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MicroRNA Regulates Hemangioendothelioma Growth by Targeting the Nox-4/MCP-1 Pathway
Gayle M. Gordillo, MD, Ayan Biswas, PhD, Savita Khanna, PhD, Sashwati Roy, PhD, Xueliang Pan, PhD, Mithun Sinha, PhD, Chandan K. Sen, PhD.
The Ohio State University, Columbus, OH, USA.
Purpose: MicroRNA (miR) are emerging as biomarkers to identify aberrant signaling pathways and potential therapeutic targets in tumors. Endothelial cell tumors are the most common soft tissue tumors in infants yet little is known about the role of miR in promoting 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. We also sought to determine how dicer activity regulates the nox-4/monocyte chemoattractant protein-1 (MCP-1) pathway, which we have previously shown is required for hemangioendothelioma formation
Methods: EOMA cells were transfected with either control or dicer lentiviral shRNA particles and injected (5 x 106 cells/100 ul PBS) subcutaneously into 6 week old 129 P3 mice. For in vitro experiments EOMA cells were transfected with control or dicer siRNA and samples collected 72 hours after transfection for measurement of miR, mRNA, or protein. Sybr green real-time PCR was used to measure miR and mRNA. Western blots and ELISA were used to measure protein. Plasmid transfections were done with reporter vectors containing firefly luciferase and co-transfected with a renilla luciferase vector as a transfection efficiency control. Luciferase levels were measured using a dual luciferase reporter assay. At least three independent replicates were conducted for all experiments. Two-sided 2 sample t-test was used to compare the difference between two groups, and ANOVA for comparison among more than 3 groups with Tukey’s adjustment for the multiple pairwise comparisons among groups. Non-parametric procedures were used when normality assumption of the data was violated even after proper data transformation. A p-value of ≤0.05 was considered statistically significant.
Results: Tumors formed in 4/4 mice injected with EOMA cells transfected with control short hairpin RNA (shRNA), but only formed in 1/5 mice injected with EOMA cells transfected with dicer shRNA and the single tumor in the dicer knockdown group was 91% smaller than the average size of tumors in the control group. This response to dicer knockdown was mediated by enhanced miR 21a-3p targeting of the nox-4 3’UTR. EOMA cells were transfected with miR 21a-3p mimics 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 versus the site mutated vectors (p<.01). Loss of nox-4 activity resulted in decreased hydrogen peroxide production and decreased production of oxidant inducible monocyte chemoattractant protein-1.
Conclusions: These are the first reported results to demonstrate the significant contribution of dicer activity and miR production in promoting hemangioendothelioma growth in vivo. These are also the first reported results of miR21a-3p targeting nox-4 mRNA and inhibiting reactive oxygen species production in endothelial cells. Collectively, these results indicate that targeting microRNA and specifically, miR-21a-3p, represent potential therapeutic strategies for the treatment of endothelial cell tumors including hemangioma and hemangioendothelioma.
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