HRH4遗传变异对组胺H4受体调控非小细胞肺癌EMT过程及恶性进展的影响及机制研究

To elucidate receptor function is one of the core scientific issues in pharmacological field. However, the factors that affect or modulate receptor function are rather complicate and not fully clarified yet. With the development of genomic research, genetic factor is gradually acknowledged as a novel and important factor affecting receptor function, which provides us new insight into this area. According to our previous investigation, the histamine H4 receptor (HRH4) mRNA and protein were expressed and exerted inhibitory effect on the epithelial-to-mesenchymal transition (EMT) progress in many kinds of non-small cell lung cancer (NSCLC) cell lines. Moreover, we also found that the polymorphism of rs11662595 in HRH4 gene was significantly associated with the clinical grade, degree of metastasis, and recurrence and death rate in NSCLC patients. Based on these findings, we rationally hypothesize that genotypes of rs11662595 may affect the gene and protein expression of HRH4 and hence affect the EMT progress of NSCLC. In order to systematically prove this hypothesis, we plan to explore the underlying mechanisms of how polymorphism of rs11662595 affects the function of HRH4 at the molecular, cellular and organizational level and demonstrate how this polymorphism affects the malignant progression of NSCLC. The accomplishment of this study will provide us in-depth understanding of the genetic effects on HRH4 function, and also provide novel rationales for personal prevention and treatment of NSCLC and new strategies for anti-cancer drug development that target HRH4.

受体功能研究是药理学研究领域的核心科学问题，但影响和调控受体功能的因素却错综复杂。随着基因组学研究深入，遗传因素对受体功能的影响为受体功能研究提供了新的思路。申请者前期研究发现组胺H4受体（HRH4）编码基因及蛋白在多种非小细胞肺癌细胞系中均有表达并在上皮细胞间质化（EMT）过程中发挥重要作用，而HRH4基因rs11662595多态性与肺癌患者临床分级、转移程度、复发率及死亡率均显著相关。基于此，申请者合理推测：rs11662595不同基因型对HRH4基因表达产物的功能有显著影响，进而极有可能改变肺癌细胞EMT过程。为系统证明上述推断，本项目拟从分子、细胞及组织水平研究rs11662595不同基因型影响HRH4功能的机制，阐明rs11662595多态性影响肺癌恶性进展的具体机理，期望深入理解遗传因素对HRH4功能影响，为以HRH4为靶点的抗肿瘤药物开发及肺癌个体化防治提供新的理论基础。

我们前期研究表明组胺H4受体（HRH4）在非小细胞肺癌（NSCLC）上皮细胞间质化（EMT）过程中有着至关重要的作用。同时，HRH4基因rs11662595多态性与包括NSCLC在内的多种肿瘤发病风险存在关联。因此我们推测：rs11662595突变可能对HRH4基因表达及功能有着显著影响，并进而影响NSCLC细胞的EMT过程。为证实上述推测，本项目从分子、细胞及组织水平对rs11662595突变影响HRH4功能的机制进行了深入研究，并结合临床样本进行验证。结果表明：尽管rs11662595突变对HRH4蛋白表达产物稳定性以及与其配体亲和力均无显著影响，但该突变能够显著抑制HRH4激活后对下游G蛋白的活化能力。离体细胞实验则表明，rs11662595突变可显著抑制HRH4激活后对NSCLC细胞EMT过程的调控作用。同时，该突变还能够显著减弱HRH4抑制NSCLC细胞增殖、侵袭等恶性行为的能力。随后的体内荷瘤小鼠实验进一步显示，HRH4激活可显著抑制野生型A549细胞接种裸鼠的肿瘤生长体积并显著延长总体生存时间，但对rs11662595突变型A549细胞接种裸鼠的肿瘤生长体积和总体生存时间无显著影响。上述结果表明rs11662595突变可以显著抑制HRH4的受体功能，是一种“失功能”型突变。我们临床研究结果同样显示，携带rs11662595突变的NSCLC患者在无进展生存时间和总体生存时间上较野生型NSCLC患者均显著缩短。同时，rs11662595突变携带患者对比较野生型患者有着更低的肿瘤分化程度、更高的TNM分级以及更严重的淋巴结转移程度。进一步从临床角度证实了rs11662595突变在NSCLC进展及恶化过程中的重要作用。本研究结果将有助于深入理解遗传因素对HRH4功能影响以及NSCLC的发病机制，为以HRH4为靶点的抗肿瘤药物开发及肺癌个体化防治提供新的理论基础。