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)inUMtumor 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±1.51mm 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±502.62mm3 vs.1694.80±904.33mm3,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.