MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes
Mohit Sachdeva, … , Diana M. Cardona, David G. Kirsch
Mohit Sachdeva, … , Diana M. Cardona, David G. Kirsch
Published September 2, 2014
Citation Information: J Clin Invest. 2014;124(10):4305-4319. https://doi.org/10.1172/JCI77116.
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Research Article

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

Abstract

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.

Authors

Mohit Sachdeva, Jeffrey K. Mito, Chang-Lung Lee, Minsi Zhang, Zhizhong Li, Rebecca D. Dodd, David Cason, Lixia Luo, Yan Ma, David Van Mater, Rebecca Gladdy, Dina C. Lev, Diana M. Cardona, David G. Kirsch

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Figure 6

miR-182 target genes cooperate to regulate metastasis.

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miR-182 target genes cooperate to regulate metastasis. (A) In silico/3′ ...
(A) In silico/3′ UTR luciferase screen used to predict miR-182 targets (top panel) and number of putative miR-182–binding sites on respective 3′ UTRs of the different genes. (B) Heat map shows differential expression of approximately 400 proteins between miR-182 WT and miR-182 deleted primary sarcoma cells from a proteomic screen. (C) Schematic shows the different approaches used to identify miR-182 target genes implicated in metastasis. (D) Western blot validates upregulation of Pai1, Timp1, Rsu1, and Mtss1 in miR-182–deleted KP cells (1–3, different KPY Mir182 WT; 4–6, different KPY Mir182f/f cell lines). (E) ELISA validates decreased uPA and increased unprocessed pro–MMP-9 in the cell-culture media from miR-182–deleted primary sarcoma cells. (F) Zymography confirms decreased activation of MMP-2 and MMP-9 in the cell-culture media from miR-182–deleted KP cells. (G and H) Kaplan-Meier curves show that ectopic expression of all 4 miR-182 targets together (H), but not individual targets alone (G) are required to rescue miR-182–mediated metastasis in an orthotopic metastasis assay. Mice were euthanized 40 days following amputation. Five mice had microscopic lung nodules despite having no clinical evidence of metastasis (Supplemental Figure 13G). MPRT, cell line expressing all 4 miR-182 targets, i.e., Mtss1, Pai1, Rsu1, and Timp1. Two-tailed Student’s t test and 1-way ANOVA were used for statistical analysis. All data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.005.