22q12.2肺癌风险染色质区增强子元件遗传变异的功能研究

Understanding the functional role and related mechanisms of genetic factors in lung carcinogenesis is critical for diagnosis and individualized treatment of the disease. Our previous study identified a new enhancer, 22q-Enh3, located within 22q12.2 lung cancer risk chromatin region. The 22q-Enh3 enhancer displayed significantly high regulatory activity in A549 lung cancer cells compared with in Beas-2B bronchial epithelial cells and other types of cancer cells, implying a function of the enhancer in lung carcinogenesis. The enhancer contained four SNPs that were highly linked with the lung cancer risk tag-SNP (rs17728461). These linked risk SNPs distributed in the binding sequences of NF-κB transcription factor, a well-known mediator for lung carcinogenesis, suggesting the enhancer regulatory activity is closely related to NF-κB. We hypothesize that the 22q-Enh3 enhancer is involved in regulation of important cell programs critical for tumor genesis and progression by promoting its targeting genes expression in NF-κB dependent way. SNP variations within the NF-κB binding sequences may increase the NF-κB binding to the enhancer and promote its regulatroy activity, thereby impacting lung carcinogenesis. The aim of this project is to test our hypothesis using cellular and molecular biological methods and chromatin function analysis based on the 22q-Enh3 enhancer knockout Beas-2B and A549 cell models and the mouse model with xenograft tumors. This study will be significant for elucidating the functional impact of single nucleotide variation on lung carcinogenesis. It will also provide a new model to investigate functional relationship between risk SNPs within gene non-coding regions and carcinogenesis.

探讨遗传因素在肺癌发生发展中的作用和机制具有重要的理论和临床意义。我们前期研究发现，22q12.2肺癌风险染色质区的22q-Enh3增强子在肺癌细胞中活性显著升高，增强子中4个与肺癌tag-SNP(rs17728461)高度连锁的SNPs分布于NF-κB转录因子结合序列中。本项目将运用染色质捕获技术、分子生物学方法和敲除22q-Enh3的细胞模型等，检验我们的研究假设：22q-Enh3增强子的调控活性、生物学功能以及对NF-κB转录因子的反应能力是一个有机联系的功能体系。增强子元件内肺癌关联SNPs位点的遗传变异，能够改变增强子元件与NF-κB的结合能力，提高增强子的调控活性，进而上调增强子元件靶基因的表达水平，促进肺癌细胞恶性表型的形成与发展。本研究将为阐述基因组非编码序列单核苷酸遗传变异在肺癌中的作用和机制提供新线索，开拓新思路。

肺癌属于人类复杂性疾病范畴，是遗传和环境因素相互作用的结果。近年来的研究显示，胚系罕见遗传变异（MAF<1%）是人类复杂性疾病的重要遗传基础，但目前对于胚系罕见遗传变异在恶性肿瘤中的生物学功能和机制却所知十分有限。本课题以位于22q12.2肺癌风险染色质区的增强子22q-Enh为切入点，综合探讨了罕见遗传变异、增强子活性及肺癌细胞恶性表型之间的功能联系。我们发现，22q-Enh在肺癌细胞中的活性远高于正常细胞，可通过形成染色质环高级结构调控下游38kb处LIF基因的活性增强肺癌细胞的增殖和成瘤能力。敲除22q-Enh可显著下调LIF基因表达，抑制肺癌细胞的平板克隆和细胞成球能力，并显著抑制裸鼠体内移植瘤的生长；恢复细胞内LIF基因表达可有效逆转敲除22q-Enh的效应，表明22q-Enh增强子可通过上调LIF基因表达在维持和促进肺癌细胞恶性表型中发挥重要作用。与之相对应，我们对101例肺腺癌病理标本中LIF基因表达水平与肿瘤分期的分析进一步证实，LIF基因表达水平与肿瘤的体积及淋巴结转移呈正相关。在上述研究基础上我们进一步分析了罕见遗传变异和22q-Enh增强子活性之间的功能联系，发现22q-Enh是一个RelA/p65 （NF - κ B 家族的重要成员） 的应答增强子，其活性受RelA/p65正向调控，而位于22q-Enh内的罕见遗传变异rs548071605可显著增强该增强子对RelA/p65的应答能力，表现为在RelA/p65高表达时突变型22q-Enh比野生型增强子的活性高出约15倍之多，表明罕见遗传变异rs548071605可显著放大22q-Enh增强子的促癌效应。我们的研究揭示了一个肺癌驱动性增强子罕见遗传变异及其作用机制，从一个新的视角为认识肺癌的遗传遗传机制提供了重要依据和研究策略。