Ouyang H, Gore J, Deitz S, Korc M
Departments of Medicine, Biochemistry and Molecular Biology, Indiana University School of Medicine, the Melvin and Bren Simon Cancer Center and the Pancreatic Cancer Signature Center, Indianapolis, IN, USA.
Oncogene. 2014 Sep 18;33(38):4664-74. doi: 10.1038/onc.2013.405. Epub 2013 Oct 7.
Increased microRNA-10b (miR-10b) expression in the cancer cells in pancreatic ductal adenocarcinoma (PDAC) is a marker of disease aggressiveness. In the present study, we determined that plasma miR-10b levels are significantly increased in PDAC patients by comparison with normal controls. By gene profiling, we identified potential targets downregulated by miR-10b, including Tat-interacting protein 30 (TIP30). Immunoblotting and luciferase reporter assays confirmed that TIP30 was a direct miR-10b target. Downregulation of TIP30 by miR-10b or siRNA-mediated silencing of TIP30 enhanced epidermal growth factor (EGF)-dependent invasion. The actions of miR-10b were abrogated by expressing a modified TIP30 cDNA resistant to miR-10b. EGF-induced EGF receptor (EGFR) tyrosine phosphorylation and extracellular signal-regulated kinase phosphorylation were enhanced by miR-10b, and these effects were mimicked by TIP30 silencing. The actions of EGF in the presence of miR-10b were blocked by EGFR kinase inhibition with erlotinib and by dual inhibition of PI3K (phosphatidylinositol 3'-kinase) and MEK. Moreover, miR-10b, EGF and transforming growth factor-beta (TGF-β) combined to markedly increase cell invasion, and this effect was blocked by the combination of erlotinib and SB505124, a type I TGF-β receptor inhibitor. miR-10b also enhanced the stimulatory effects of EGF and TGF-β on cell migration and epithelial-mesenchymal transition (EMT) and decreased the expression of RAP2A, EPHB2, KLF4 and NF1. Moreover, miR-10b overexpression accelerated pancreatic cancer cell (PCC) proliferation and tumor growth in an orthotopic model. Thus, plasma miR-10b levels may serve as a diagnostic marker in PDAC, whereas intra-tumoral miR-10b promotes PCC proliferation and invasion by suppressing TIP30, which enhances EGFR signaling, facilitates EGF-TGF-β cross-talk and enhances the expression of EMT-promoting genes, whereas decreasing the expression of several metastasis-suppressing genes. Therefore, therapeutic targeting of miR-10b in PDAC may interrupt growth-promoting deleterious EGF-TGF-β interactions and antagonize the metastatic process at various levels.
胰腺导管腺癌（PDAC）癌细胞中微小RNA-10b（miR-10b）表达增加是疾病侵袭性的一个标志。在本研究中，我们确定与正常对照相比，PDAC患者血浆miR-10b水平显著升高。通过基因谱分析，我们鉴定出被miR-10b下调的潜在靶点，包括Tat相互作用蛋白30（TIP30）。免疫印迹和荧光素酶报告基因检测证实TIP30是miR-10b的直接靶点。miR-10b下调TIP30或siRNA介导的TIP30沉默增强了表皮生长因子（EGF）依赖性侵袭。通过表达对miR-10b有抗性的修饰TIP30 cDNA可消除miR-10b的作用。miR-10b增强了EGF诱导的表皮生长因子受体（EGFR）酪氨酸磷酸化和细胞外信号调节激酶磷酸化，TIP30沉默也模拟了这些效应。在存在miR-10b的情况下，EGF的作用被厄洛替尼抑制EGFR激酶以及双重抑制PI3K（磷脂酰肌醇3'-激酶）和MEK所阻断。此外，miR-10b、EGF和转化生长因子-β（TGF-β）共同作用显著增加细胞侵袭，这种效应被厄洛替尼和I型TGF-β受体抑制剂SB505124联合阻断。miR-10b还增强了EGF和TGF-β对细胞迁移和上皮-间质转化（EMT）的刺激作用，并降低了RAP2A、EPHB2、KLF4和NF1的表达。此外，在原位模型中，miR-10b过表达加速了胰腺癌细胞（PCC）增殖和肿瘤生长。因此，血浆miR-10b水平可能作为PDAC的诊断标志物，而肿瘤内miR-10b通过抑制TIP30促进PCC增殖和侵袭，TIP30可增强EGFR信号传导、促进EGF-TGF-β串扰并增强促进EMT的基因表达，同时降低几种转移抑制基因的表达。因此，在PDAC中靶向miR-10b进行治疗可能会中断促进生长有害的EGF-TGF-β相互作用，并在多个层面拮抗转移过程。
