miRNAs作为潜在生物学标志在CYP2A13代谢活化AFB1所致呼吸系统肿瘤中的作用研究

Cytochrome P450 2A13 (CYP2A13) enzymes is an extrahepatic CYP enzymes that mainly expressed in human respiratory tract. The data from our previous studies showed that CYP2A13 was highly efficient in the metabolic activation of aflatoxin B1 (AFB1), and enhanced AFB1-induced the neoplastic transformation in human bronchial epithelial (BEAS-2B) cells. Thus, it is reasonable to speculate that CYP2A13 can metabolically activate airborne AFB1 in situ in pulmonary cells to form active metabolites which subsequently contribute to the tumorigeneicity in human respiratory tract. However, to date, few studies were found to focus on this effect. Besides, our recent study selected five potential microRNAs (miRNAs) which were involved in epithelial-mesenchymal transition (EMT) or DNA damage repair in AFB1-induced the cellular neoplastic transformation using the microRNA array, but the critical roles as well as the molecular mechanisms are needed to be further investigated. In the present program, we will construct HBE cell stably expressing CYP2A13 (H-2A13) and transgenic mouse expressing CYP2A13 controlled by a tetracycline (Tet)-on/off system to illustrate the effects of CYP2A13 on AFB1-induced respiratory tumor, and try to explore the roles of the selected miRNAs in AFB1-induced carcinogenesis, and identify the target miRNAs which will be further confirmed to be as potential biomarkers for AFB1 exposure and human lung cancer through a small samples cohort study from occupational population and a case-control study of lung cancer from the areas with high or low AFB1 contamination. The program will help provide a new strategy to identify the relationship between airborne AFB1 and human lung cancer, and also help provide the important technical support and practical warranty for the health risk assessment of the environmental carcinogens.

CYP2A13是主要在人呼吸系统表达肝外代谢酶。前期研究表明，CYP2A13可代谢活化AFB1，并增强其对支气管上皮细胞的恶转能力。因此推测，经呼吸道吸入的AFB1可以被CYP2A13原位代谢成活性致癌物，从而诱发肿瘤，但迄今未见报道。近期利用miRNA芯片，初步筛选了5个与AFB1诱导的细胞恶转相关的miRNAs，但作用机制有待阐明。本研究利用构建稳定表达CYP2A13的HBE细胞（H-2A13）和肺脏可控性表达CYP2A13的转基因小鼠（Tet-on系统），系统阐明CYP2A13对AFB1所诱发的呼吸系统肿瘤的影响，初步探讨miRNAs在其过程中的关键作用，并逐步筛选关键的miRNAs。再利用AFB1职业人群队列研究和AFB1高/低污染区的肺癌病例对照研究，验证候选miRNAs作为AFB1暴露及肺癌罹患的潜在生物学标志。研究结果将为解析空气AFB1污染与人群肺癌的关系提供新的研究策略。

miRNA是当前肿瘤研究的热点之一，具有稳定性好、组织特异性强、灵敏度高等优点，是极具发展前景的肿瘤生物标志。鉴此，课题组前期利用AFB1处理的第50代恶转细胞进行了miRNA芯片分析，发现了一些有价值的线索，为本研究的顺利开展奠定了基础。围绕课题的研究目标和任务，尤其是miRNAs的筛选、调控机制、生物标志方面开展了系列工作；此外，还根据当前miRNA领域的研究进展不断丰富研究内容，有利于全面系统地认识miRNA在CYP2A13代谢活化AFB1致呼吸系统肿瘤中的重要作用。在miRNA芯片结合生物信息学分析的基础上，筛选出miR-138-1*作为候选miRNA进行功能研究，发现miR-138-1*可通过对PDK1的调控从而抑制PI3K/PDK1/Akt信号通路的活化而影响AFB1所致B-2A13细胞的恶性转化及其过程。基于miRNA芯片及数据库，筛选了以miR-487a/b为代表的外泌体miRNA簇，发现外泌体miR-487a/b可促进正常B-2A13细胞的增殖能力，干预后，P50代恶转细胞的增殖、迁移和克隆形成能力显著下降，提示miR-487a/b簇可能是发挥重要作用的外泌体miRNAs，有望成为AFB1致呼吸系统肿瘤的潜在生物标志。鉴于circRNA和miRNA与靶基因（mRNA）调控关系，开展了circRNA芯片和mRNA芯片分析，利用生物信息学结合肿瘤生物学行为和机制分析，探索cirRNA-miRNA-mRNA通路在AFB1致细胞恶性转化中的作用，筛选出了以ID3和FGF5为代表的靶基因、以miR-3663-3p和miR-3138为代表的miRNAs、以circ_0041714和circ_0088466为代表的circRNAs，并已证明ID3和FGF5与AFB1所致的细胞恶性转化有关。此外，构建了cyp2a5（与cyp2a13同源）敲除和cyp2a13嵌入小鼠，并利用cyp2a5敲除小鼠验证了AFB1可经CYP2A5代谢介导了呼吸系统炎症及组织细胞的不典型增生，初步观察了miRNAs在其中的调控作用，为进一步明确miRNAs作为AFB1致呼吸系统肿瘤的生物标志提供了依据。研究结果为空气AFB1污染的风险评估和防控对策提供了理论依据和技术支撑。相关成果已发表在本领域国际主流期刊并被多次引用，受到了国内外同行的一致认可。