LncRNA-LINC01181经外泌体传输调控TAM极化重塑口腔鳞癌微环境的机制研究

The M2 phenotype of tumor-associated macrophages (TAM) promotes the formation of tumor immunosuppressive microenvironment. It has been reported that the polarization of TAM is associated with multiple factors in the tumor microenvironment (TME) However, the regulatory role of long non-coding RNAs (LncRNA) in the polarization of TAM has remained poorly understand. Our previous studies indicated that the number of TAM with M2 phenotype in the tumor stroma was closely correlated with poor prognosis of patients with oral squamous cell carcinoma (OSCC). Through high-throughput sequencing, we identified that a functional unknown LncRNA- LINC01181 was upregulated aberrantly in OSCC samples compared with adjacent histological normal tissues. Moreover, the expression level of LINC01181 in the tumor nest was significantly higher than that in the tumor stroma. Intriguingly, we found abundant expression of LINC01181 in the exosomes derived from OSCC cells. Bioinformatics analysis indicated that LINC01181 was co-expressed with important M2 polarization-associated genes including PPARγ and IRF4. In addition, LINC01181 was predicted to function as a ceRNA for miR-27a to facilitate PPARγ and IRF4 expression. Therefore, we speculate that OSCC-derived LINC01181 may be transferred by exosomes to TAM, regulates miR-27a/ PPARγ&IRF4 axis to facilitate its M2 polarization, and then leads to the formation of immunosuppressive microenvironment to promote OSCC progression. In this study, we tend to confirm the correlation between LINC01181 and the progression of OSCC, and also the correlation between LINC01181 and the polarization of TAM　in　 tissues of OSCC　patients; to analysis the influences of LINC01181 on TAM polarization and function in　vitro and in vivo; and finally, to elucidate the mechanisms of TAM polarization involving exosome-transmitted LINC01181 regulating miR-27a/ PPARγ&IRF4 axis. This study hopes to provide a novel candidate target for the diagnosis and treatment of OSCC.

M2型肿瘤相关巨噬细胞（TAM）可促进肿瘤免疫抑制微环境的形成。已知TAM的极化受微环境中诸多因素影响，但LncRNA如何调控TAM极化的机制迄今不明。我们前期发现口腔鳞癌（OSCC）组织中M2型TAM增多与不良预后相关；测序发现LINC01181在OSCC组织中异常高表达；且OSCC细胞来源exosome中富含该LncRNA。生物信息学分析预测LINC01181可作为ceRNA 结合miR-27a上调M2极化相关重要基因PPARγ和IRF4。故推测该LncRNA可经exosome传输至TAM调控miR-27a/PPARγ&IRF4轴，诱导TAM向M2极化，塑造免疫抑制微环境。本项目拟在临床样本中确认LINC01181与OSCC进程及TAM极化的相关性；细胞和动物水平分析该LncRNA对TAM极化和功能的影响；揭示该LncRNA调控TAM极化的分子机制，为OSCC的诊断和治疗提供新靶标。

在口腔鳞状细胞癌（OSCC）中，肿瘤细胞、免疫细胞、成纤维细胞以及其它胞外成分构成的肿瘤微环境掣肘肿瘤治疗，探明微环境中细胞间相互作用的成分和机制，为OSCC的诊断和治疗提供新靶标仍是亟待解决的关键任务之一。因此，本项目围绕OSCC细胞、肿瘤相关巨噬细胞（TAMs）、肿瘤相关成纤维细胞（CAFs）及其长链非编码RNAs，分别从临床标本、细胞、分子和动物水平开展了以下几方面研究：1.探讨了OSCC细胞产生的LncRNA-LINC01181对巨噬细胞极化的调控作用；2. 分析了OSCC患者外周血中单核细胞（巨噬细胞前体）不同亚型的比例及临床意义；3. 研究了OSCC来源成纤维细胞（CAFs）中异常高表达的LncRNA-LOC100506114对OSCC生长和迁移的作用及机制。研究结果证实：1. OSCC肿瘤组织中LINC01181等几种LncRNA的表达水平显著高于对应的癌旁组织，且肿瘤细胞产生的LINC01181可通过外泌体传递给巨噬细胞，促进巨噬细胞向M2型极化。2. OSCC患者外周血单核细胞中一种能分化成M2型巨噬细胞的中间型单核细胞（CD14++CD16+）的比例显著升高，与OSCC临床病理参数的相关性分析发现，其与OSCC的侵袭性呈正相关；且ROC曲线分析显示，这群细胞比例对于区分OSCC和健康人具有一定的诊断意义。3.LncRNA- LOC100506114在CAFs中的表达水平显著高于正常成纤维细胞，可促进CAFs表型，进而促进OSCC增殖和迁移，其机制是LOC100506114通过靶向Runx2促进CAFs表达GDF10，进而激活肿瘤细胞的TGFβR1/Smad3/ERK通路。这些研究结果为OSCC的诊断和治理提供了潜在的新分子靶标。