适应素耳结合外壳相关蛋白2高表达对肝细胞癌生物学功能的影响

Effect of high expression of adaptin-ear-binding coat-associated protein 2 on the biological function of hepatocellular carcinoma

目的:探讨适应素耳结合外壳相关蛋白2(NECAP2)在肝细胞癌中的表达与肝细胞癌(HCC)患者预后的关系,观察NECAP2在肝癌细胞MHCC-97H中的表达及其对HCC增殖及侵袭的影响。方法:从肿瘤基因组图谱(TCGA)数据库下载并整理TCGA-LIHC的数据进行分析;选取16例肝癌样本及其配对的癌旁样本进行研究,用荧光实时定量聚合酶链反应(RT-qPCR)检测这16对样本、正常肝细胞LO2以及肝癌细胞MHCC-97H中的差异表达水平;用小干扰RNA转染MHCC-97H细胞以沉默NECAP2的表达,实验分为实验组(si-NECAP2组)和对照组(si-NC组)。并采用细胞计数试剂盒(CCK-8)、5-乙炔基-2’脱氧尿嘧啶核苷(EdU)实验、Transwell实验和蛋白质印迹法(Western blot)分别检测实验组和对照组的细胞活力、增殖、迁移和侵袭能力以及表皮生长因子受体(EGFR)蛋白的表达量。两组间比较采用t检验。结果:基于TCGA数据库中的肝癌细胞组和正常肝细胞组,癌旁组织中NECAP2的表达水平明显低于肝癌组织(3.747±0.474比4.367±0.593),差异有统计学意义(t=7.102,P<0.05);NECAP2在正常肝组织中的表达(3.746±0.474比4.263±0.503)要显著低于配对的肝癌组织,差异有统计学意义(t=5.152,P<0.05)。ROC曲线下面积(AUC)值为0.802,表明NECAP2对HCC有很好的诊断价值,Kaplan-Meier分析结果显示,NECAP2高表达组患者的生存期更短;实验组细胞吸光度(A)值、EdU阳性细胞占比、迁移细胞数量和侵袭细胞数量[3.201±0.300、(15.06±8.90)%、(103.000±7.550)个、(112.333±5.508)个]明显低于对照组[4.852±0.462、(49.070±10.520)%、(141.000±8.888)个、(146.333±4.041)个],差异有统计学意义(t=5.995、4.269、5.664、8.621,P<0.05)。此外,EGFR的表达量更低,与NECAP2的表达呈正相关。结论:NECAP2在HCC中的表达显著上调,NECAP2高表达组的患者预后更差;细胞实验证明下调NECAP2的表达可能通过下调EGFR的表达来抑制HCC的增殖、迁移及侵袭能力。

Objective To investigate the relationship between the expression of adaptin-ear-binding coat-associated protein 2(NECAP2)in hepatocellular carcinoma(HCC)and the prognosis of HCC patients,and to study the expression of NECAP2 in MHCC-97H and its effect on HCC proliferation and invasion.Methods The cancer genome atlas(TCGA)-LIHC were downloaded from TCGA database.A total of 16 HCC samples and their paired adjacent samples were selected and the differential expression levels of normal liver cells and MHCC-97H were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).MHCC-97H cells were transfected with small interfering RNA to silence NECAP2 expression,and si-NECAP2 group and si-NC group were set up.Cell viability,proliferation,migration,invasion,and epidermal growth factor receptor(EGFR)protein expression were determined by cell counting kit(CCK-8),5-ethynyl-2′-deoxyuridine(EdU),Transwell,and Western blotting.Comparison between the two groups was performed by using the t-test.Results Based on the HCC cell group and normal hepatocyte group in TCGA database,the expression level of NECAP2 in adjacent tissues was significantly lower than that in HCC tissues(3.747±0.474 vs.4.367±0.593,t=7.102,P<0.05);The NECAP2 expression in normal liver tissues was significantly lower than that in paired HCC tissues(3.746±0.474 vs.4.263±0.503,t=5.152,P<0.05).Area under the ROC curve(AUC)value of 0.802 showed that NECAP2 had a good diagnostic value for HCC.The results of the Kaplan-Meier analysis showed that NECAP2 patients in the high expression group had a shorter survival period.The absorbance values,proportion of EdU positive cells,number of migrating cells and number of invasive cells[3.201±0.300,(15.06±8.90)%,(103.000±7.550)cells,(112.333±5.508)cells]were significantly reduced in si-NECAP2 group as compared with those in si-NC group[(4.852±0.462),(49.070±10.520)%,(141.000±8.888)cells,(146.333±4.041)cells,t=5.995,4.269,5.664,8.621,P<0.05].Moreover,EGFR expression was even lower and positively correlated with NECAP2 expression.Conclusion The expression of NECAP2 was significantly upregulated in HCC,and prognosis of the patients with higher NECAP2 expression was worse.The cell experiments proved that downregulation of NECAP2 expression may inhibit the proliferation,migration and invasion of HCC by downregulation of EGFR expression.