Effect of high expression of Zwint on the proliferation of hepatoma cells and the prognosis of liver transplantation for hepatocellular carcinoma
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摘要:
目的 探讨Zw10结合因子（Zwint）在原发性肝癌（肝癌）中的表达情况及其对肝癌肝移植预后的影响。 方法 收集50例肝癌肝移植受体的肝癌组织、癌旁组织及临床资料。采用实时荧光定量聚合酶链反应（PCR）、蛋白免疫印迹（Western Blot）法和免疫组织化学（免疫组化）法，分别比较20例肝癌肝移植受体的20对肝癌组织及癌旁组织中Zwint信使核糖核酸（mRNA）表达情况和Zwint蛋白的表达水平；选取两个成功干扰Zwint表达的肝癌细胞系HepG-2（si-Zwint-1组和si-Zwint-2组），以空白对照作为si-NC组，采用细胞计数试剂盒（CCK）-8实验、平板克隆实验和细胞周期实验，比较各组细胞的增殖能力和细胞周期；采用Western Blot法和免疫组化法，分析肝癌组织和肝癌细胞中Zwint与细胞周期蛋白D1（cyclin D1）表达的一致性；以Zwint蛋白表达水平的中位数为分割点，将入组病例分高表达组（22例）和低表达组（28例），分析Zwint蛋白表达水平与肝癌肝移植受体临床特征、总体存活率及无复发存活率的关系。 结果 实时荧光定量PCR结果提示肝癌组织中Zwint mRNA表达水平高于癌旁组织（P=0.03）。Western Blot及免疫组化结果均提示肝癌组织中Zwint蛋白的表达水平高于癌旁组织（均为P < 0.05）。干扰肝癌细胞系HepG-2的Zwint基因后，CCK-8及平板克隆实验显示细胞增殖能力明显减弱（均为P < 0.01），细胞周期阻滞于G1期（均为P < 0.05）。Zwint蛋白表达水平与肿瘤直径和肿瘤、淋巴、转移（TNM）分期密切相关（均为P < 0.05）。Zwint高表达组肝癌肝移植受体的总体存活率低于低表达组（P=0.02）。 结论 Zwint高表达于肝癌组织，其通过调节细胞周期促进肝癌细胞增殖，且Zwint表达水平与肝癌肝移植后预后呈负相关。 -
关键词:
- Zw10结合因子（Zwint） /
- 原发性肝癌 /
- 肝移植 /
- 细胞增殖 /
- 细胞周期 /
- 细胞周期蛋白D1（cyclin D1） /
- 细胞周期蛋白依赖激酶（CDK）4 /
- 肿瘤、淋巴、转移（TNM）分期 /
- 存活率
Abstract:Objective To investigate the expression of zeste white 10 interactor (Zwint) in primary hepatocellular carcinoma (HCC) and its effect on the prognosis of liver transplantation for HCC. Methods HCC tissues, paracancerous tissues and clinical data of 50 liver transplant recipients for HCC were collected. The expression levels of Zwint messenger RNA (mRNA) and Zwint protein in 20 pairs of HCC tissues and paracancerous tissues of 20 liver transplant recipients for HCC were compared using real-time fluorescence quantitative polymerase chain reaction (PCR), Western Blot and immunohistochemistry (IHC). Two HCC cell lines HepG-2 which interfered with the expression of Zwint successfully were selected as si-Zwint-1 group and si-Zwint-2 group, and the blank control was taken as si-NC group. The cell proliferation and cell cycle of various groups were compared using cell counting kit (CCK) -8 experiment, flatcloning assay and cell cycle experiment. The consistency of the expression of Zwint and cyclin D1 in HCC tissues and cells was analyzed using Western Blot and IHC. The enrolled patients were divided into high expression group (22 cases) and low expression group (28 cases) based on the median of Zwint protein expression level, and the relationship of the expression level of Zwint protein and clinical characteristics, overall survival rate and disease free survival rate of liver transplant recipients for HCC was analyzed. Results The results of real-time fluorescence quantitative PCR showed that the expression level of Zwint mRNA in HCC tissues was higher than that of paracancerous tissues (P=0.03). The results of Western Blot and IHC showed that the expression level of Zwint protein in HCC tissues was higher than that of paracancerous tissues (both P < 0.05). After the Zwint gene of HCC cell line HepG-2 was interfered, CCK-8 and flatcloning assay showed that the cell proliferation potential was significantly weakened (all P < 0.01), and the cell cycle arrested at stage G1 (all P < 0.05). The expression level of Zwint protein was closely related to tumor diameter and tumor, node, metastasis (TNM) staging (all P < 0.05). The overall survival rate of liver transplant recipients for HCC in the high Zwint expression group was lower than that of the low expression group (P=0.02). Conclusions Zwint is highly expressed in HCC tissues, and it can promote the proliferation of HCC cells through regulating cell cycle. The expression level of Zwint is negatively correlated with the prognosis of liver transplantation for HCC. -
图 1 肝癌组织及癌旁组织Zwint mRNA和Zwint蛋白表达情况
A图为实时荧光定量PCR分析肝癌组织及其配对的癌旁组织Zwint mRNA表达情况；B图为Western Blot法分析肝癌组织及其配对的癌旁组织Zwint蛋白表达情况，T为肝癌组织，N为癌旁组织；C图为免疫组化法分析肝癌组织及癌旁组织Zwint蛋白表达情况（核内呈褐色的为Zwint阳性细胞，呈蓝色的为Zwint阴性细胞）
Figure 1. Expression of Zwint mRNA and Zwint protein in liver cancer tissues and paraneoplastic tissues
图 2 各组肝癌细胞的增殖能力和细胞周期情况
A图为验证干扰效率；B图为CCK-8实验分析各组细胞的增殖能力；C图为平板克隆实验分析各组细胞的单细胞成团能力，与si-NC组比较，aP<0.01；D图为细胞周期实验分析各组细胞的细胞周期情况，与si-NC组比较，aP<0.05
Figure 2. Cell proliferation and cell cycle of hepatoma carcinoma cells in each group
图 3 Zwint、cyclin D1及其相关蛋白在肝癌组织和细胞内的表达情况
Figure 3. Expression of Zwint, cyclin D1 and correlative protein in liver cancer tissues and cells
图 4 Zwint高表达组和低表达组的总体生存曲线和无复发生存曲线
Figure 4. Total survival curve and recurrence free survival curve of high Zwint expression group and low Zwint expression group
表 1 Zwint表达与肝癌受体临床特征指标的关系
Table 1. Relationship between Zwint expression and clinicopathological characteristics of hepatocellular carcinoma recipients[n(%)]
指标 n Zwint表达 P值 低表达组（n=28） 高表达组（n=22） 性别 男 36 20（40） 16（32） 0.919 女 14 08（16） 06（12） 年龄 ≤55岁 35 21（42） 14（28） 0.384 > 55岁 15 07（14） 8（16） 肝功能Child-Pugh分级 A级 29 19（38） 10（20） 0.278 B级 16 07（14） 09（18） C级 05 02（4）0 3（6） 甲胎蛋白 ≤400 μg/L 34 22（44） 12（24） 0.071 > 400 μg/L 16 6（12） 10（20） 肿瘤直径 ≤5 cm 36 26（52） 10（20） < 0.0010 > 5 cm 14 2（4） 12（24） 微血管侵犯 有 09 5（10） 4（8） 0.976 否 41 23（46） 18（36） TNM分期 < 3期 36 24（48） 12（24） 0.015 ≥3期 14 4（8） 10（20） 
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