硒蛋白P抑制CAFs促进乳腺癌细胞转移的作用及其机制研究

As one of the crucial cellular components of tumor microenvironment, cancer-associated fibroblasts（CAFs）are not only involved in microvascular and lymphatic vessel formation, but also in extracellular matrix remodeling, as well as in promoting the proliferation, invasion and metastasis of cancer cells. Selenoproteins P (SEPP1) belongs to a class of proteins mediating metabolism, many of which have been shown to have redox functions, acting as antioxidants to decrease oxidative stress. Our previous studies have found that the expression of SEPP1 in CAFs was lower than that in normal fibroblasts Whirly. Using siRNA technology to suppress the expression of SEPP1 in Whirly can increase the migration of tumor cells cocultured with the SEPP1 downregulated Whirly, also Upregulateing PDGF-bb expression in CAFs induce the SEPP1 downregulated. Therefore, we suppose that SEPP1 was highlikely associated with the invasion and metastasis of breast cancer in tissues. However, the function and mechanism of SEPP1 in regulating the interactions between CAFs and breast cancer cells in tumor microenvironment are not clear. The aim of this study is to establish an knockout mice and zebrafish model of nornal fibroblasts, CAFs and breast cancer cells in vivo and in vitro, to explore the regulatory roles of EMT and PDGF-BB/PDGFRβ signaling, and to elucidate the mechanisms how SEPP1 inhibit cancer metastasis, Provided theoretical evidence for clinical diagnosis and treatment of breast cancer.

肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs)是肿瘤微环境重要成分之一，能促进癌细胞增殖、侵袭和转移。硒蛋白P（selenoprotein P, SEPP1）是一类具有代谢功能的蛋白质，参与氧化还原作用，然而该蛋白在肿瘤方面的作用不明。本研究组发现CAFs的SEPP1表达明显低于正常纤维细胞Whirly，下调Whirly的SEPP1表达后，共培养的肿瘤细胞迁移增加，上调PDGF-BB表达可使SEPP1表达下调。由此可见，SEPP1可能与乳腺癌的侵袭和转移密切相关，然而SEPP1在调控CAFs促进乳腺癌细胞转移机制尚不清楚。本研究拟通过基因敲除鼠和斑马鱼模型，探索SEPP1对EMT的作用以及PDGF-BB/PDGFRβ通路的影响，阐明SEPP1抑制CAFs促进肿瘤迁移的作用机制，为临床上诊治乳腺癌提供新的理论依据。

硒蛋白P（selenoprotein P, SEPP1）是一类具有代谢功能的蛋白质，参与氧化还原作用，然而该蛋白在肿瘤方面的作用不明。本研究组发现肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs)的SEPP1表达明显低于正常纤维细胞Whirly，下调Whirly的SEPP1表达后，共培养的肿瘤细胞迁移增加，上调PDGF-BB表达可使SEPP1表达下调。由此可见，SEPP1可能与乳腺癌的侵袭和转移密切相关，然而SEPP1在调控CAFs促进乳腺癌细胞转移机制尚不清楚。本研究拟通过基因敲除鼠和斑马鱼模型，探索SEPP1对EMT的作用以及PDGF-BB/PDGFRβ通路的影，阐明SEPP1抑制CAFs促进肿瘤迁移的作用机制，为临床上诊治乳腺癌提供新的理论依据。通过基础研究和临床标本相关检测对其SEPP1调控抑制肿瘤转移的机制有了重要发现：（1）我们通过斑马鱼模型以及体外试验证实敲除SEPP1后成纤维细胞对肿瘤迁移的影响发生改变，应用SEPP1基因敲除鼠模型进一步证实了肿瘤的侵袭转移的发生是与SEPP1的下降密切相关。（2）进一步我们通过生信分析和测序发现SEPP1可能与调控aPOER2的一个名为REELIN的蛋白相互竞争影响aPOER2下游通路的激活，从而影响肿瘤发生转移。我们通过shRNA下调肿瘤细胞中LRP8（apOER2）的表达，REELIN刺激肿瘤转移的作用会减少。（3）我们分别用不同的CAFs分泌促进肿瘤转移的蛋白TGF-β，TGF-2，PDGF-BB刺激成纤维细胞，发现SEPP1的表达亦会随之减少，同时REELIN表达升高。提示SEPP1和REELIN之间可能存在相互作用，进一步通过蛋白互作作用证实其两者相关性。（4）通过蛋白结构分析和western blot分析证实，我们看到REELIN和SEPP1相互作用可能通过Reelin-Dab1-cofilin/WASP轴参与其中有关。（5）我们通过使用生物提纯硒蛋白可以在一定程度上逆转由于PDGF促进SEPP1下调所致的肿瘤转移增加。（6）在临床标本中同样也看到了SEPP1表达下降与肿瘤预后不良，复发转移有着密切相关。提示SEPP1可能是与预后相关的一个重要标志物。同时结合前期研究有望应用于临床。