TEMs来源的外泌体传递ceRNA调控上皮性卵巢癌微环境中CTSL1高表达促进腹膜转移的机制研究

Peritoneal dissemination metastasis involvement are main routes for tumor metastasis of epithelial ovarian cancer (EOC) in over 70% patients，thus, patients with peritoneal metastasis always end in poor prognosis. Previous research revealed that Tie2 expressing monocytes/macrophages (TEMs) were aberrantly higher infiltrated EOC tissue. Moreover, the increased TEMs in EOC promote tumor metastasis through angiogenesis. However, the transcript pattern of TEMs remains unclear. According to the microarray analysis, the lncRNA CTSL1P8 was significantly up-regulated in TEMs. Its homolog gene Cathepsin L1 (CTSL1) was also up-regulated by CTSL1P8 in a competing endogenous RNA (ceRNA)-liked manner. RT-PCR assay showed that the CTSL1P8 was further enriched in TEMs derived exosomes. Based on the preliminary results, in this study, we would focus on the pathway in which TEMs derived exosomes transfer CTSL1P8 to the targeting cells. The study would also reveal the mechanism by which CTSL1P8 served as a ceRNA that promotes the expression of CTSL1 and promote the peritoneal metastasis in EOC. By elucidating this process, the research might shed new lights to the mechanism study of EOC metastasis and provide new therapeutic target.

70%上皮性卵巢癌(EOC)患者确诊时已进展至晚期，表现为腹膜广泛转移，预后较差。课题组前期研究发现，酪氨酸激酶受体阳性单核/巨噬细胞(TEMs)在EOC微环境中浸润密度上升，并且通过促进血管再生参与EOC腹膜转移。然TEMs作用机制所依赖的具体基因组表达模式尚不明确。基因芯片筛选结果显示，TEMs中长链非编码RNA分子CTSL1P8表达升高，且与其同源基因，组织蛋白酶L1（CTSL1）之间存在类似竞争性内源（ceRNA）调控模式，进一步RT-PCR检测发现， CTSL1P8在TEMs来源的外泌体中富集。基于上述工作，本研究拟深入探索：CTSL1P8是否经TEMs来源外泌体在EOC微环境中传递，进入靶细胞后发挥分子海绵效应，通过ceRNA机制上调CTSL1表达，并最终促进EOC腹膜转移。对上述通路的阐明，将揭示TEMs促进EOC腹膜转移具体分子机制，并为EOC诊治新靶点提供重要理论依据。

上皮性卵巢癌（EOC）微环境中巨噬细胞对肿瘤进展及转移具有重要调控作用，项目前期发现一类EOC微环境中浸润增加的特殊类型巨噬细胞（Tie2+macrophages, TEMs）。TEMs具有特异性的RNA表达模式，并可能通过长链非编码RNA（lncRNA）调控EOC腹膜转移过程，然而这一通路中的具体分子机制尚不明确。.在本项目中，我们通过免疫荧光染色和流式细胞检测了TEMs在EOC肿瘤微环境、患者腹水以及外周血中表达数量，结果显示与良性卵巢囊肿患者相比，EOC肿瘤微环境及外周血中TEMs比例显著升高，且患者腹水中TEMs进一步富集。同时通过生存曲线分析发现，TEMs浸润增加与患者不良预后显著相关。体内外功能试验表明，TEMs通过激活IGF1-IGF1R及下游Erk1/2-Akt通路，促进血管新生，进而加速EOC腹膜转移。另一方面，我们利用基因过表达系统，直接验证了TEMs中表达升高的lncRNA分子CTSL1P8对EOC肿瘤细胞功能的调控作用，研究结果表明，CTSL1P8分子可直接促进EOC细胞增殖、迁移及侵袭转移能力。在具体分子机制的探索过程中，我们利用双荧光素酶报告检测发现，CTSL1P8分子通过ceRNA机制上调靶基因CTSL1分子的表达水平，这一发现提示lnc-CTSL1P8促进EOC转移可能是由表达升高CTSL1蛋白所介导。.本项目研究结果揭示，EOC腹膜微环境中特殊分子表型巨噬细胞可通过增加组织血管新生，为肿瘤转移提供有利条件。同时可通过长链非编码RNA分子直接调控EOC细胞自噬等功能通路，促进肿瘤增殖转移。因此，我们的研究结果为EOC腹膜转移过程阐明了新的分子机制通路，也为EOC诊治提供了新的干预靶点。