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 UMin 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 angiogenesisin vivo. Group data comparisons were performed using analysis of Student'sttest. A two-tailedP Results The results of Western blotting analysis indicated that the expressions of N-RAS (1.10 +/- 0.35vs.0.41 +/- 0.36,t = 3.997,P = 0.012), p-AKT (1.16 +/- 0.22vs.0.57 +/- 0.03,t = 7.05,P = 0.001), and VEGF (0.97 +/- 0.32vs.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 mmvs.42.91 +/- 0.94 mm,t = 6.603,P = 0.003) and less branch points (350.00 +/- 19.97vs.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 +/- 3.3vs.279.1 +/- 4.9,t = 273.75,P vs.95.6 +/- 4.7,t = 21.27,P < 0.001, respectively).In vivo, xenografts expressin