肿瘤细胞释放外泌体miRNA向微环境CAF传递自噬表型促进肺腺癌侵袭转移的机制研究

Autophagy in tumor and microenvironment is still need to be clarified. We have found: ①Autophagy in CAFs and Lung adenocarcinoma (LC) cells was positively related, high autophagy in both CAFs and LC was associated with poor survival.②High-content image revealed autophagy of CAFs was increased when co-cultured with LC cell line, LC-facilitated CAFs autophagy upregulation was reduced after blocking LC autophagy. ③Bioinformatic analysis revealed high levels of miR155 and miR19b in exosomes isolated from LC. Functional studies showed that miR155 and miR19b was transferred from LC to CAFs, where they conferred autophagy by co-binding to multiple mTOR signaling nodes, including RHEB and RICTOR. We therefore hypothesized that miR155/19b delivered by exosomes derived from LC to confer autophagy in neighboring CAFs, which further promote LUAD metastasis and invasion. The current project aims to assess the relationship between autophagy conferred from LC to neighboring CAFs and aggressive phenotype in LUAD cells. Secondly, the mechanism of exosomal miRNAs in autophagy confer between LC and CAFs will be further demonstrated in over-expression/knockdown manners. Finally, the correlation between exosomal miR155/19b-RHEB/RICTOR-autophagy pathway and LUAD progression will be further evaluated in clinical samples. To date, no data is available about the function and mechanisms of autophagy transmission in tumor microenvironment promoting LUAD progression. In summary, our study is the original source of innovation and can provide potential biomarkers for the prevention and treatment of LUAD.

肿瘤与微环境细胞两者自噬的关系尚不清楚。前期发现：①临床组织芯片中肿瘤相关成纤维细胞（CAF）与肺腺癌（LC）自噬水平显著正相关，CAF及LC自噬水平均高者预后最差；②共培养及高内涵成像示：CAF自噬可促进LC侵袭转移，LC自噬可促进CAF自噬，沉默LC自噬相关基因则CAF自噬降低；③生信分析及实验发现LC上清外泌体中miR155/19b显著富集，可靶向CAF中自噬抑制基因RHEB、RICTOR。故提出LC可通过释放含miR155/19b的外泌体向CAF传递自噬表型，进而促进自身侵袭转移的科学假说。本项目拟体内外证实LC可向CAF传递自噬表型；以外泌体miRNA为线索，阐明LC向CAF传递自噬表型的机制；临床评价LC及CAF自噬、外泌体miRNA及其靶基因与LC侵袭转移的相关性。有关LC向CAF传递自噬表型促进自身侵袭转移的现象与机制未见报道，本研究可为LC防治提供新思路。

肿瘤细胞自噬与肿瘤发生、发展密切相关，但肿瘤微环境细胞自噬与肿瘤的关系尚不清楚。本研究发现营养缺乏条件下，肿瘤细胞可向微环境肿瘤相关成纤维细胞（CAFs）传递自噬以维持其在低营养条件下的增殖与侵袭迁移。结合临床样本我们发现肺腺癌细胞及肿瘤相关成纤维细胞（CAFs）自噬水平正相关，与淋巴结分期正相关，癌细胞与CAFs自噬均高表达预后差。体外实验发现葡萄糖饥饿诱导的高自噬肺腺癌细胞株A549可释放外泌体至CAFs，并时间依赖性增加CAFs自噬, CAFs分泌IL-6/IL-8，增强肺腺癌侵袭转移。 circRNA表达谱芯片结合转录组测序筛选获得肺腺癌外泌体内circ008891可传递至CAFs，绑定ANXA2，抑制其入核，从而促进ANXA2介导的ATG16L+吞噬体的生成及自噬形成。本研究结果提示肺腺癌细胞向CAFs传递自噬是肿瘤细胞应对营养缺乏的有效策略，且circ008891与肺腺癌预后负相关，靶向circ008891抑制自噬传递是潜在的肺腺治疗靶点。