miRNA-200s/205对乳癌CAF活化和活性维持促进ECM重塑及癌细胞增殖侵袭的机制研究

Cancer-associated fibroblasts (CAFs) are a major member in the tumor stroma and play an important role in the tumor development and metastasis. The activity, especially its maintenance of CAFs is a key for understanding CAFs’ functions. Our previous studies found that miR-200 family and miR-205 are down-regulated in breast cancer CAFs compared with NFs by miRNA-array, and those miRNAs possibly play a role in the activation of CAFs and ECM remodeling. Some of important transcriptors (TCF4、TCF12、Fli-1、KLF12) may associated with CAFs’ function. Here, we hypothesis that miR-200s/205 activate and maintain the activity of CAFs to remodel the ECM and enhance tumor growth and invasion. The activity of CAFs and the relationship between miRNAs and their targets will be detected under either the overexpression and siRNA silence of miR-200s/205 or their key targets. The remodeling of ECM will be checked by 3D co-culture, fibroblast contraction assay, and Picric acid Sirius red staining. The tumor cells proliferation and invasion will be analysis by 3D co-culture, nude mouse, Transwell assay. Our results will disclose a novel potential mechanism of miR-200s/205 in activation and maintenance of CAFs, remodeling of ECM and promoting development of breast cancer.

CAFs是肿瘤微环境的最主要成员，对肿瘤发展、远处转移产生重要影响。研究CAFs的活化、活性维持是揭示CAFs功能的关键。前期工作发现乳癌CAFs中miR-200s/205明显下调，关键靶转录因子TCF4、TCF12、Fli-1、KLF12可能与CAF功能有关，且CAFs存在ECM基因异常。故，本项目提出miR-200s/205调控TCF4等关键转录因子、促进并维持CAFs活性、参与ECM重塑，推动乳腺癌细胞增殖和侵袭。拟采用过表达和干扰技术明确miR-200s/205与靶基因正反调控关系、维持CAFs活性；3D共培养、胶收缩、ECM成分分析、天狼星红染色等研究miR-200s/205及靶基因参与ECM重塑；3D共培养、裸鼠成瘤、Transwell等研究ECM对不同亚型乳癌细胞增殖、浸润。研究结果将揭示miR-200s/205对乳癌CAFs活性维持、ECM重塑、促肿瘤发展新机制

经过3年来艰辛工作，圆满完成了本课题的研究内容，取得了丰硕的成果，主要成果总结如下：1. 揭示miR-200s家族在CAF中普遍下调，是CAF活化的启动因素； miR-200的靶基因Fli-1和TCF-12这两个与细胞分化密切相关的转录因子，直接参与了CAF的活化调控。miR-200s与靶基因Fli-1和TCF-12协同直接或间接上调与ECM重建相关的纤连蛋白（FN）、赖氨酰氧化酶编码基因（FOX）的表达，参与ECM的重构，从而引起癌细胞在体外、体内环境中的侵袭、转移。2. 发现miR-205在CAF普遍下调，是CAF活化的另一个重要miRNA；miR-205通过直接靶向YAP1，上调IL11/LI15的表达，促进VEGF-非依赖的肿瘤血管形成，对癌细胞的侵袭转移起调控作用。3. 揭示miR-200b/c在癌前病变非典型增生组织来源的成纤维细胞（AHFs, atypical ductal hyperplasia associated fibroblasts）低表达，诱导了乳腺癌癌前病变组织成纤维细胞活化；miR-200b/c靶向IKKb促进NF-kB信号的活化，上调PAI-1(Plasminogen activator Inhibitor-1)的异常表达，从而在MCF-7细胞模型中促进癌细胞增殖与极性改变对。这一研究首次揭示肿瘤微环境在乳腺癌起始与早期发展中的重要作用。.本项目已发表SCI收录论文3篇，IF值全部>5.0，其中2篇论文IF>8.0。已协助培养博士研究生2名。