核孔蛋白43在肝细胞癌组织中的表达及其对患者预后和肝癌细胞增殖与迁移的影响

顾永鹏; 刘洁; 朱贞宝; 吴迪; 谢传明; 张雷达

doi:10.3760/cma.j.cn115610-20241026-00468

核孔蛋白43在肝细胞癌组织中的表达及其对患者预后和肝癌细胞增殖与迁移的影响

Expression of nucleoporin 43 in hepatocellular carcinoma tissues and its impact on prognosis of patients and proliferation and migration of hepatocellular carcinoma cells

摘要

摘要:
目的探讨核孔蛋白43（NUP43）在肝细胞癌组织中的表达及其对患者预后和肝癌细胞增殖与迁移的影响。
方法采用回顾性队列研究方法和实验研究方法。收集2008年1月至2012年12月陆军军医大学第一附属医院收治的102例肝细胞癌患者的临床病理资料；男83例,女19例；年龄为56（19~87）岁。采用免疫组织化学染色分析NUP43在肝细胞癌组织中的表达。体外培养肝癌HepG2和SK‑HEP‑1细胞,采用蛋白质印迹法验证细胞转染Flag‑NUP43过表达质粒后的效果。采用CCK‑8、细胞体外迁移实验分析过表达NUP43对HepG2和SK‑HEP‑1细胞功能的影响。正态分布的计量资料以x±s表示,组间比较采用独立样本t检验。偏态分布的计量资料以M（范围）表示。计数资料以绝对数表示,组间比较采用配对χ²检验。采用Kaplan‑Meier法计算生存时间,Log‑rank检验进行生存分析。采用R 4.2.1软件绘制生存曲线。单因素和多因素分析采用COX比例风险回归模型。
结果（1）NUP43在肝细胞癌与癌旁组织中的表达情况及高低表达患者临床病理特征分析。免疫组织化学染色结果显示：NUP43主要表达于细胞质和细胞核膜。102例肝细胞癌组织样本中,NUP43低表达49例,NUP43高表达53例；102例肝细胞癌癌旁组织样本中,NUP43低表达80例,NUP43高表达22例；肝细胞癌与癌旁组织中NUP43表达比较,差异有统计学意义（χ²=16.505,P<0.05）。102例肝细胞癌组织样本中,NUP43低表达和高表达患者肿瘤长径、病理学分级、肝内转移比较,差异均有统计学意义（χ²=5.104,23.217,4.169,P<0.05）。（2）肝细胞癌患者生存情况及影响预后的因素分析。102例肝细胞癌患者随访时间为17.9（0.1~107.9）个月。NUP43低表达和高表达患者术后1、3、5年总生存率分别为79.59%、53.06%、34.69%和52.83%、18.87%、9.43%,两者生存情况比较,差异有统计学意义（χ²=27.071,P<0.05）。多因素分析结果显示：性别、NUP43表达、TNM分期、病理学分级是肝细胞癌患者术后生存的独立影响因素（风险比=1.846,2.206,2.040,2.177,95%可信区间为1.231~2.768,1.419~3.429,1.322~3.148,1.377~3.254,P<0.05）。（3）过表达NUP43对肝癌HepG2和SK‑HEP‑1细胞增殖和迁移能力的影响。蛋白质印迹法检测结果显示：转染Flag‑NUP43过表达质粒可显著上调HepG2和SK‑HEP‑1细胞中NUP43的表达。CCK‑8实验结果显示：HepG2细胞转染对照质粒和Flag‑NUP43过表达质粒后,细胞增殖指数分别为0.79±0.07和1.47±0.05,两者比较,差异有统计学意义（t=19.402,P<0.05）；SK‑HEP‑1细胞转染对照质粒和Flag‑NUP43过表达质粒后,细胞增殖指数分别为0.59±0.05和0.95±0.05,两者比较,差异有统计学意义（t=15.753,P<0.05）。细胞体外迁移实验结果显示：HepG2细胞转染对照质粒和Flag⁃NUP43过表达质粒后,细胞迁移数分别为（188±8）个和（595±13）个,两者比较,差异有统计学意义（t=46.192,P<0.05）；SK‑HEP‑1细胞转染对照质粒和Flag‑NUP43过表达质粒后,细胞迁移数分别为（136±10）个和（447±20）个,两者比较,差异有统计学意义（t=24.721,P<0.05）。
结论 NUP43在肝细胞癌组织中的表达显著高于癌旁组织。性别、NUP43表达、TNM分期、病理学分级是肝细胞癌患者术后生存的独立影响因素。过表达NUP43可显著促进肝癌HepG2和SK‑HEP‑1细胞的增殖和迁移能力。

Abstract:
Objective To investigate the expression of nucleoporin 43 (NUP43) in hepato-cellular carcinoma tissues and its impact on prognosis of patients and proliferation and migration of hepatocellular carcinoma cells.
Methods The retrospective cohort study and experi-mental study were conducted. The clinicopathological data of 102 hepatocellular carcinoma patients who were admitted to The First Affiliated Hospital of Army Medical University from January 2008 to December 2012 were collected. There were 83 males and 19 females, aged 56(range, 19-87)years. The expression of NUP43 in hepatocellular carcinoma tissues was analyzed by immunohistochemical staining. HepG2 and SK‑HEP‑1 hepatocellular carcinoma cells were cultured in vitro. The Western blot was used to verify the effects of Flag-NUP43 overexpression plasmid in transfected cells. The CCK‑8 and cell migration experiments were used to analyze the effect of NUP43 overexpression on HepG2 and SK‑HEP‑1 hepa-tocellular carcinoma cells. Measurement data of normal distribution were expressed as Mean±SD, and comparison between groups was conducted using the independent sample t test. Measurement data of skewed distribution were represented as M(range). Count data were expressed as absolute numbers, and comparisons between groups was conducted using the paired chi‑square test. The Kaplan‑Meier method was used to calculate survival time, and Log‑rank test was used for survival analysis. The R 4.2.1 software was used to draw survival curves. The COX proportional hazards regre-ssion model was used for univariate and multivariate analyses.
Results (1) Expression of NUP43 in hepatocellular carcinoma and adjacent tissues, and analysis of clinicopathological characteristics of patients with high and low expression of NUP43. Results of immunohistochemical staining showed that NUP43 was mainly expressed in the cytoplasm and nuclear membrane of cells. Of 102 hepatocellular carcinoma tissue samples, there were 49 samples with low expression of NUP43 and 53 samples with high expression of NUP43. Of 102 hepatocellular carcinoma adjacent tissue samples, there were 80 samples with low expression of NUP43 and 22 samples with high expression of NUP43. There was a significant difference in the expression of NUP43 between hepatocellular carcinoma and adjacent tissues (χ²=16.505, P<0.05). Of 102 hepatocellular carcinoma tissue samples, there were significant differences in tumor diameter, pathological grading, and intrahepatic metastasis between the patients with low expression of NUP43 and the patients with high expression of NUP43 (χ²=5.104, 23.217, 4.169, P<0.05). (2) Survival of hepatocellular carcinoma patients and prognostic factors analysis. The follow‑up time of 102 hepatocellular carcinoma patients was 17.9(range, 0.1-107.9)months. The postoperative 1‑, 3‑, and 5‑year overall survival rates were 79.59%, 53.06% and 34.69% for the patients with low expression of NUP43, versus 52.83%, 18.87%, and 9.43% for the patients with high expre-ssion of NUP43, showing a significant difference between them (χ²=27.071, P<0.05). Results of multi-variate analysis showed that gender, NUP43 expression, TNM staging, and pathological grading were independent influencing factors for postoperative survival in patients with hepatocellular carcinoma (hazard ratio=1.846, 2.206, 2.040, 2.177, 95% confidence interval as 1.231-2.768, 1.419-3.429, 1.322-3.148, 1.377-3.254, P<0.05). (3) Effects of NUP43 overexpression on the proliferation and migration of HepG2 and SK‑HEP‑1 hepatocellular carcinoma cells. Western blot analysis showed that transfection of Flag‑NUP43 overexpression plasmid significantly increased the expression of NUP43 in HepG2 and SK‑HEP‑1 cells. Results of CCK‑8 experiment showed that after transfection with the control plasmid and Flag‑NUP43 overexpression plasmid, the cell proliferation indices of HepG2 were 0.79±0.07 and 1.47±0.05, respectively, showing a significant difference between them (t=19.402, P<0.05). After transfection with the control plasmid and Flag‑NUP43 overexpression plasmid, the cell proliferation indices of SK‑HEP‑1 cells were 0.59±0.05 and 0.95±0.05, respectively, showing a significant difference between them (t=15.753, P<0.05). Results of cell migration experiments showed that after transfection with the control plasmid and Flag‑NUP43 overexpression plasmid, the number of cell migrations in HepG2 was 188±8 and 595±13, respectively, showing a significant difference between them (t=46.192, P<0.05). After transfection with the control plasmid and Flag‑NUP43 overexpre-ssion plasmid, the number of cell migrations in SK‑HEP‑1 cells were 136±10 and 447±20, respectively, showing a significant difference between them (t=24.721, P<0.05).
Conclusions The expression of NUP43 in hepatocellular carcinoma tissues is significantly higher than that in adjacent tissues. Gender, NUP43 expression, TNM staging, and pathological grading are independent influen-cing factors for postoperative survival of hepatocellular carcinoma patients. Overexpression of NUP43 can significantly promote the proliferation and migration of HepG2 and SK‑HEP‑1 hepatocellular carcinoma cells.

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