Identification of genes and molecular pathways associated with anti-miR-182 treatment

To identify genes differentially modulated by anti-miR-182 treatment in a liver melanoma metastasis mouse model. Targeting oncogenic microRNAs is emerging as a promising strategy for cancer therapy. Here we provide proof-of-principle for the safety and efficacy of miRNA targeting against metastatic tumors. We tested the effect of targeting miR-182, a pro-metastatic miRNA frequently overexpressed in melanoma, whose silencing represses invasion and induces apoptosis in vitro. In particular, we assessed the effect of anti-miR-182 oligonucleotides synthesized with 2’ sugar modifications and a phosphorothioate backbone in a mouse model of melanoma liver metastasis. Luciferase imaging showed that mice treated with anti-miR-182 had an appreciably lower burden of liver metastases compared to the control. We confirmed that miR-182 levels were effectively downregulated in the anti-miR treated tumors relative to the scrambled treated tumor both in the liver and in the spleen. This downregulation was accompanied by an upregulation of miR-182 direct targets. Transcriptome analysis of mouse tissues treated with anti-miR-182 or scramble oligonucleotides revealed an enrichment for genes controlling survival, adhesion and migration modulated in response to anti-miR-182 treatment. These data indicate that in vivo administration of anti-miRs allows for efficient miRNA targeting and concomitant upregulation of target levels. Our results suggest that the use of anti-miR-182 is a promising therapeutic strategy for metastatic melanoma and provide solid proof-of-principle for similar strategies against other metastatic tumors. Keywords: Differentially expressed genes (mRNAs) in response to miRNA inhibition Quadruplicate (n=4) samples of anti-miR-182 treated human melanoma metastasis compared to quadruplicate control treated metastasis.

本研究旨在鉴定肝脏黑色素瘤转移小鼠模型中，经抗miR-182（anti-miR-182）处理后发生差异调控的基因。靶向致癌性微小RNA（microRNA, miRNA）正成为癌症治疗领域极具潜力的策略。本研究为靶向微小RNA治疗转移性肿瘤的安全性与有效性提供了原理验证依据。

我们针对miR-182开展了靶向效果验证：miR-182作为一种促转移微小RNA，在黑色素瘤中常呈高表达，沉默该基因可在体外抑制肿瘤侵袭并诱导细胞凋亡。具体而言，我们在黑色素瘤肝脏转移小鼠模型中，评估了经2’糖修饰与硫代磷酸酯骨架合成的抗miR-182寡核苷酸（oligonucleotide）的作用效果。荧光素酶成像结果显示，与对照组小鼠相比，经抗miR-182处理的小鼠肝脏转移瘤负荷显著降低。

我们证实，相较于乱序对照寡核苷酸处理的肿瘤，经抗miR-182处理的肿瘤中，miR-182的表达水平在肝脏与脾脏中均得到有效下调。该下调过程同时伴随miR-182直接靶基因的表达上调。对经抗miR-182或乱序对照寡核苷酸处理的小鼠组织开展转录组分析后发现，受抗miR-182处理调控的、参与细胞存活、黏附与迁移过程的基因呈现富集。

上述数据表明，体内施用抗miR可实现高效的微小RNA靶向，并伴随靶基因表达水平的同步上调。本研究结果显示，抗miR-182的应用是治疗转移性黑色素瘤的极具潜力的策略，并为针对其他转移性肿瘤的同类靶向策略提供了可靠的原理验证依据。

关键词：响应微小RNA抑制的差异表达基因（信使RNA, mRNA）；抗miR-182处理的人黑色素瘤转移灶与对照处理的转移灶的四份重复（n=4）样本。