CAF窖蛋白-1下调在乳腺癌迁移运动中的作用及其调控机制研究

Amounts of clinical studies reveal that caveolin 1 (Cav1) of tumor stromal fibroblasts as cancer associated fibroblasts (CAFs) is an important biological marker for the prognosis of patients with breast cancer. Our previous research also confirmed that when fibroblasts and breast cancer cells were co-cultured, the downregulation of cav1 in fibroblasts could promote the expression of growth factors in fibroblasts, thereby promoting the proliferation and anti-apoptotic characteristics of tumor cells. Based on practical clinical questions and preliminary research results, we will further study the effects of CAFs on tumor cells invasion and migration and the interaction between CAFs and tumor cells. Primary cav1+ and cav1- CAFs cells from patients with breast cancer and human breast cancer cell MDA-MB435 will be co-cultured in 3D model. Transwell assays, real-time quantitative PCR, western blot are to be implemented. The immunofluorescence laser confocal microscopy, kinase activity experiments will be used to observe microstructures related to tumor cells migration, as well as clarifying the molecular pathway. In addition, we will adopt high throughput sequencing to detect discrepant expressed lncRNAs related to cav1 level in CAFs and then reveal their internal regulatory mechanism. Aimed at breast cancer patients with cav1- CAFs, this study is to investigate the mechanism of CAFs and tumor cells promoting malignant progression of cancer, which will provide evidence for precise treatment of breast cancer.

临床发现，肿瘤基质成纤维细胞（CAFs）窖蛋白1（Cav1）与乳腺癌患者预后有关。我们前期研究发现在成纤维细胞和乳腺癌细胞共培养中，成纤维细胞Cav1下调可促进肿瘤细胞的增殖和抗凋亡特性。在此基础上，本课题将构建乳腺癌患者Cav1阳性和阴性的CAFs和人乳腺癌细胞3D共培养模型；采用PCR、Transwell实验、激酶活性实验、Western blot、免疫荧光激光共聚焦观察等方法，研究CAFs Cav1对肿瘤细胞侵袭、迁移的作用及肿瘤细胞对CAFs的反馈调控、观察EMT和细胞运动相关超微结构变化，并阐明其分子通路。另外，将研究lncRNA分子NKILA在CAFs中对Cav1的调控和采用高通量测序检测CAFs中与Cav1相关差异表达的lncRNA并揭示其内在调控机制。本课题着眼于CAFs Cav1对乳腺癌微环境的影响，研究CAFs与肿瘤细胞在肿瘤恶性进展中的机制，为乳腺癌的精准治疗提供依据

乳腺癌是女性最常见的恶性肿瘤，发病率高，侵袭性强，极易转移和复发，给女性健康和生活质量造成了严重危害。窖蛋白-1（CAV-1）是细胞膜上重要的功能蛋白，参与包括细胞周期、细胞增殖及信号传导等多种细胞活动。研究表明，乳腺癌组织中原发性的CAV-1表达缺失与患者的临床分期和预后密切相关。而成纤维细胞作为组织间质中最常见的细胞，能调节分泌多种细胞因子（如TGF-β）重塑肿瘤微环境，促进乳腺癌的进一步发展。本项目从两方面进行研究，一方面探讨肿瘤微环境成纤维细胞中CAV-1的下调对乳腺癌细胞TGF-β1/Smad信号通路及其侵袭、转移和细胞干性的影响；另外通过生物信息学分析与基础实验结合提出一种新机制：lncRNA HOTAIR/miR-203/CAV1 轴促进乳腺癌细胞的增殖、迁移和侵袭。项目通过收集临床癌组织标本，检测乳腺癌旁组织与乳腺癌组织中、成纤维细胞ESF-1与乳腺癌细胞MDA-MB-231、MCF-7中的CAV-1 、EMT标记蛋白和细胞干性标记蛋白的表达水平，构建乳腺癌细胞3D共培养模型，采用PCR、流式细胞术、Western blot、干细胞成球实验、免疫荧光等多种实验方法，发现乳腺癌旁组织中CAV-1表达量明显高于癌组织，TGF-β表达量、EMT标记蛋白和细胞干性标记蛋白表达量明显低于乳腺癌组织；发生淋巴结转移的乳腺癌间质组织中CAV-1表达量明显低于未发生淋巴结转移的乳腺癌间质组织；成纤维细胞中CAV-1表达下调能够促进其分泌TGF-β1，激活乳腺癌细胞TGF-β1/Smad信号通路，增强乳腺癌细胞干性以及EMT过程，促进乳腺癌细胞的侵袭和迁移；并且发现LncRNA HOTAIR通过行使竞争性内源性 RNA（ceRNA）的功能，与miR-203结合以此下调CAV1 mRNA 的表达，进而影响乳腺癌细胞的增殖、迁移和侵袭。本项目为乳腺癌创新分子诊疗靶点提供了理论和实践基础。