核蛋白14对黑素瘤新生血管形成的影响及机制研究

Effect of nucleolar protein 14 on angiogenesis in melanoma and its mechanism

目的分析核蛋白14(NOP14)对黑素瘤血管形成的影响。方法收集2016年1月至2018年12月在广州市第一人民医院经病理确诊的40例黑素瘤患者的黑素瘤组织,免疫组化检测NOP14和CD31[以微血管密度(MVD)表示]的表达。将黑素瘤细胞A375和SK-MEL-1细胞分别分为4组,分别转染空载体(空载体组)、NOP14过表达载体(NOP14组)、靶向NOP14的siRNA(siNOP14组)和阴性对照siRNA(siNC组)。荧光定量PCR和Western印迹检测各组NOP14 mRNA和蛋白的表达,Western印迹和ELISA检测细胞及其培养基中血管内皮生长因子(VEGF)和血管内皮生长因子受体(VEGFR)的表达。利用Transwell小室构建以上各组A375、SK-MEL-1细胞与人脐静脉内皮细胞(HUVEC)共培养模型,利用CCK8、Transwell小室和Matrigel血管拟态实验检测共培养后各组HUVEC的增殖、迁移、侵袭和管腔形成能力。采用线性回归模型分析黑素瘤组织中NOP14表达水平与MVD的关系,多因素方差分析检验细胞增殖活性的差异,其余实验指标的两组之间比较采用独立样本t检验。结果NOP14高表达组(20例)黑素瘤组织MVD为44±13,中表达组(17例)为58±16,低表达组(3例)为62±11,且NOP14表达和MVD呈负相关(r=-0.525,P=0.017)。与空载体组相比,NOP14组A375和SK-MEL-1细胞及培养基中VEGF和VEGFR的表达水平均显著降低(P<0.05)。与siNC组相比,siNOP14组A375和SK-MEL-1细胞及培养基中VEGF和VEGFR的表达水平均显著升高(P<0.05)。在A375与HUVEC共培养模型中,与空载体共组相比,NOP14组HUVEC的增殖活性(F=131.85,P<0.05)和迁移细胞数[22±5比63±8,t=7.07,P=0.002)、侵袭细胞数(14±5比45±10,t=4.94,P=0.008)及分支节点数(8±2比14±3,t=5.06,P<0.001)均显著下降;与siNC组相比,siNOP14组HUVEC的增殖活性(F=79.92,P<0.01)和迁移细胞数(152±30比59±4,t=5.36,P=0.006)、侵袭细胞数(134±21比50±8,t=6.40,P<0.001)及分支节点数(27±3比15±4,t=6.10,P<0.001)显著升高。在SK-MEL-1细胞与HUVEC共培养模型中,各组HUVEC的增殖、迁移和侵袭活性以及管腔形成能力和与各组A375细胞共培养的HUVEC变化趋势一致。结论黑素瘤组织中NOP14表达和MVD呈负相关,NOP14具有抑制黑素瘤血管新生的作用。

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,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.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.