探讨13q12.12肺癌风险染色质区增强子遗传变异的生物学效应及机制

Understanding lung cancer genetic mechanisms is critical for the disease diagnosis and individualized treatment, while elucidating biological consequences of risk SNPs is an attractive and challenging way to reveal cancer genetic mechanisms. SNP rs753955 within non-coding region of the 13q12.12 risk locus is highly associated with lung cancer risk of Chinese population. In our previous work we identified a new enhancer named 13q-Enh2 harboring three common genetic variations in high LD with the risk SNP rs753955. These three linked risk SNPs distributed within the flanking sequences of p53 binding sites. eQTL analysis in 117 Chinese NSCLC samples and GTEx data suggested suppressive effects of mutant enhancer alleles on the TNFRSF19 expression that was closely related to high tumor staging and short survival time. We hypothesize that the regulatory activity on target gene, biological functions and reactivity to p53 protein of the 13q-Enh2 enhancer form a functional system to help maintain normal phenotype of bronchial epithelia cells. The risk SNPs within the enhancer can interfere with the normal response of the enhancer to p53, inhibit its regulatory activity on TNFRSF19 gene, and thus impact on lung carcinogenesis. The aim of this project is to test our hypothesis by integrating cellular and molecular biological methods, gene editing and chromatin function analysis techniques and mouse model with xenograft tumors. The study will provide an illuminating new clue to the genetic mechanism of lung cancer.

阐述肺癌的遗传机制具有重要的理论和临床意义。我们前期实验和临床研究发现，13q12.12肺癌风险染色质区的13q-Enh2增强子在支气管上皮细胞中的活性显著升高；增强子中3个与肺癌Tag-SNP rs753955高度连锁的SNPs与肺癌组织中TNFRSF19基因表达下调紧密关联，且TNFRSF19表达水平与肺癌TNM分期显著负相关；这3个SNPs均位于增强子p53结合位点的侧翼序列中。本项目将系统运用细胞分子基因编辑和染色质分析等技术，验证我们的研究假设：13q-Enh2增强子对靶基因的调控活性，生物学功能及其对p53蛋白的反应性构成一个紧密联系的功能体系，在保护支气管上皮细胞正常表型中发挥重要生物学功能。增强子元件内的肺癌风险SNPs能够干扰增强子对p53的正常反应，抑制其对TNFRSF19基因的调控活性，促进肺癌发生与发展。该研究将为阐述肺癌的遗传机制提供富有启发性的新线索新思路。

遗传因素在肺癌的发生发展中发挥重要作用，揭示肿瘤风险染色质区遗传变异的生物学意义是阐述肿瘤遗传机制富有前景和挑战性的策略。位于13q12.12的Tag-SNP rs753955（A>G）与中国人群的肺癌风险高度相关。本研究综合利用基因编辑技术、遗传学、细胞生物学和分子生物学实验，结合临床样本分析，在rs753955的49kb强连锁不平衡区中发现了一个具有高度肺组织特异性的活性增强子。该增强子通过形成染色质环结构与相距150kb的TNFRSF19基因启动子相互作用上调其表达水平，进而有效保护正常肺支气管上皮细胞免受肺特异性强致癌物NNK诱导的DNA损伤和细胞恶性转化。进一步研究发现，该增强子能够与p53蛋白结合，其活性受p53蛋白调节。增强子内3个与Tag-SNP rs753955高度连锁的种系遗传变异rs17336602 (G>C), rs4770489 (A>G), and rs34354770 (A>C)，则可通过显著削弱该增强子与p53蛋白的结合抑制其增强子活性，这一效应在细胞暴露于致癌物NNK时尤其突出。对117例中国人肺癌临床样本的分析结果以及GTEx数据进一步证实上述增强子遗传变异的促癌效应。这项研究揭示了13q12.12风险染色质非编码区遗传变异在肺癌发生中的生物学功能和机制，为阐述肺癌的遗传机理提供了重要实验依据和新的研究思路。