腹膜微环境改变诱导胃癌细胞表型重塑促进胃癌腹膜转移的机制研究

Peritoneal metastasis is the result of cross-talking between peritoneal free cancer cells and peritoneal microenvironment. Our previous studies revealed that cytokines including TGF-β1 secreted by peritoneal cancer cells recruit and activate peritoneal macrophages to M2 tumor-associated macrophages, which induce a series of pathological changes, including mesothelial-fibrosis transformation and apoptosis of peritoneal mesothelial cells, exposure of submesothelial connective tissue, and angiogenesis, by increasing expression of TGF-β1, IL-10, VEGF and MMPs. All these changes result in the enhancement of adhesion and invasion of peritoneal free cancer cells to peritoneum, and promoting the development of peritoneal metastasis. In addition, free cancer cells in the alterative peritoneal microenvironment experience phenotype remodeling, which characterized by the enhancement of the ability of adhesion and invasion, epithelial-mesenchymal transition (EMT), and the enhancement of "cancer stem cell" characteristics. Whereas, miR-139 downregulation in cancer cells may be involved in the procedure of phenotype remodeling of free cancer cells. The aim of the current study is to further validate the effect of the alterative peritoneal microenvironment in inducing downregulation of miR-139 expression in free cancer cells and the consequence of remodeling cell phenotype and promoting peritoneal metastasis, moreover, to clarify the upstream and downstream molecules signaling pathways involves in the procedure of phenotype remodeling correlated with miR-139. The results of the present study will be valuable for further investigations of effective prevention and treatment of peritoneal metastasis for gastric caners.

胃癌腹膜转移是腹腔游离癌细胞与腹膜微环境双向互动的结果。申请者前期发现，癌细胞分泌的以TGF-β1为主的细胞因子募集并活化腹腔巨噬细胞为M2型肿瘤相关巨噬细胞，后者通过高表达TGF-β1、IL-10 、VEGF、MMPs等细胞因子一方面诱导间皮细胞成纤维转化及凋亡、间皮下结缔组织暴露、腹膜纤维化，一方面诱导血管生成，促进了癌细胞粘附侵袭及腹膜转移的形成。此外，癌细胞在改变的腹膜微环境中发生了表型重塑，其特点为粘附侵袭能力增强、EMT改变及肿瘤细胞"干性"的强化。其中，miR-139在癌细胞中表达下调可能参与了上述过程。本项目拟进一步明确腹膜微环境改变诱导腹腔游离癌细胞miR-139表达异常，导致细胞表型重塑及促进腹膜转移的作用，并深入探讨miR-139调控癌细胞表型重塑的上、下游分子信号通路。研究结果将为腹膜转移防治提供新的思路与靶点。

本项目重点探讨了在改变的腹膜微环境中，腹腔游离癌细胞以粘附侵袭能力增强及“干性”强化为特点的表型重塑的分子机制。研究发现，腹膜转移阳性患者腹水可以诱导原代培养胃癌原发灶癌细胞多种粘附侵袭、EMT及“干性”相关标志物表达上调，癌细胞粘附侵袭能力增强，发生一定程度的“干性”转化；癌细胞miR-148-3p/miR-152-3p低表达可能是上述改变的分子机制。上调miR-148-3p/miR-152-3p表达，可以诱导MKN-45细胞多种粘附侵袭、EMT及“干性”标志物表达下调，降低癌细胞粘附侵袭能力。缺氧在胃癌腹膜转移过程中发挥了重要作用，可能是游离癌细胞“干性”强化的使动因素之一。缺氧条件下，HIF-1α在癌细胞中表达上调，与胃癌腹膜转移及患者预后密切相关。HIF-1α表达水平与干细胞标志物OCT4、Nestin表达呈正相关；下调癌细胞HIF-1α表达，可逆转癌细胞在缺氧条件下EMT及“干性”标志物表达的上调，并可逆转癌细胞在缺氧条件下粘附侵袭能力、侧群细胞比例、细胞成球能力的增强。生物信息学预测发现，HIF-1α启动子区域存在miR-148-3p/miR-152-3p靶向结合位点，该结果经表达相关性分析及荧光素酶报告基因实验证实。此外，lncRNA SNHG14可能通过ceRNA机制竞争性结合miR-148-3p/miR-152-3p，影响后者调控HIF-1α介导癌细胞粘附侵袭能力及“干性”强化的作用，该假设有待今后研究确认。上述研究成果从“种子”-“土壤”双向互动的“种子”层面对胃癌腹膜转移的机制进行了全新的解释，丰富了胃癌腹膜转移发生发展的理论研究，为胃癌腹膜转移的早期诊断或阻断治疗提供了新的靶点。