外泌体中3'末端2'-O-甲基化修饰miRNA作为非小细胞肺癌新型液体活检分子标志物的价值及作用机制研究

外周血甲基化修饰RNA具备成为新型肿瘤标志物潜能，但外泌体（exosome）甲基化修饰miRNA临床价值未见报道。我们最新结果显示，非小细胞肺癌（NSCLC）细胞培液、患者血清exosome中部分3'末端2'-O-甲基化修饰miRNA水平与癌组织一致升高，有望成为NSCLC新型分子标志物；生物信息学预测这些miRNA可调节NSCLC细胞及肿瘤微环境细胞功能。据此提出假说：exosome 中3'末端2'-O-甲基化修饰miRNA是NSCLC潜在分子标志物，其较未修饰miRNA可能更具价值和功能，可通过调节肿瘤及微环境相关细胞功能，参与NSCLC发生发展。为验证假说，我们拟筛选NSCLC患者血清exosome显著、稳定变化的3'末端2'-O-甲基化修饰miRNA；探明其修饰、调节肿瘤及微环境细胞作用机制；构建其上游修饰、下游靶基因间调控网络，为NSCLC精准诊断和发病机制研究提供新思路和策略。

Methylated RNAs in circulation have been recognized as novel potential biomarkers for tumor detection, however, study on the clinical significance of methylated miRNAs in exosome has never been reported. In our recent study, we found that several miRNAs with 2'-O-methylation modification at the end of 3’-UTR were significantly upregulated in exosomes from cell culture medium of NSCLC cell line and from serum of NSCLC patients; Moreover, 2'-O-methylated miRNAs were also also elevated in NSCLC. Bioinformatics analysis suggested that these methylated miRNAs might be involved in the alteration in cancer associated function of NSCLC cells as well as cells in tumor microenvironment, such as vascular endothelial cells. Thus, we hypothesize that dysregulated 2'-O-methylated miRNAs in serum exosome are potential molecular biomarkers of NSCLC, and 2'-O-methylated miRNAs may have more valuable and functional than unmethylated miRNAs; Additionally, they may regulate the biological functions of NSCLC, and enter into tumor microenvironment-associated cells, change their phenotype and function and be involved in the development and progression of NSCLC. In order to verify this hypothesis, we will adopted clinical samples, cell lines and tumor-bearing mouse model, to identify significantly altered 2'-O-methylated miRNAs in exosomes that have clinical value in the diagnosis of NSCLC; to explore the potential mechanism for miRNA methylation, the role and mechanism of these miRNAs in regulating NSCLC cells and tumor microenvironment; and to construct the regulatory network between the upstream methylase, 2'-O-methylated miRNAs and their downstream target genes. The purpose of our study is to open up a new idea and new strategies for the diagnosis, mechanism and molecular targeting treatment of NSCLC.