The metastatic potential of osteosarcoma cells is inversely correlated to cell surface FAS expression. Downregulation of FAS allows osteosarcoma cells to escape FAS ligand–mediated apoptosis when they enter a FAS ligand–positive microenvironment such as the lung. We have previously demonstrated that miR-20a, encoded by the miR-17-92 cluster, downregulates FAS expression in osteosarcoma. We further demonstrated an inverse correlation between FAS expression and miR-20a expression. However, the mechanism of FAS regulation by miR-20a was still unclear. The purpose of the current study was to evaluate the mechanism of FAS regulation by miR-20a in vitro and test the effect of targeting miR-20a in vivo. We investigated whether miR-20a's downregulation of FAS was mediated by binding to the 3′-untranslated region (3′-UTR) of FAS mRNA with the consequent induction of mRNA degradation or translational suppression. We identified and mutated two miR-20a binding sites on the FAS mRNA 3′-UTR. Using luciferase reporter assays, we demonstrated that miR-20a did not bind to either the wild-type or mutated FAS 3′-UTR. In contrast, overexpression of miR-20a resulted in downregulation of FAS promoter activity. Similarly, the inhibition of miR-20a increased FAS promoter activity. The critical region identified on the FAS promoter was between −240 bp and −150 bp. Delivery of anti-miR-20a in vivo using nanoparticles in mice with established osteosarcoma lung metastases resulted in upregulation of FAS and tumor growth inhibition. Taken together, our data suggest that miR-20a regulates FAS expression through the modulation of the FAS promoter and that targeting miR-20a using anti-miR-20a has therapeutic potential. Mol Cancer Ther; 17(1); 130–9. ©2017 AACR.

中文翻译：

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miR-20a 通过调节 Fas 启动子活性来调节骨肉瘤细胞中 Fas 的表达，并可在治疗上靶向抑制肺转移

骨肉瘤细胞的转移潜能与细胞表面 FAS 表达呈负相关。FAS 的下调允许骨肉瘤细胞在进入 FAS 配体阳性微环境（如肺）时逃避 FAS 配体介导的细胞凋亡。我们之前已经证明，由 miR-17-92 簇编码的 miR-20a 下调骨肉瘤中的 FAS 表达。我们进一步证明了 FAS 表达和 miR-20a 表达之间的负相关。然而，miR-20a对FAS的调控机制尚不清楚。本研究的目的是在体外评估miR-20a对FAS的调控机制，并在体内测试靶向miR-20a的效果。我们研究了 miR-20a' FAS 的下调是通过与 FAS mRNA 的 3'-非翻译区 (3'-UTR) 结合，随后诱导 mRNA 降解或翻译抑制来介导的。我们鉴定并突变了 FAS mRNA 3'-UTR 上的两个 miR-20a 结合位点。使用荧光素酶报告基因检测，我们证明 miR-20a 不与野生型或突变的 FAS 3'-UTR 结合。相反，miR-20a 的过表达导致 FAS 启动子活性的下调。类似地，miR-20a 的抑制增加了 FAS 启动子的活性。在 FAS 启动子上鉴定的关键区域介于 -240 bp 和 -150 bp 之间。在患有骨肉瘤肺转移的小鼠体内使用纳米颗粒体内递送抗 miR-20a 导致 FAS 上调和肿瘤生长抑制。综合起来，我们的数据表明 miR-20a 通过调节 FAS 启动子来调节 FAS 表达，并且使用抗 miR-20a 靶向 miR-20a 具有治疗潜力。摩尔癌症治疗; 17(1); 130-9。©2017 AACR。