Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4

Background: Endothelial cell tumors are the most common soft tissue tumor in infants. Results: Dicer knockdown up-regulated miR-21a-3p that targeted nox-4 mRNA preventing endothelial cell tumor formation in vivo. Conclusion: Nox-4 silencing inhibits endothelial cell tumor formation. Dicer knockdown up-regulates miR 21a-3p, which targets the Nox-4 3-UTR. Significance: Novel drivers of endothelial cell tumor formation are reported. MicroRNAs (miR) are emerging as biomarkers and potential therapeutic targets in tumor management. Endothelial cell tumors are the most common soft tissue tumors in infants, yet little is known about the significance of miR in regulating their growth. A validated mouse endothelial cell (EOMA) tumor model was used to demonstrate that post-transcriptional gene silencing of dicer, the enzyme that converts pre-miR to mature miR, can prevent tumor formation in vivo. Tumors were formed in eight of eight mice injected with EOMA cells transfected with control shRNA but formed in only four of ten mice injected with EOMA cells transfected with dicer shRNA. Tumors that formed in the dicer shRNA group were significantly smaller than tumors in the control group. This response to dicer knockdown was mediated by up-regulated miR 21a-3p activity targeting the nox-4 3-UTR. EOMA cells were transfected with miR 21a-3p mimic and luciferase reporter plasmids containing either intact nox-4 3-UTR or with mutation of the proposed 3-UTR miR21a-3p binding sites. Mean luciferase activity was decreased by 85% in the intact compared with the site mutated vectors (p < 0.01). Attenuated Nox-4 activity resulted in decreased cellular hydrogen peroxide production and decreased production of oxidant-inducible monocyte chemoattractant protein-1, which we have previously shown to be critically required for endothelial cell tumor formation. These findings provide the first evidence establishing the significance of dicer and microRNA in promoting endothelial cell tumor growth in vivo.