ABSTRACT
Objective To evaluate the effect of nucleolar protein 14 (NOP14) on angiogenesis in melanoma.Methods 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,NOPI4 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.Coculture 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.Results 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.Conclusion The NOP14 expression is negatively correlated with MVD in melanoma tissues,and NOP14 can inhibit angiogenesis in melanoma.
ABSTRACT
Objective@#To investigate expression of nucleolar protein 14(NOP14) and CD31 in pancreatic cancer mouse model and its correlation with tumor progression.@*Methods@#Clinicopathological data of 5 patients with pathologically confirmed pancreatic ductal adenocarcinoma(PDAC) and hepatic metastasis between January 2013 and December 2015 was collected in Department of General Surgery, Peking Union Medical College Hospital. Immunohistochemistry staining was employed to detect the expression of NOP14 in matched primary PDAC and relevant metastasis.Pancreatic cancer cells with NOP14 stably knocked down were established by transfecting lentivirus with NOP14 targeted silencing RNA.The inhibition efficacy was detected by quantitative real time PCR and western blot.Microvascular density(MVD) in pancreatic cancer transplantation mouse model was determined by CD31 immunohistochemistry staining analysis and correlated with NOP14 expression and tumor progression.@*Results@#NOP14 had a significant higher expression in liver metastasis than primary pancreatic adenocarcinoma (2.09±0.45 vs. 1.31±0.27, P=0.028). NOP14 was knocked down 86 percent on mRNA level determined by qPCR and 78 percents on protein level detected by western blot. MVD was significantly decreased in NOP14-inhibited tumor from both pancreatic cancer cells subcutaneously and orthotopically grafted tumor mouse model with the value of 61.40±13.85 vs. 85.53±14.59 (P=0.041) and 38.33±10.91 vs. 59.33±15.37(P =0.037), respectively. Besides, MVD was positively associated with tumor volume(r=0.842, P<0.01) and metastasis (r=0.726, P=0.008).@*Conclusion@#NOP14 presents higher expression in hepatic metastasis of pancreatic adenocarcinoma and might promote tumor progression by increasing microvascular density.