TEAD4新靶RBM8A通过IGF1R-PI3K-AKT和Wnt通路调节乳腺癌生长转移

RBM8A is a kind of RNA-binding protein (RBP) that participates in RNA shearing and transporting. However, the role and the molecular mechanisms of RBM8A in breast cancer remain unknown. Our previous TCGA data and bioinformatic analysis showed that RBM8A and TEAD4 were over-expressed in breast cancer tissues, and a significant positive correlation was identified between them. TEAD4 could bind to the promoter region of RBM8A, and silencing of TEAD4 led to the down-regulation of RBM8A. RIP-seq data suggested that RBM8A involved in IGFIR-AKT and Wnt signaling pathways. Based on these findings, we suppose that TEAD4 regulates breast cancer growth and metastasis through activation of IGFIR-PI3K-AKT and Wnt signaling pathways by upregulating RBM8A expression. In the present study, firstly, the clinical significance of TEAD4 and TEAD4 in breast cancer is analyzed by using immunohistochemical, immunoblotting and quantitive real-time PCR analysis. Then, RNAi, ChIP, RIP, Pull-down, dual-luciferase reporter assay, cellular and molecular biological technique are applied to explore the network regulation mechanisms of RBM8A on the mature and transport of mRNA in breast cancer, and authenticate TEAD4 targeting regulation of RBM8A expression. To use slow virus pack LV-sh-RBM8A and LV-sh-TEAD4 expression vector, then, we will transfect breast cancer cell line, construct permanent cell line, establish bearing cancer hairless mouse model, dynamic state detect the size and metaptosis of tumor through small animal living body imaging system. Finally, the influences of TEAD4-RBM8A-IGFIR-PI3K-AKT/Wnt signaling pathways are verified in vivo. These findings will provide a novel perspective on diagnosis and treatment of breast cancer.

RBM8A是一种参与RNA剪切转运的RNA结合蛋白，在乳腺癌中的作用及调控机制不明。前期工作结合生物信息学分析表明RBM8A和TEAD4在乳腺癌中高表达，并呈正相关；TEAD4在RBM8A启动子区有结合位点，其沉默导致RBM8A下调；RIP-seq结果提示RBM8A参与IGF1R-AKT和Wnt通路的调节，提出“TEAD4调节RBM8A表达，促进IGF1R-AKT和Wnt通路激活，参与乳腺癌生长转移”的假说。本研究拟收集乳腺癌组织样本分析TEAD4和RBM8A表达的临床意义；应用RNAi、ChIP、RIP、Pull-down、荧光报告基因以及常规细胞和分子生物学技术，分析RBM8A对乳腺癌mRNA成熟与转运的网络调控；TEAD4对RBM8A靶向调控。构建TEAD4或RBM8A沉默细胞系，建立荷瘤动物模型，进行在体验证。研究结果将为RBM8A在乳腺癌诊断与治疗中的应用提供新视角。

RBM8A是一种参与RNA剪切转运的RNA结合蛋白，在乳腺癌中的作用及调控机制不明。本研究前期通过收集临床样本进行检测显示RBM8A在乳腺癌组织中高表达，通过生物信息学分析表明RBM8A和TEAD4在乳腺癌中高表达，并呈正相关；通过细胞增殖，流式细胞术、划痕实验、transwell及荷瘤小鼠实验验证RBM8A和TEAD4促进乳腺癌生长及迁移的生物学现象；并且通过RNAi、ChIP、RIP、Pull-down、荧光报告基因以及常规细胞和分子生物学技术，验证TEAD4可以结合在RBM8A的启动子区促进其转录；此外，RBM8A与EIF4A3形成复合体并IGF1-R和IRS-2 mRNA调控两者翻译；为了进一步验证RBM8A/EIF4A3的生物学效应，本研究通过MTT实验、流式细胞术、划痕沉默EIF4A3显著抑制乳腺癌细胞的增殖，及迁移。本研究通过以上实验证明TEAD4调节RBM8A表达，RBM8A/EIF4A3复合物促进IGF1-R和IRS翻译，参与乳腺癌生长转移。研究结果将为RBM8A在乳腺癌诊断与治疗中的应用提供新视角。