p16启动子DNA甲基化诱导细胞永生化和恶性转化作用及其机制

The p16 gene is one of the most frequently inactivated genes in cancer genomes, mainly through DNA methylation. It is highly supposed that epigenetic changes of cancer related genes, including p16 methylation, may induce malignant transformation and drive cancer development. However, a direct experimental evidence to support this hypothesis has not been reported yet. In our recent studies, we have constructed an artificial p16-specific methyltransferase, and found that p16 promoter methylation directly inactivates transcription of both p16 and ANRIL. Most importantly, we have observed that p16 methylation obviously blocks the cell senescence and prolongs the lifespan of primary normal fibroblasts. In this project, we are going to study the potential of p16 methylation to immortalize and induce malignant transformation of normal fibroblasts. The effects of p16 methylation on malignant transformation induced by oncogene genes, including KRas, will also be investigated. Furthermore, whether inactivation of lncRNAs including ANRIL transcription by p16 methylation may contribute to the cell immortalization and transformation will be studied as well. The signal pathways and crucial genes involve in the cell immortalization and transformation will be characterized too. These studies will provide direct evidences to support whether epigenetic alterations are driver factors in cancer development.

P16基因是肿瘤基因组中失活频率最高的位点，DNA甲基化是其失活的主要途径。虽然P16甲基化等表观遗传变异可能是肿瘤发生驱动因素的假说早已提出，至今缺乏直接实验证据的支持。我们前期已构建p16基因特异性DNA甲基化酶，首次发现p16CpG岛甲基化不仅能够抑制P16基因和ANRIL转录，而且还能抑制正常成纤维细胞衰老，显著延长细胞寿命。在本项目中我们将进一步研究p16CpG岛甲基化本身是否具有诱导细胞永生化和恶性转化作用，是否能够协同KRAS等基因诱导细胞恶性转化，为表观遗传变异是否能够驱动肿瘤发生提供科学依据；研究ANRIL等lncRNAs转录调控是否参与p16启动子甲基化诱导细胞永生化和恶性转化过程，同时从染色质构象变化角度探讨p16甲基化对ANRIL等转录调控的机制，分析P16甲基化的下游信号分子。该研究对揭示p16基因甲基化是否直接启动细胞恶性转化及其分子机制有重要科学意义。

DNA甲基化等表观遗传变异可能是肿瘤发生驱动因素的假说早已提出，至今缺乏直接实验证据的支持。本项目中我们利用前期构建的p16基因特异性DNA甲基化酶（P16-Dnmt），证实P16基因甲基化本身能够促进正常成纤维细胞CCD-18Co增殖，延长其寿命，但不足以使其永生化和恶性转化。进一步的联合诱导方案显示，P16基因甲基化与KRAS突变体、P53突变体也没能诱导细胞永生化和恶性转化，但联合hTERT过表达能够诱导正常成纤维细胞永生化，并向肿瘤细胞方向演化。本课题还完成了P16基因甲基化对细胞染色质构象的影响分析，完成了P16基因甲基化前后以及永生化细胞的表达谱改变分析，并对相关基因的差异表达和生物学功能进行验证，还探索了相关lncRNA的调控作用，为阐明正常细胞发生永生化并向恶性转化发展的分子机制提供了线索。