MKK7基因编码区罕见遗传变异对人群肺癌发病和预后的影响

"Common diseases-Rare variant" is recently proposed rationale for screening susceptible gene of complex diseases, and tumor is considered to be the result of cumulative events. Our previous study have found that a rare variant (E116K) in coding region of MKK7 gene contributed to a significantly increased risk and poor survival of lung cancer. As we known, MKK7 is a core molecule of mitogen-activated protein kinase pathway (MAPK) that plays decisive role in regulation of multiple cell process including cell proliferation, differentiation, senescence, apoptosis and inflammation. By inhibiting the tumorgenesis role of oncogenes like H-ras1, MKK7 is considered to be a tumor suppressor gene. Further bioinformatics analysis shown that there are five putative functional rare genetic variants in coding region of MKK7, these variants may influence the function of MKK7, therefore, we hypothesize that the cumulative genetic effect of MKK7 rare variants may harbor role in the occurrence and development of lung cancer. Here, this project intends to investigate the association between MKK7 rare variants and lung cancer risk as well as prognosis by genotyping these variants and detecting genotype-phenotype (i.e., kinase activity) correlation in two independent case-control studies with large sample size (3000 cases and 3000 controls); and to observe the genetic effect of these variants on influencing the kinetic alterations of lung canceration under the stimulation of carcinogens or X-ray through construction of a series human bronchial epithelial model cell lines comprising the above variants; with the model cell lines and constructed mammalian two-hybrid system, to detect the protein-protein interaction to reveal the effect of above variant on carcinogenic function of H-ras1. Eventually, to illustrate the biological mechanism of MKK7 rare variations on lung carcinogenesis, the project will help to clarify the mechanism of lung cancer pathology, and provide scientific evidences for prevention and treatment of lung cancer.

"罕见变异引起常见疾病"是探讨疾病易感基因的新思路，肿瘤被认为是多个罕见事件累积的结果。课题组前期发现MKK7基因罕见的遗传变异E116K与肺癌发病高度关联且使患者生存期缩短；MKK7是丝裂原活化蛋白激酶(MAPK)信号通路的核心分子，能抑制H-ras1等癌基因的作用，广泛参与调控细胞增殖、分化、衰老、凋亡及炎症等过程，被认为是肿瘤抑制基因。生物信息学分析发现该基因还有4个具潜在功能的编码区罕见变异，其累积效应极可能影响肺癌的发生发展。本项目拟在两中心、大样本人群（3000对）检测上述变异及酶活性等表型，探讨其与肺癌发病和预后的关联；构建系列含上述变异的人支气管上皮模式细胞系，观察致癌物作用下其癌变的动态过程；建立哺乳动物双杂交系统检测蛋白-蛋白相互作用，并利用模式细胞系研究上述变异对H-ras1致癌作用的影响；揭示MKK7罕见变异的生物学功能，阐明肺癌变机制，为肺癌防治提供科学依据。

本研究基于“罕见变异-常见疾病”的理论假说，采用大样本，双中心的病例对照研究，探索了MKK7编码区多个潜在功能性罕见变异与我国人群肺癌发生发展的关系及关联位点的分子生物学功能。项目共新收集肺癌病例3000余例和性别相同，年龄相近的对照3000余例。利用直接测序法，在共计5016例病例和5181例对照中检测了MKK7基因5个罕见变异位点p. Glu116Lys（rs28395770G>A）、p.Asn118Ser （rs56316660A>G）、p.Arg138Cys（rs56106612C>T）、p.Ala195Thr （rs55800262G>A）和p.Leu259Phe（rs1053566 C>T）的基因型，仅发现p.Glu116Lys位点与我国人群肺癌发生发展存在显著相关，而其他四个位点不存在显著关联。相较于116Glu纯合基因型携带者，116Lys变异基因型携带者发生肺癌的风险显著上升（OR=3.29; 95%CI=2.70-4.01）。并且，116Lys基因型能显著增加肺癌发生转移的概率，缩短携带肺癌患者的中位生存期（-5个月），提高肺癌病死率（HR=1.53; 95%CI=1.32-1.78）。位点组合分析同样显示p. Glu116Lys在我国人群肺癌易感性中占主要效应。系列功能研究证实MKK7蛋白116位氨基酸由Glu变异为Lys，在体内和体外水平，均能显著增加MKK7蛋白的促肿瘤细胞生长、增殖、转移和侵袭能力，但其不影响MKK7蛋白的激酶活性。基因芯片分析显示该变异能显著改变多个肿瘤关联基因的表达及信号通路的活性。此外，两种携带不同氨基酸的MKK7蛋白均未检测到与H-ras1蛋白存在交互效应。上述结果表明MKK7基因编码区p.Glu116Lys罕见变异能显著影响我国人群肺癌的发病风险和肺癌患者的预后，是我国人群肺癌易感和预后效果评估的潜在生物标志。本结果后续可用于我国人群肺癌风险和预后效果评估，为肺癌高危人群的筛选和肺癌的靶向防治提供科学依据。