环状RNA靶向RTK信号通路介导EMT参与亚硝胺联合藻毒素致食管癌的作用机制研究

Circular RNA (circRNA), a novel type of endogenous RNA, target various aspects of epigenetic regulation including transcriptional regulation, post-transcriptional regulation, and etc., which implicates that circRNA might be the node connecting factor with stabilization and is supposed as a new biomarker for tumor. Esophageal cancer has recently raised great concerns since N-nitrosamines, as a group of emerging disinfection by-products (DBPs) in drinking water, show long-term exposure as well as microcystins. And the mechanism of tumor heterogeneity in esophageal cancer induced by N-nitrosamines and microcystins are suggested to be involved in circRNA regulation. The present study will identify the candidate circRNAs associated with tumor heterogeneity through multicenter cooperation in the South and North high incidence area of esophageal cancer. Esophageal cancer model of cells and mice are constructed with N-nitrosamines and microcystins exposure. The mechanism of the stem cell-like and aggressive malignancy induced by N-nitrosamines and microcystins in esophagus is recognized with respect to candidate circRNA targeting RTK signaling pathway involved in epithelial-mesenchymal transition. Receiver operating characteristic curve (ROC) analysis is carried out to evaluate sensitivity and specificity of candidate plasma circRNAs diagnosis model, followed by screening test in high-risk population of esophageal cancer. The specific plasma circRNAs are suggested as available biomarkers for grading screening and precision diagnosis of esophageal cancer with high malignancy, as well as potential targets for early treatment of esophageal cancer.

环状RNA（circular RNA，circRNA）是近年来新发现的一类内源性RNA，在表遗传调控方面参与了多种基因调控的新机制。食管癌作为环境相关肿瘤，由环境危险因素的分布及作用差异影响肿瘤异质性的机制中，circRNA参与的表遗传机制成为新的突破。本次研究通过南北方食管癌高发区的多中心合作，筛选饮用水中的亚硝胺消毒副产物与藻毒素诱导食管癌的肿瘤异质性相关的circRNA。从细胞和动物整体水平探讨亚硝胺联合藻毒素暴露下，候选circRNA靶向RTK信号通路参与EMT介导的食管癌干性特征和转移潜能的可能调控机制和关键靶点。并基于候选食管癌血浆circRNA诊断模型的ROC分析和食管癌高危人群的筛检试验，识别具有干性潜能和侵袭特征的癌细胞亚群的分子特征，作为食管癌高危人群筛检和临床诊疗重点人群的生物标志，有利于早期发现恶性程度高的食管癌患者，为建立食管癌的分级筛检和精准诊疗方案提供依据。

食管癌作为常见的环境相关性肿瘤，而饮用水中亚硝胺和微囊藻毒素暴露是其高发的一重要危险因素。近年来，随表观遗传学迅速发展，环状RNA (circRNAs)作为一潜在的分子生物标志物，在肿瘤发生及发展中作用引起学者广泛关注，但目前关于circRNA参与亚硝胺联合微囊藻毒素致食管癌理论尚存空缺。本研究以亚硝胺联合微囊藻毒素成功构建了正常食管上皮恶转模型，补充了亚硝胺联合微囊藻毒素致癌的分子生物学证据。在此基础上，于食管癌高发区江苏淮安筛选了食管癌相关circRNAs，探讨了hsa_circ_0006867、hsa_circ_0063865和hsa_circ_0001707参与亚硝胺联合微囊藻毒素致癌的分子机制，并进一步明确了食管癌细胞源外泌体hsa_circ_0002557激活癌相关成纤维细胞重塑肿瘤微环境异质性的特异性作用。研究应用RNA-pull down/MS、RIP技术等识别了hsa_circ_0063865可作为分子桥梁引导 eEF1A2 结合 MYH9介导细胞骨架重排的这一新分子机制；而结合双荧光素报告系统和Ago2 RIP等明确了hsa_circ_0063865和hsa_circ_0006867可分别通过miR-450b-3p/RCN1/PERK/CHOP 和miR-499a-3p/MEF2C参与亚硝胺联合微囊藻毒素致癌的新机制；而hsa_circ_0001707可作为 miR-203a-3p分子海绵来调控Snail2信号通路轴参与调控食管鳞癌细胞EMT过程。Exo-hsa_circ0002557显示可重构食管癌肿瘤微环境异质性，研究发现Exo_circ0002557调控hsa-miR-607/TGF-βII促进食管成纤维细胞活化为肿瘤相关成纤维细胞，重塑细胞外胶原纤维重排促进食管癌进展的新机制。本研究综合探讨了hsa_circ_0006867、hsa_circ_0063865和hsa_circ_0001707和Exo_hsa_circ0002557等circRNAs参与藻毒素促进亚硝胺致食管癌的作用机制，发现这些候选circRNAs在细胞骨架重排，内质网应激及胞外胶原纤维重塑等方面发挥重要功能，可作为食管癌高危人群筛检和早期诊断的候选分子标志，为食管癌的早期预防和靶向治疗提供了有应用前景的技术指标和潜在作用靶点。