Department of Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
J Biol Chem. 2011 Jan 14;286(2):1429-35. doi: 10.1074/jbc.M110.146530. Epub 2010 Nov 8.
Tissue factor has been recognized as a regulator of tumor angiogenesis and metastasis. The tissue factor gene is selectively expressed in highly invasive breast cancer cells, and the mechanisms regulating tissue factor expression in these cells remain unclear. This study demonstrates that microRNA-19 (miR-19) regulates tissue factor expression in breast cancer cells, providing a molecular basis for the selective expression of the tissue factor gene. Tissue factor protein was barely detectable in MCF-7, T47D, and ZR-75-1 cells (less invasive breast lines) but was expressed at a significantly higher level in MDA-MB-231 and BT-20 cells (invasive breast lines) as assayed by Western blot. The tissue factor gene promoter was activated, and forced expression of tissue factor cDNA was achieved in MCF-7 cells, implying that the 3'-UTR of the tissue factor transcript is responsible for the suppression of tissue factor expression. Bioinformatics analysis predicted microRNA-binding sites for miR-19, miR-20, and miR-106b in the 3'-UTR of the tissue factor transcript. Reporter gene assay using the TF-3'-UTR luciferase reporter construct confirmed that the 3'-UTR negatively regulates gene expression in MCF-7 cells, an effect reversed by deletion of the miR-19-binding site. Application of the miR-19 inhibitor induces endogenous tissue factor expression in MCF-7 cells, and overexpression of miR-19 down-regulates tissue factor expression in MDA-MB-231 cells. RT-PCR analysis using cDNA made from Ago2-immunoprecipitated RNA samples confirmed that Ago2 binds preferentially to tissue factor 3'-UTR in MCF-7 cells, as compared with MDA-MB-231 cells, consistent with the observation that miR-19 levels are higher in MCF-7 cells.
组织因子已被认为是肿瘤血管生成和转移的调节剂。组织因子基因在高度侵袭性乳腺癌细胞中选择性表达，而这些细胞中调节组织因子表达的机制仍不清楚。本研究表明，microRNA-19（miR-19）调节乳腺癌细胞中的组织因子表达，为组织因子基因的选择性表达提供了分子基础。Western blot 检测结果显示，组织因子蛋白在 MCF-7、T47D 和 ZR-75-1 细胞（侵袭性较低的乳腺癌细胞系）中几乎检测不到，但在 MDA-MB-231 和 BT-20 细胞（侵袭性较高的乳腺癌细胞系）中表达水平显著升高。组织因子基因启动子被激活，并且在 MCF-7 细胞中实现了组织因子 cDNA 的强制表达，这表明组织因子转录本的 3'-UTR 负责抑制组织因子的表达。生物信息学分析预测了 miR-19、miR-20 和 miR-106b 在组织因子转录本 3'-UTR 中的结合位点。使用 TF-3'-UTR 荧光素酶报告基因构建体的报告基因检测证实，3'-UTR 负向调节 MCF-7 细胞中的基因表达，miR-19 结合位点缺失可逆转该效应。miR-19 抑制剂的应用可诱导 MCF-7 细胞中内源性组织因子表达，而过表达 miR-19 可下调 MDA-MB-231 细胞中的组织因子表达。使用 Ago2 免疫沉淀 RNA 样本进行的 RT-PCR 分析证实，与 MDA-MB-231 细胞相比，Ago2 更倾向于与 MCF-7 细胞中的组织因子 3'-UTR 结合，这与 miR-19 水平在 MCF-7 细胞中较高的观察结果一致。
