Targeting of syndecan-1 by microRNA miR-10b promotes breast cancer cell motility and invasiveness via a Rho-GTPase- and E-cadherin-dependent mechanism
Targeting of syndecan-1 by microRNA miR-10b promotes breast cancer cell motility and invasiveness via a Rho-GTPase- and E-cadherin-dependent mechanism
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DOI：
10.1002/ijc.27629
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发表时间：
2012-09-15
影响因子：
6.4
通讯作者：
Goette, Martin
中科院分区：
文献类型：
--
作者：
Ibrahim, Sherif A.;Yip, George W.;Goette, Martin
microRNA 是小的内源性非编码 RNA，可在转录后调节基因表达。在乳腺癌中，跨膜硫酸乙酰肝素蛋白聚糖 syndecan-1（oncomiR miR-10b 的预测靶标）的过度表达与不良临床结果相关。为了研究 miR-10b 和 syndecan-1 的潜在功能关系，分别用 pre-miR-10b、syndecan-1 siRNA 或对照试剂瞬时转染 MDA-MB-231 和 MCF-7 乳腺癌细胞。通过增殖、迁移和侵袭室测定以及延时视频显微镜监测改变的细胞行为。定量实时 PCR (qPCR)、流式细胞术和 3'UTR 荧光素酶检测表明，miR-10b 过表达诱导 syndecan-1 转录后下调，导致癌细胞迁移和基质胶侵袭性增加。 Syndecan-1 沉默产生了该表型的副本。对纤连蛋白和层粘连蛋白的粘附以及基底细胞增殖增加。 Syndecan-1 与粘着斑激酶共免疫沉淀，显示 syndecan-1 耗尽后激活增加。 Syndecan-1 缺陷细胞的 Affymetrix 筛选和验证性 qPCR 以及蛋白质印迹分析揭示了 ATF-2、COX-2、cadherin-11、vinculin、actin 的上调？ 2、MYL9、transgelin-1、RhoA/C、基质金属蛋白酶 2 (MMP2) 和乙酰肝素酶，以及 AML1/RUNX1、E-钙粘蛋白、CLDN1、p21WAF/CIP、细胞周期蛋白依赖性激酶 6、TLR-4、PAI1/2、Collagen1alpha1、JHDM1D、Mpp4、MMP9、matrilin-2 的下调和 ANXA3/A10。视频显微镜显示 Syndecan-1 耗尽的细胞的 Rho 激酶依赖性运动大幅增加，显示丝状伪足形成增加。我们得出结论，syndecan-1 是 oncomiR miR-10b 的新靶标。细胞骨架功能的 Rho-GTPase 依赖性调节和 E-钙粘蛋白表达的下调被确定为 miR-10b-syndecan-1 轴的相关效应子，这成为开发乳腺癌新治疗方法的有希望的目标。
microRNAs are small endogenous noncoding RNAs, which post-transcriptionally regulate gene expression. In breast cancer, overexpression of the transmembrane heparan sulfate proteoglycan syndecan-1, a predicted target of the oncomiR miR-10b, correlates with poor clinical outcome. To investigate the potential functional relationship of miR-10b and syndecan-1, MDA-MB-231 and MCF-7 breast cancer cells were transiently transfected with pre-miR-10b, syndecan-1 siRNA or control reagents, respectively. Altered cell behavior was monitored by proliferation, migration and invasion chamber assays, and time-lapse video microscopy. miR-10b overexpression induced post-transcriptional downregulation of syndecan-1, as demonstrated by quantitative real-time PCR (qPCR), flow cytometry, and 3'UTR luciferase assays, resulting in increased cancer cell migration and matrigel invasiveness. Syndecan-1 silencing generated a copy of this phenotype. Adhesion to fibronectin and laminin and basal cell proliferation was increased. Syndecan-1 coimmunoprecipitated with focal adhesion kinase, which showed increased activation upon syndecan-1 depletion. Affymetrix screening and confirmatory qPCR and Western blotting analysis of syndecan-1-deficient cells revealed upregulation of ATF-2, COX-2, cadherin-11, vinculin, actin ? 2, MYL9, transgelin-1, RhoA/C, matrix metalloproteinase 2 (MMP2) and heparanase, and downregulation of AML1/RUNX1, E-cadherin, CLDN1, p21WAF/CIP, cyclin-dependent kinase 6, TLR-4, PAI1/2, Collagen1alpha1, JHDM1D, Mpp4, MMP9, matrilin-2 and ANXA3/A10. Video microscopy demonstrated massively increased Rho kinase-dependent motility of syndecan-1-depleted cells, which displayed increased filopodia formation. We conclude that syndecan-1 is a novel target of the oncomiR miR-10b. Rho-GTPase-dependent modulation of cytoskeletal function and downregulation of E-cadherin expression are identified as relevant effectors of the miR-10b-syndecan-1 axis, which emerges as a promising target for the development of new therapeutic approaches for breast cancer.