新型疾病分子标志物循环miRNA分离富集、高灵敏检测新技术研究

MicroRNAs are small (typically ≈22 nt in size) regulatory RNA molecules that play important roles in a wide range of physiologic and pathologic processes. Circulating MicroRNAs (cmiRNAs), first discovered in human blood in 2008, are found to be significantly correlated with the occurance and development of cancer, metabolic disorder, and cardiovascular disease, and can serve as a novel class of blood-based disease biomarkers. This project focuses on the separation and detection of hepatocellular carcinoma-related cmiRNAs. We will define distribution of these cmiRNAs in clinical samples of plasma and serum in different stable forms, including encapsulation in membrane-bound vesicles and miRNA-protein complexes, and develop the novel methods based on micro-size-exclusion chromatography and magnetic bead immunoaffinity for efficiently purifying various populations of cmiRNAs (cmiRNAs associated with exosomes, microvesicles, and apoptotic bodies; miRNA- Ago2; miRNA-NPM1; miRNA-HDL) in peripheral blood. The separation and identification of distinct cmiRNA populations, originating from different cell types and reflecting cell type-specific miRNA expression and/or release mechanisms, may increase biomarker sensitivity and provide more precise information for clinical diagnosis or prognosis. We will also establish the ultrasensitive detection methods of cmiRNAs based on target assisted enzyme-catalyzed signal amplification and nanoparticle-enhancement, and construct integrated high-throughput microfluidic chip for sample processing, purification and quantitative determination of distinct cmiRNA populations in human blood. Our researches lay the foundation for the development of circulating miRNAs as a novel class of blood-based disease biomarkers.

可以稳定地存在于外周血的循环miRNA（cmiRNA）与肿瘤、代谢和心血管等重要疾病发生、发展进程有关，是理想的非侵袭性早期疾病诊断分子标志物。本项目针对肝癌特异性cmiRNAs的分离富集、高灵敏检测问题开展研究，评估相关cmiRNA在外周血不同组分不同亚群中的表达差异、定量分布特点及临床诊断意义；建立cmiRNA不同组分（如囊泡包裹形式、蛋白复合体形式等）提取纯化新技术，采用微流控体积排阻色谱及免疫磁珠技术设计多通道微流控芯片，实现cmiRNA不同组分的高通量、自动化、高效分离富集；建立多种基于酶催化靶标循环及纳米强化信号放大的cmiRNA高灵敏检测新方法，设计构筑外周血样本处理、cmiRNA组分纯化及分类检测的一体化微流控芯片。本项目研究为肿瘤早期诊断、分型、预后、疗效预测及疾病复发预测等提供一种简单有效的基于新型肿瘤标志物cmiRNAs的非损伤性检测手段。

本项目针对新型肿瘤分子标志物循环miRNA 的分离富集、高灵敏检测问题开展研究，分析肝癌特异性外周循环 miRNA在外周血不同组分不同亚群中的表达差异、定量分布特点及临床诊断意义。从酶介导和纳米粒子介导的信号放大两方面开发了多种新型高效能的酶催化信号放大机制和纳米增强信号策略，建立了12种不同信号检测方式的高灵敏度miRNA检测分析新方法，开发了2种miRNA分析试剂盒；构建了新型高效的DNA分子机器拓展应用于活细胞内特定miRNA的定量分析及成像；建立了不同亚群循环miRNA，外泌体包裹形式和Ago2蛋白复合体形式，分类提取、富集纯化新技术，设计构筑了微流控芯片及96孔板两种技术平台，实现了循环miRNA不同存在形式的高通量、自动化、高效分离富集及检测；通过肝癌患者临床样本分析，获得了一系列具有不同等级诊断价值的miRNA；基于上述研究成果，拓展了循环核酸cfDNA的磁珠高效富集及EGFR突变检测，为非小细胞肺癌患者EGFR-TKI治疗提供基因分型手段。该项目研究成果的科学意义和应用价值在于通过开发高灵敏特异性miRNA检测方法和微流控芯片平台，系统地分析了循环miRNA不同组分的浓度，与肿瘤的发生、发展、术后疗效的相关性，评估了疾病诊断敏感性和特异性的平衡，建立了肿瘤相关循环miRNA在血液中分布模式与疾病的对应关系，提高了循环miRNA标志物的灵敏度及专一性，为肝癌早期诊断提供更加准确的信息。项目实施以来，已发表项目标注 SCI 论文 53篇；已申请中国发明专利9项（授权3项）；项目组成员获批国家基金委优秀青年项目资助1项；获2017年度教育部高等学校科学研究优秀成果自然科学奖二等奖等。