血清外泌体circRNAs作为结直肠癌肝转移诊断标志物的筛选及功能验证

Colorectal liver metastases (CRLM) is a difficult point for the treatment of colorectal cancer Because of its difficulty to find early. The early diagnosis of molecular biomarker is still missing. Our previous study found that multiple miRNAs associated with CRLM. Circular RNA(circRNA) , found to be "miRNA sponge" , bind to miRNAs to play a regulatory role after transcription. It is more stable with the circular structure and expresses different in tissuses and enriches in exosomes of serum. Exosomes may be carriers that transport the regulating circRNAs to metastatic organs. For these reasons, we intend to identify CRLM-related circRNAs in serum by using exosomal RNA sequencing and deduce the circRNAs that interact with miRNAs associated with CRLM, derived from previous studies, followed by detection of large clinical samples by ddPCR to analyze the association between circRNA expression abundance and CRLM clinicopathological data and follow-up data. This study will investigate the potential of exosomal circRNA in serum as a diagnostic biomarker of CRLM, and explore its function and regulation mechanism and provide a basis for CRLM diagnosis, treatment and metastases monitoring.

结直肠癌肝转移（CRLM）因其难以早发现而成为结直肠癌治疗的难点，目前尚缺早期诊断分子标志物。我们前期研究发现多个miRNA可能与CRLM相关，而环状RNA（circRNA）被发现是“miRNA海绵”，其与miRNA结合发挥转录后调控作用，且组织特异性强、环状结构更稳定，在血清外泌体中大量富集；外泌体则可能是运输circRNA到转移器官的载体;因此，我们拟以血清外泌体全转录组测序来鉴定CRLM相关的circRNA，也从前期研究得到的可能与CRLM相关的miRNA推导出与其相互作用的circRNA，以生物信息学分析，筛选差异表达的circRNA。并通过数字PCR检测大临床样品的回顾性实验来分析circRNA表达丰度与CRLM临床病理数据和随访数据的关联。本课题探讨血清外泌体circRNA作为CRLM诊断标志物的潜能，并初步探讨其功能及调控机理。研究将为CRLM的诊断、治疗及转移监控提供依据。

背景：结直肠癌肝转移（CRLM）是导致结直肠癌（CRC）高死亡率的主要原因。近年来研究发现，肿瘤来源的外泌体环状RNA可释放并稳定存在于血液中，有作为肿瘤标志物的潜质。本项目旨在探讨血液外泌体circRNA作为CRLM诊断标志物的潜能，并探讨其功能和调控机理。. 主要研究内容：通过全转录组测序和基因芯片技术，筛选在结直肠肝转移（CRLM）患者血液外泌体中差异表达的环状RNA（circRNA），并用real-time PCR进行验证。用微滴数字PCR进一步扩大临床标本检测，与患者的临床指标作关联分析，制作ROC曲线，并评价其诊断效能。通过生物信息学分析和细胞功能实验初步探讨circRNA在结直肠癌发生发展过程中的作用及分子机制。. 重要结果及关键数据：本课题筛选出一组在CRLM患者血液外泌体中差异表达的环状RNA。微滴数字PCR检测了其中的外泌体Circ_0001296的表达，结果表明Circ_0001296在结直肠癌肝转移患者血浆外泌体中高表达，其鉴别原发性结直肠癌和结直肠癌肝转移的诊断效能优于传统标志物CEA、CA19-9、CA125和TK1。当血浆外泌体中Circ_0001296与以上四个标志物联合，其诊断效能可提高至0.900。进一步的研究发现，Circ_0001296高表达可以促进直肠癌细胞的迁移和侵袭，Circ_0001296有可能通过miR-509-3p调控AXL的表达。. 科学意义：本项目研究发现该成果具有潜在的临床应用价值，为CRLM的诊断和精准治疗提供理论依据。