小鼠Pold4介导的基因组稳定性在肺癌发生发展中的功能和机制研究

Cancer, especially lung cancer, is one of the leading causes of death. Genome instability is considered a hallmark of cancer cells, which increases the variation of genetic information in normal cells, thus gain the ability of tumorigenesis. Defects associated with DNA replication or replication-related repair genes can lead to replication stress, which result in genome instability. Pold4 is a structural subunit of Pol delta complex, which is an DNA polymerase and DNA repair enzyme. Through analysis of patient tissue samples, previous studies have shown that reduced expression of Pold4 is associated with the occurrence and poor prognosis of lung cancer, though the mechanism remains unclear. More importantly, there is no straight evidence linking Pold4 to tumor formation so far. Therefore, a detailed in vivo study on the correlation between Pold4 and tumorigenesis will be crucial in unstanding Pold4’s function in tumorigenesis and its potential clinical application in cancer therapy. We intend to construct Pold4 knockout lung cancer cell lines and Pold4 knockout mice by CRISPR/Cas9 technology. The mouse models will be established through both subcutaneous transplantation of lung cancer cells and spontaneous or drug-induced lung tumor formation in C56BL/6 mice. Using these models, we will further explore the role and molecular mechanism of Pold4 in genomic stability and, genesis and progression of tumor.

癌症，尤其是肺癌，是导致人类死亡的主导因素之一。基因组不稳定性是癌细胞的标志，它使得正常细胞遗传信息变异的增多，从而获得肿瘤发生的能力。DNA复制或复制相关修复的基因缺陷可导致复制压形成，进而使基因组不稳定。Pold4是DNA聚合酶和DNA修复酶Polδ复合体的结构性亚基。之前的研究表明病人组织中Pold4低表达与肺癌发生、不良预后相关。然而相关的功能机制研究还不够深入，而且目前没有相关证据直接表明Pold4与肿瘤的关系。因此探究Pold4在体内与肿瘤的联系对基础研究和临床应用方面意义十分重大。本项目拟采用CRISPR/Cas9技术构建Pold4完全敲除肺癌细胞系和Pold4敲除小鼠，通过细胞皮下成瘤及小鼠自发成瘤或药物诱导肺癌模型，来进一步深入探讨其在调控基因组稳定性及肿瘤发生发展的作用和分子机制。

肺癌是全球肿瘤发病率和死亡率最高的癌种。肿瘤细胞的重要特征之一就是基因组不稳定，DNA复制或损伤缺陷可能会促使基因组不稳定发生。Pold4是DNA聚合酶delta复合体的最小亚单元，然而Pold4是否影响基因组稳定性和肺癌发生及其机制仍是未知的。我们通过构建Pold4敲除小鼠和敲除肺癌LLC细胞，发现Pold4敲除不影响小鼠正常发育，也不影响正常细胞的细胞增殖、DNA复制和DNA损伤修复能力。然而，当肺癌LLC细胞中缺失Pold4，细胞增殖明显减慢，转录组也显示调控细胞生长的基因显著下调，其次表观调控基因表达和翻译起始通路基因也显著下调，细胞DNA损伤增加，基因组不稳定，细胞皮下成瘤能力也减弱。更重要的是通过分析发现Pold4低表达的肺癌病人无病生存率高于Pold4高表达的肺癌病人，与我们观察的Pold4敲除小鼠肝瘤形成机率低于WT小鼠结论较一致。因此，Pold4很可能作为今后肺癌甚至其他癌症的潜在分子靶点，为治疗肺癌提供重要的理论基础。

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