LncRNA调控P53信号通路参与藻毒素促进亚硝胺致食管癌的作用机制研究

Long non-coding RNAs (lncRNAs), non-protein coding transcript longer than 200 nucleotides, target various aspects of gene expression processing including epigenetic regulation, transcriptional regulation, post-transcriptional regulation, and etc. which implicates that lncRNA might be the node connecting different biomarkers 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 carcinogenesis of esophageal cancer induced by N-nitrosamines and microcystins were suggested to be involved in lncRNA regulation. In our study, esophageal cancer model of cells and rats are induced by N-nitrosamines and microcystins. The mechanism of long non-coding RNAs affecting the carcinogenesis of N-nitrosamines promoted by microcystins in esophagus is determined with respect to P53 activation and stabilization, and downstream gene regulation involved in P53 signaling pathway. Receiver operating characteristic curve (ROC) analysis is carried out to evaluate sensitivity and specificity of candidate plasma lncRNA diagnosis model, followed by screening test in high-risk population of esophageal cancer. The specific plasma lncRNAs is suggested as available biomarkers for screening and early diagnosis of esophageal cancer, as well as potential target for early treatment of esophageal cancer.

LncRNA是近年来识别的新型非编码RNA，已发现在表遗传调控方面参与多种基因调控的新机制。食管癌作为环境相关肿瘤，由环境危险因素诱导肿瘤发生的过程中lncRNA参与的表遗传调控机制可能发挥着重要的作用。由于工农业和生活活动对水环境的污染，生活饮用水中亚硝胺类消毒副产物和藻毒素长期持续暴露，可能成为食管癌的重要环境危险因素来源。本研究以亚硝胺为启动剂、藻毒素为促癌剂，分析食管上皮细胞暴露于亚硝胺和藻毒素后的MAPK通路及P53通路等胞内应答通路，探讨候选lncRNA在P53的活化与稳定性维持、下游基因调控等方面的功能与调控机制，识别lncRNA参与藻毒素促进亚硝胺致食管癌的可能作用机制。并基于候选食管癌血浆lncRNA诊断模型的ROC分析和食管癌高危人群的筛检试验，构建灵敏、特异的食管癌人群筛检分子标志，并为食管癌的早期诊断和治疗提供科学的技术指标和潜在作用靶点。

饮用水中亚硝胺和藻毒素等的污染对消化道肿瘤发病风险的影响日益受到关注。本研究以亚硝胺和微囊藻毒素联合染毒大鼠和正常食管上皮细胞，初步明确亚硝胺与藻毒素联合暴露可诱导正常食管上皮恶转和动物食管癌，增加人群食管癌发病风险，为亚硝胺和藻毒素致癌作用补充了流行病学和动物研究证据。在此基础上，在淮安食管癌高发现场筛选了食管癌相关的lncRNA标志物，并深入探讨了LncRNA UCA1、ROR和LincRNA-p21在食管癌发生中的分子作用机制。研究应用RNA-pull down/MS、RIP技术等识别了核内定位的lncRNA-UCA1通过结合hnRNPF参与调控FGFR2可变剪接的这一新的作用机制，发现核内lncRNA-UCA1可通过FGFR2 IIIc/PI3K-AKT信号通路参与调控食管癌EMT进程；而结合双荧光素报告系统、MS2-RIP和WB等实验发现胞质定位的lncRNA-UCA1可作为miR-18a-5p的“分子海绵”参与竞争性调节靶基因SORBS2，抑制食管癌细胞的增殖能力。而ROR可作为包括miR-145、miR-204-5p等多个miRNAs的分子海绵来调控FSCN1和MGM2等下游信号分子，发挥癌基因样的作用，参与P53信号通路的调控。LincRNA-p21显示是p53的直接靶标，研究发现lincRNA-p21参与下游p53依赖性转录抑制，并揭示了食管癌细胞中lincRNA-p21通过p21调控细胞周期的机制。本研究综合探讨了lncRNA-UCA1、ROR、lincRNA-p21等长链非编码RNA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lncRNAs在P53的活化与稳定性维持、下游基因调控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。