miR-671-5p/FGFR2/uPA通路在食管鳞癌肿瘤相关成纤维细胞中的作用机制研究

FGFR2 plays an essential role in growth and development. High expression level of FGFR2 was found in some tumors, indicating that FGFR2 was associated with tumor development and progression. High expression level of FGFR2 was found in cancer-associated fibroblasts (CAFs), but not in tumor-surrounding stromal tissues of Esophageal carcinoma (ESCC), making it a CAF-specific marker. But there is no detailed knowledge about why and how FGFR2 is overespressed. In our previous study, we found that miR-671-5p directly bound to 3'UTR of FGFR2, leading to FGFR2 mRNA degradation. Antibody array indicated that expression levels of some secreted proteins, including Activin A, IFN-gamma and uPA, were significantly higher in the supernantant of FGFR2-positive CAFs than in NFs (normal fibroblasts). uPA was shown higher expression level in stably FGFR2-expressing cells (293A-FGFR2) than in empty vector transfected cells (293A), suggesting uPA might be an downstream protein of FGFR2. In the study, we committed to confirm the relationship between FGFR2 and miR-671-5p, through analyzing activity of 3'-UTR of FGFR2, and influence of miR-671-5p on ESCC cells; confirm the correlation of FGFR2 and uPA, by detecting expression and phosphorylation of key proteins in FGFR2 and uPA signaling networks. Thus, how miR-671-5p-FGFR2-uPA axis provides a suitable microenvironment for tumor development and progression in ESCC. Based on our study, we will provide further knowledge on functions of FGFR2 and relationship between FGFR2 and ESCC progression, supporting for future development of anticancer drugs.

FGFR2与肿瘤的发生发展重要相关。它被发现是食管鳞癌肿瘤相关成纤维细胞（CAF）的标志蛋白，但相关分子调控机制却没有进一步报道。我们前期研究已发现FGFR2的3'UTR是miR-167-5p的靶向序列，且CAF比癌旁的正常成纤维细胞更多地分泌ActivinA、IFN-γ和uPA等，并发现过表达FGFRR2的293细胞模型中uPA的表达水平显著上调，提示uPA可能位于FGFR2的下游。本研究将分析FGFR2的3'UTR中特定序列的活性，并在细胞中过表达和干扰miR-167-5p，明确二者的调控关系；检测并抑制uPA及FGFR2相关通路，分析关键节点蛋白的作用，探讨二者的调控机制；并深入研究FGFR2是如何经由miRNA分子和蛋白因子促进了食管鳞癌的恶化，分析其分子作用机制。本课题的新发现将进一步解析FGFR2与食管鳞癌发生的关系，获得FGFR2分子调控和作用的新知识，补充肿瘤发生机制。

成纤维细胞生长因子受体2（FGFR2）与肿瘤的发生发展重要相关。它被发现是食管鳞癌肿瘤相关成纤维细胞（Cancer-associated fibroblasts ,CAF）的标志蛋白。本项目前期研究已发现FGFR2的3'UTR含有miR-167-5p的靶向序列，且CAF比癌旁的正常成纤维细胞更多地分泌uPA等蛋白，并发现过表达FGFRR2的293细胞模型中uPA的表达水平显著上调，提示uPA可能位于FGFR2的下游，故本项目研究miR-671-5p、FGFR2及uPA之间的相互调控关系及作用机制。结果发现，除CAF高表达uPA外，其条件培养基也显著促进癌细胞生长；类似的，FGFR2高表达的293细胞模型中，uPA表达水平显著上调并且具有明显促癌作用；同时临床数据分析结果显示肿瘤基质中uPA的阳性表达与ESCC病人的肿瘤侵润程度和总生存率显著相关；信号通路检测发现uPA依赖于PI3K-AKT和ERK信号通路促进ESCC细胞的增殖、侵袭和转移，抑制这两条通路也会明显抑制细胞的上述现象；相应的，在移植瘤裸鼠模型中发现使用uPA抗体能显著抑制在体瘤的生长。另一方面，通过双荧光报告系统发现miR-671-5p确实能靶向FGFR2的3’-UTR并调控其表达；且双变量分析发现miR-671-5p在大多数FGFR2阳性ESCC临床组织样品中低水平表达；临床ESCC-FGFR2组织中通过miRNA芯片分析和QPCR验证发现miR-671-5p的表达确实较低；过表达miR-671-5p下调FGFR2的表达并显著抑制癌细胞生长，而干扰miR-671-5p能上调FGFR2的表达并显著促生长;同样的，过表达miR-671-5p抑制裸鼠移植瘤的生长，反之则显著促进瘤体生长；信号通路检测发现miR-671-5p通过靶向FGFR2降低其磷酸化从而抑制PI3K-AKT和ERK信号通路。总而言之，研究揭示了miR-671-5p靶向FGFR2并影响下游uPA蛋白的表达水平进而影响PI3K-AKT和ERK信号通路最终引起食管鳞癌发生发展的变化的分子作用机制。上述结果也补充了FGFR2的分子调控和作用机制，为进一步解析食管鳞癌的发病机制及其治疗提供了有价值的理论依据。