线粒体自噬功能障碍；④中国人IPF易感基因变异与欧美人群具有显著差异，ABCA3基因杂合型突变在中国人IPF患者中频繁检出，与中国人IPF发病高风险存在明显相关性。本项目进展进一步加深对衰老及相关疾病发展过程中线粒体功能异常的认识，项目结果揭示的MUTYH功能缺陷相关mtDNA特征性改变可能应用于衰老及相关疾病发生及表型发展的评估和监测，而ABCA3基因突变可能成为IPF发病高风险的诊断分子。