miR-1290作为肺腺癌预后标志物及其调控巨噬细胞极化的研究

There are a large number of tumor-associated macrophages (TAMs) in the microenvironment of lung adenocarcinoma. TAMs in M2 polarized state is the main phenotype in advanced patients, and the number of which is related to prognosis. However, the mechanism of M2 polarization in macrophages in the lung adenocarcinoma microenvironment remains unclear. Previously, we found that miR-1290 was highly expressed in tumor tissue and serum of patients with lung adenocarcinoma and correlated with prognosis. In addition, miR-1290 can down-regulate the target gene SOCS3 expression, promoting lung adenocarcinoma progression. Since SOCS3 is a key protein in regulation of macrophage polarization, the following hypothesis is proposed: lung adenocarcinoma cells secrete miR-1290, which acts on macrophages in the tumor microenvironment via exosomes, inhibiting SOCS3 expression, promoting M2 polarization, leading to rapid tumor progression and poor prognosis. Pre-experiments showed that there were a great deal of miR-1290 in the exosomes from serum of patients with lung adenocarcinoma, and could induce M2 phenotype polarization. This study will explore the molecular mechanism of miR-1290 promoting M2 phenotype polarization in the microenvironment of lung adenocarcinoma and its value as a prognostic marker from three levels including cell, animal and clinical specimen study. The aim of the study is to understand the molecular mechanism of lung adenocarcinoma progression from the perspective of tumor cells and macrophages interaction, and to provide evidence for miR-1290 detection applying for lung adenocarcinoma prognosis.

肺腺癌微环境中存在大量肿瘤相关巨噬细胞（TAM），中晚期患者TAM以M2型为主，且M2型数量越多预后越差。然而，肺腺癌微环境中TAM向M2型极化的机制不明。前期课题组发现肺腺癌肿瘤组织和血清中miR-1290高表达，且与预后相关。此外，miR-1290可下调靶基因SOCS3，促进肺腺癌进展。由于SOCS3是调控巨噬细胞极化的关键蛋白，提出如下假说：肺腺癌细胞分泌miR-1290，由外泌体介导作用于微环境中巨噬细胞，抑制SOCS3表达，促进M2型极化，推动肿瘤进展，预后更差。预实验表明肺腺癌外泌体存在大量miR-1290，且能诱导M2型极化。研究从细胞、动物和临床标本三个层次，探索微环境中miR-1290促进M2型极化的机制；通过临床大样本检测和随访，评估其作为肺腺癌预后标志物的价值。课题有助从肿瘤与巨噬细胞互作角度理解肺腺癌进展的分子机制，也为将miR-1290检测用于预后判断提供依据。

肺腺癌微环境中存在大量肿瘤相关巨噬细胞（TAM），中晚期肺腺癌患者肿瘤组织中的TAM以M2型为主，且M2型TAM的数量与预后相关。然而，肺腺癌微环境中TAM向M2型极化的机制不明。本课题旨在阐明肺腺癌来源的驱动TAM向M2型极化的分子及其机制。.首先，分离鉴定肺腺癌细胞株和肺腺癌患者血清来源的外泌体，分别将其加入巨噬细胞THP-1培养体系中。激光共聚焦显微镜证实外泌体被THP-1细胞摄取；流式细胞术和RT-PCR结果表明THP-1细胞向M2型极化。接着，通过肺腺癌患者与健康体检者血清外泌体miRNA的差异分析，筛选到miR-1290是外泌体中差异最为显著的miRNA分子。而在肺腺癌外泌体处理的THP-1细胞中，miR-1290水平增高。在THP-1细胞中过表达和敲低miR-1290，可以改变其极化的转态。机制研究发现，巨噬细胞极化的关键调控蛋白SOCS3是miR-1290的靶基因。miR-1290能通过靶向抑制SOCS3的表达，促进STAT3通路活化，从而推动M2型极化。构建稳定敲低miR-1290的肺腺癌细胞系，其外泌体对于THP-1 M2型极化的作用显著降低，处理后巨噬细胞培养上清促肺腺癌细胞侵袭转移的能力也减弱。动物实验中，敲低肺腺癌外泌体miR-1290可减小裸鼠皮下移植瘤的体积和肿瘤中浸润的CD163+巨噬细胞数量。进一步的研究证实，缺氧是诱导肺腺癌细胞外泌体中miR-1290水平升高的重要原因。缺氧微环境上调了肺腺癌细胞HIF-1α的水平，进而miR-1290在外泌体中的水平显著升高。此外，临床研究结果证实血清外泌体miR-1290是影响肺腺癌患者预后的独立危险因素。血清外泌体miR-1290水平高的肺腺癌患者的无进展生存期明显缩短，预后不良。.本研究发现肺腺癌细胞分泌高水平的miR-1290，由外泌体介导作用于肿瘤微环境中的巨噬细胞，通过抑制靶基因SOCS3的表达，促进巨噬细胞向M2型极化，导致肿瘤进展。本研究初步揭示了肺腺癌免疫微环境中巨噬细胞极化的机制，有望为肺腺癌的免疫治疗和预后判断提供新的分子靶标。