Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA.
1] Department of Cancer Biology and Pharmacology, University of Illinois College of Medicine at Peoria, Peoria, IL, USA [2] Department of Neurosurgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA.
Oncogene. 2014 Apr 10;33(15):1922-33. doi: 10.1038/onc.2013.151. Epub 2013 Jun 3.
Matrix metalloproteinase-9 (MMP-9) represents one of the most prominent proteins associated with tumorigenesis and is a modulator of the tumor microenvironment during angiogenesis. Recently, syndecan-1 (SDC1), a transmembrane heparan sulfate-bearing proteoglycan, was also speculated to have a critical role in contributing to angiogenesis when associated with MMP-9. However, the mechanism behind their synergistic regulation is not fully understood. In the current study, we report for the first time that ionizing radiation (IR)-induced MMP-9 enhances SDC1 shedding, corroborating to tube-inducing ability of medulloblastoma (MB) cells. Furthermore, we observed that the tumor angiogenesis is associated with higher MMP-9-SDC1 interactions on both the cell surface and extracellular medium. Our results also revealed the existence of a novel regulatory mechanism where MMP-9 drives the suppression of miR-494, resulting in enhanced SDC1 shedding and angiogenesis. From the in situ hybridization analysis, we found that MMP-9-specific shRNA (shMMP-9) treatment of mouse intracranial tumors resulted in elevated expression of miR-494. This negative correlation between MMP-9 and miR-494 levels was observed to be dependent on the methylation status of a miR-494 promoter-associated CpG island region (-186 to -20), which was confirmed by bisulfite-sequencing and methylation-specific PCR (MSP) analysis. Further, validation of MMP-9 and SDC1 3'-untranslated region (3'-UTR) targets with luciferase reporter assay provided a more favorable result for miR-494-mediated regulation of SDC1 but not of MMP-9, suggesting that the 3'-UTR of SDC1 mRNA is a direct target of miR-494. Overall, our results indicate that angiogenesis induced by radiotherapy is associated with an MMP-9-miR-494-SDC1 regulatory loop and that MMP-9-SDC1 activity creates a negative feedback loop by regulating the expression of miR-494.
基质金属蛋白酶-9（MMP-9）是与肿瘤发生最相关的蛋白之一，是血管生成过程中肿瘤微环境的调节剂。最近，跨膜硫酸乙酰肝素蛋白聚糖连接蛋白-1（SDC1）也被推测在与 MMP-9 相关时在促进血管生成中具有关键作用。然而，它们协同调节的机制尚不完全清楚。在本研究中，我们首次报道电离辐射（IR）诱导的 MMP-9 增强 SDC1 的脱落，这与成神经管细胞瘤（MB）细胞的管诱导能力一致。此外，我们观察到肿瘤血管生成与细胞表面和细胞外基质中更高的 MMP-9-SDC1 相互作用有关。我们的结果还揭示了一种新的调控机制，即 MMP-9 驱动 miR-494 的抑制，导致 SDC1 脱落和血管生成增强。通过原位杂交分析，我们发现 MMP-9 特异性 shRNA（shMMP-9）处理小鼠颅内肿瘤导致 miR-494 表达升高。MMP-9 和 miR-494 水平之间的负相关关系被观察到依赖于 miR-494 启动子相关 CpG 岛区域（-186 到-20）的甲基化状态，这通过亚硫酸氢盐测序和甲基化特异性 PCR（MSP）分析得到证实。进一步通过荧光素酶报告基因检测验证 MMP-9 和 SDC1 3'-UTR 靶标，结果更有利于 miR-494 对 SDC1 的调节而不是对 MMP-9 的调节，表明 SDC1 mRNA 的 3'-UTR 是 miR-494 的直接靶标。总之，我们的研究结果表明，放射治疗诱导的血管生成与 MMP-9-miR-494-SDC1 调节环有关，MMP-9-SDC1 活性通过调节 miR-494 的表达形成负反馈回路。
