microRNA 调控网络介导的癌基因Src诱导乳腺癌细胞上皮-间质转化

Oncogene Src is frequently overexpressed in more than 50% breast cancer inducing uncontrolled cell proliferation, EMT and metastasis. The mechanisms remain unclear, and there is still no good way to treat Src-induced breast cancer. EMT is a key step for the cancer metastasis, which is regulated by miRNAs and transcriptional factors. Our preliminary work has successfully established a cell model for Src induction of EMT in breast epithelial cells. Based on our analysis on the cell model, we hypothesize it is the upregulation of miR-29a and downregulation of miR-205 that mediate at least partly the oncogenic function of Src. We found that miR-29a promotes cellular proliferation and migration in breast cancer, while miR-205 inhibits. Target gene analysis indicate miR-29a suppresses indirectly the expression of miR-200c through targeting Sp1, a transcriptional factor which is required to maintain miR-200c expression level in epithelial cells. As such, miR-29a promotes EMT; while miR-205 inhibits EMT through targeting ZEB1 and ZEB2, two activators of EMT. In this proposal we will demonstrate two regulatory networks, Src-miR29a-Sp1-miR200c-EMT and Src-miR205-ZEB1/ZEB2-EMT, for regulating Src-induced EMT through analysis on breast cancer cells, cancer stem cells, clinical samples and breast tumor animal model as well. Our purpose is to determine the mechanisms through which miR-29a and miR-205 regulate Src-induced breast cancer EMT and metastasis, providing experimental basis to develop new therapeutic approach for treating breast cancer by targeting EMT regulators.

Src在50%以上乳腺癌异常高表达，诱导癌细胞上皮-间质转化（EMT）及恶性转移，其机制尚不明确。EMT作为癌转移的关键，受miRNA及转录因子等调控。前期工作建立了Src诱导乳腺上皮细胞EMT的细胞模型。通过该细胞模型的间质特征研究和miRNA分析，提出假说：miR-29a特异上调及miR-205表达丢失可介导Src的致癌作用。靶基因分析提示miR-29a通过调控转录因子Sp1间接抑制miR-200c表达，促进EMT；miR-205通过抑制ZEB1/ZEB2表达，抑制EMT。本项目将通过乳腺癌细胞、癌干细胞、临床样本及荷瘤动物的分析，证实Src-miR29a-Sp1-miR200c-EMT及Src-miR205-ZEB1/ZEB2-EMT两条调控网络，阐明miR-29a及miR-205调控Src诱导的乳腺癌EMT和转移分子机制，为靶向EMT调控因子探索乳腺癌治疗新措施提供实验基础。

Src在50%以上乳腺癌异常高表达，Src 表达越高，乳腺癌患者疾病特异性生存期越短.研究证明，Src 基因的表达诱导癌细胞上皮-间质转化（EMT）及恶性转移，其机制尚不明确。EMT作为癌转移的关键，受miRNA及转录因子等调控。Src诱导的乳腺癌EMT及恶变，是否与非编码miRNA调控通路有关？带着这个问题，开展了本项目研究。首先建立的Src永生化的三阴性乳腺癌细胞模型MCF-10A-Src, 其次从细胞水平揭示了SRC促进细胞增殖，诱导EMT和细胞迁移、侵袭等生物学功能，在调控机制上通过miRNA芯片筛选到下游特异下调的miR-205、miR-200c以及特异上调的miR-29a，进一步功能学实验证明，这些miRNA及下游靶基因介导了Src促进三阴性乳腺癌发生和发展转移的作用，比如miR-29a-KLF4通路、miR-205-Notch通路均参与了三阴性乳腺癌干细胞及癌细胞转移的调控。miR-29a通过PTEN-AKT信号通路促进癌细胞转移，miR-205抑制乳腺癌细胞干性，同时抑制乳腺癌细胞迁移和侵袭能力。利用荷瘤动物模型、临床标本及TCGA数据库分析，验证了 Src及下游miRNA对乳腺癌的发生发展的特异调控功能和机制。最后，为了推进Src下游miRNA向临床医用的转化，我们自主设计并优化了一种miRNA抑制剂：微小RNA拉链，该结构能够高效敲低致病性miRNA，比如抑制miR-221/222，与化疗药联合使用，对抑制三阴性乳腺癌发生及发展，具有重要的协同效应。本项目研究成果，不仅揭示了SRC诱导三阴性乳腺癌发生及转移的的作用机制，更重要的是为抑制三阴性乳腺癌干细胞以及抑制三阴性乳腺癌细胞转移，提供了新的潜在治疗靶点和miRNA靶向抑制的新工具，具有临床应用前景。