MicroRNA-145 suppresses uveal melanoma angiogenesis and growth by targeting neuroblastoma RAS viral oncogene homolog and vascular endothelial growth factor

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. It has been demonstrated that microRNA-145 (miR-145) is correlated with the progression of various cancers by regulating the expression of multiple target genes, especially a number of genes that regulate angiogenesis and proliferation. However, the underlying mechanisms of miR-145 in tumor angiogenesis of UM are still not well illustrated. Thus, we aimed to explore the potential target genes or pathways regulated by miR-145 in UM and the effect of miR-145 on invasion and angiogenesis. METHODS: Totally, 24 choroid samples were collected in our study, including 12 UM samples and 12 normal uveal tissues. The expression of neuroblastoma RAS viral oncogene homolog (N-RAS), phosphorylated protein kinase B (p-AKT), and vascular endothelial growth factor (VEGF) in UM tissues and normal uveal tissues was analyzed using Western blotting analysis. Lentivirus expression system was used to construct MUM-2B and OCM-1 cell lines with stable overexpression of miR-145. Transwell and endothelial cell tube formation assay were used to measure the effects of miR-145 on the invasion and angiogenesis of UM in vitro. The downstream target genes of miR-145 were predicted by bioinformatics and confirmed using a luciferase assay. BALB/c nude mice models were established to investigate the mechanisms of miR-145 on tumor growth and angiogenesis in vivo. Group data comparisons were performed using analysis of Student's t test. A two-tailed P < 0.05 was considered as statistically significant. RESULTS: The results of Western blotting analysis indicated that the expressions of N-RAS (1.10 ± 0.35 vs. 0.41 ± 0.36, t = 3.997, P = 0.012), p-AKT (1.16 ± 0.22 vs. 0.57 ± 0.03, t = 7.05, P = 0.001), and VEGF (0.97 ± 0.32 vs. 0.45 ± 0.21, t = 3.314, P = 0.008) in UM tumor tissues were significantly higher than those in normal uveal tissue. Luciferase assay demonstrated N-RAS and VEGF as downstream targets of miR-145. Moreover, tube formation assay revealed that miR-145-transfected human microvascular endothelial cell line formed shorter tube length (36.10 ± 1.51 mm vs. 42.91 ± 0.94 mm, t = 6.603, P = 0.003) and less branch points (350.00 ± 19.97 vs. 406.67 ± 17.62, t = 3.685, P = 0.021) as compared with controls. In addition, the numbers of invaded MUM-2B and OCM-1 cells with miR-145 overexpression were significantly lower than the controls (35.7 ± 3.3 vs. 279.1 ± 4.9, t = 273.75, P < 0.001 and 69.5 ± 4.4 vs. 95.6 ± 4.7, t = 21.27, P < 0.001, respectively). In vivo, xenografts expressing miR-145 had smaller sizes (miR-145 vs. miR-scr, 717.41 ± 502.62 mm(3)vs. 1694.80 ± 904.33 mm(3), t = 2.314, P = 0.045) and lower weights (miR-145 vs. miR-scr, 0.74 ± 0.46 g vs. 1.65 ± 0.85 g, t = 2.295, P = 0.045). CONCLUSION: Our results indicated that miR-145 is an important tumor suppressor and the inhibitory strategies against N-RAS/VEGF signaling pathway might be potential therapeutic applications for UM in the future.