血浆分泌型miRNA作为非小细胞肺癌分子标志物及参与肿瘤微环境调节的机制研究

NSCLC is difficulty in early detection and has a poor prognosis; however, no specific diagnostic marker has been discovered. Secreted miRNAs are a class of novel intercellular communication signal molecules that have physiologic functions. Our previous studies found that several miRNAs were significantly upregulated in exosomes from plasma from NSCLC patients and also from supernatant of NSCLC cell lines; NSCLC-secreted exosome was able to carry specific miRNAs into fibroblasts and macrophage; Bioinformatics analysis suggested that these miRNAs might be involved in the alteration in phenotype and function of these two kinds of cells. Thus, we hypothesize that plasma secreted miRNAs are potential molecular biomarkers of NSCLC and they may enter into tumor microenvironment-associated fibroblasts and macrophage, 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 select secreted miRNAs that have clinical value in the diagnosis of NSCLC; to explore the role and mechanism of secreted miRNAs in regulating tumor microenvironment related fibroblasts and macrophage; and to construct the regulatory network between miRNAs and their downstream target genes. The purpose of our study is to open up a new idea and new ways for the diagnosis, mechanism and molecular targeting treatment of NSCLC.

非小细胞肺癌（NSCLC）早期诊断困难，预后差， 但无理想的分子诊断标志物。分泌型miRNA是一类具备生理功能的新型细胞间信号交流分子，与肿瘤密切相关。我们前期研究发现，一些miRNA在NSCLC血浆和癌细胞培液外泌体（exosome）中均高表达；肺癌细胞分泌的exosome可携带特定miRNA进入相关成纤维细胞和巨噬细胞；生物信息学预测它们参与这两种细胞表型和功能变化。据此提出假说：血浆分泌型miRNA是NSCLC潜在的分子标志物，它们以exosome为载体进入肿瘤微环境相关成纤维细胞和巨噬细胞，改变其表型和功能，参与NSCLC发生发展。为验证假说，我们拟通过临床标本、细胞及动物研究，筛选对NSCLC有诊断价值的分泌型miRNA；探明其调节肿瘤微环境相关成纤维细胞和巨噬细胞的作用机制；构建其与下游靶基因间的调控网络。为NSCLC的诊断、发病机制、分子靶向治疗提供新思路和新途径。

非小细胞肺癌（NSCLC）早期多无症状且复发率高，发病机制不详, 且缺乏NSCLC 特异的血清学标志物。本项目运用高通量低密度芯片技术初筛和RT-qPCR验证相结合的方法发现miR-520c-3p和miR-1274b在NSCLC患者血清/血浆外泌体中的水平显著高于良性结节患者和正常对照，而在患者术后明显下降。两种miRNA联合检测对早期NSCLC有较高的诊断准确性。miR-1274b、miR-520c-3p在人肺腺癌细胞培液exosome中的含量也显著高于正常对照HBE细胞株培液exosome。体外细胞实验显示，肺腺癌细胞分泌的exosome可显著促进人脐静脉内皮细胞（HUVEC）的侵袭、迁移和血管生成能力，miR-1274b、miR-520c-3p可能在其中起主要作用。生物信息学分析结果显示，这两种miRNA共同靶向基因在癌症信号通路中富集。体内动物模型实验显示，分泌型miR-1274b、miR-520c-3p可明显促进NSCLC肿瘤的生长和浸润。结论：本研究结果为NSCLC寻找到了潜在的新的分子标志物，也为NSCLC提供了新的潜在的治疗靶点。. 本项目累计发表12篇论文(SCI论文10篇)；参加国内外学术交流7人次；培养博硕士生4名。项目投入经费57万元，支出47.1106万元，各项支出基本与预算相符；剩余经费9.8894万元，计划用于本项目后续研究支出。