血浆环状RNA作为肺癌早期生物标志物的分子流行病学研究

Recently the latest research results for circular RNA (circular RNA, circRNA), which were published at some important academic journals such as Nature, Science, Cell etc., told us new attention should be paid on it. Plasma circRNA has many features as abundant, stable, easy to detect, etc., it is expected to become a new biomarker for early stage lung cancer. This research will intend to use next-generation sequencing technologies to preliminarily screen lung cancer-specific plasma circRNA spectrum, further taking independent and cross validations to optimize the lung cancer plasma circRNA, and using multi-stage lung cancer samples to evaluate the value for taking plasma circRNA as a biomarker of early stage lung cancer, further apply bioinformatics methods to explore the biological mechanism of related circRNAs. This project will establish lung cancer specific plasma circRNA profile; obtain plasma circRNAs which can be used for early lung cancer screening; explore circRNA potential biological mechanisms for lung cancer; finally to provide theoretical and technical foundation for determining the simple and inexpensive biomarkers for early stage cancer screening.

近期环状RNA（circular RNA, circRNA）的最新研究成果陆续在Nature、Science、Cell等重要学术期刊上发表，使circRNA重新受到大家的关注和重视。血浆circRNA具有含量丰富、性质稳定、易于检测等特征，有望成为肺癌等恶性肿瘤的早期发现生物标志物。本课题拟依托二代测序技术初步筛选肺癌特异circRNA谱，进一步通过独立验证和交叉验证筛选和优化肺癌血浆circRNA；通过已经收集的肺癌发病早期多阶段病变样本，评价血浆circRNA作为肺癌早期生物标志物的价值；应用生物信息学技术初步探讨相关circRNA潜在生物学功能和作用机制。本课题的顺利实施有助于建立肺癌血浆circRNA表达谱数据库，获得能用于肺癌早期筛查的血浆circRNA及circRNA在肺癌发生中的潜在生物学机制，为确定简单易行微创价廉的肺癌早期筛查生物标志物提供理论和技术基础。

本课题依托二代测序平台（Illumina HiSeq 1500）对30例肺癌病人的癌和癌旁组织进行全转录组测序，通过30例新发肺癌患者的癌和癌旁组织进行全转录组测序，进一步对total-RNA-seq数据进行数据分析和整合，共识别出5471个候选circRNA，进一步通过癌组织和癌旁组织的差异表达分析，挑选了120个肺癌组织特异的circRNA进入下一步验证；采用 Real-Time RT-PCR 的方法，对参与测序的30例肺癌病人血浆circRNA进行单个逐一检测，验证并进一步筛选肺癌血浆水平差异表达circRNA，10个circRNA能在血浆中稳定表达，初步确定这10个circRNA（circCCNB1、cirVRK1、circCCDC134、circZCCHC6、circC1orf116、circFARSA、circPMS1、circDNA2、circPSD3和circSMAD2，P值分别为：0.0019、0.002、0.01、0.01、0.01、0.016、0.03、0.04、0.04、0.05）可作为肺癌诊断血浆生物标志物; 根据上述验证筛选结果，对另外选择的100对肺癌病例对照进行交叉验证，进一步筛选优化肺癌诊断血浆circRNA生物标志物，采用三种circRNA识别算法提高circRNA识别的准确性，结合生物信息学网站功能预测，鉴定出circFARSA在组织和血浆中稳定表达，并存在差异（P值为0.016）；进一步比较肺癌前期病变各阶段血浆circRNA，功能实验表明circFARSA在验证样本中稳定表达（癌组织和血浆样本）；受试者工作曲线和曲线下面积初步评估其诊断价值，AUC为0.71，进一步净得分改善值为65%，说明circFARSA可作为肺癌发生的早期生物标志物。本课题获得了能用于肺癌早期筛查的血浆circRNA，探讨了circRNA在肺癌发生中的潜在生物学机制，为确定简单易行微创价廉的肺癌早期筛查生物标志物提供理论和技术基础。

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