To evaluate the effect of nucleolar protein 14 (NOP14) on angiogenesis in melanoma.

Melanoma tissues were collected from 40 patients with pathologically diagnosed melanoma in Guangzhou First People′s Hospital from January 2016 to December 2018, and immunohistochemical study was conducted to determine the expression of NOP14 and CD31 (expressed as microvessel density [MVD]) . Melanoma cell lines A375 and SK-MEL-1 were both divided into 4 groups: empty vector group transfected with the empty vector, NOP14 group transfected with a NOP14-overexpressing vector, siNOP14 group transfected with the siRNA targeting NOP14, and siNC group transfected with a negative control siRNA. Fluorescence-based quantitative PCR and Western blot analysis were performed to determine the mRNA and protein expression of NOP14 respectively, and Western blot analysis and enzyme-linked immunosorbent assay (ELISA) to measure the expression of vascular endothelial growth factor (VEGF) and VEGF receptor (VEGFR) in cells and their culture media. Co-culture models of human umbilical vein endothelial cells (HUVECs) and A375/SK-MEL-1 cells in the above groups were established in Transwell chambers, and cell counting kit-8 (CCK8) assay, Transwell migration and invasion assays and Matrigel-based vasculogenic mimicry assay were performed to evaluate the cellular proliferative, migratory, invasive activity and tube formation capacity respectively. A linear regression model was used to analyze the relationship between NOP14 expression and MVD in melanoma tissues, multi-way analysis of variance to analyze the difference in cellular proliferative activity, and independent-sample t test to compare other experimental indices between 2 groups.

The expression of CD31 (MVD) was 44 ± 13 in the group with high NOP14 expression (n = 20) , 58 ± 16 in that with moderate NOP14 expression (n = 17) , and 62 ± 11 in that with low NOP14 expression (n = 3) . The NOP14 expression was negatively correlated with MVD (r = - 0.525, P = 0.017) . Compared with the empty vector group, the expression of VEGF and VEGFR in A375 and SK-MEL-1 cells and their culture media significantly decreased in the NOP14 group (all P < 0.05) . Compared with the siNC group, the expression of VEGF and VEGFR in the A375 and SK-MEL-1 cells and their culture media significantly increased in the siNOP14 group (all P < 0.05) . In the co-culture models of A375 cells and HUVECs, the NOP14 group showed significantly decreased proliferative activity of HUVECs (F = 131.85, P < 0.05) , and numbers of migratory cells (22 ± 5 vs. 63 ± 8, t = 7.07, P = 0.002) , invasive cells (14 ± 5 vs. 45 ± 10, t = 4.94, P = 0.008) and branch points (8 ± 2 vs. 14 ± 3, t = 5.06, P < 0.001) compared with the empty vector group; compared with the siNC group, the siNOP14 group showed significantly increased proliferative activity of HUVECs (F = 79.92, P < 0.01) , and numbers of migratory cells (152 ± 30 vs. 59 ± 4, t = 5.36, P = 0.006) , invasive cells (134 ± 21 vs. 50 ± 8, t = 6.40, P < 0.001) and branch points (27 ± 3 vs. 15 ± 4, t = 6.10, P < 0.001) . In the co-culture models of SK-MEL-1 cells and HUVECs, the 4 groups showed the same trend of changes in the cellular proliferative, migratory, invasive activity and tube formation capacity of HUVECs as the above groups in the co-culture models of A375 cells and HUVECs.

The NOP14 expression is negatively correlated with MVD in melanoma tissues, and NOP14 can inhibit angiogenesis in melanoma.

Nevi and melanomas; Melanoma, experimental; Nuclear proteins; Angiogenesis inhibitors; Cell proliferation; Cell movement; Nucleolar protein 14