人锌指蛋白23在原发性肝细胞肝癌中的表达及临床意义

目的 分析hZNF23在人HCC组织及HepG2细胞系中的表达情况,以探讨其与HCC临床病理特征和细胞凋亡的关系.方法 采用实时荧光定量RT-PCR法检测37例HCC患者(按Edmondson分为两组,Ⅰ+Ⅱ级17例,Ⅲ+Ⅳ级20例;按临床分为3组,HCC合并肝硬化和HBV感染组31例,HCC合并肝硬化和HCC合并HRV感染组各3例)癌组织和其癌旁肝组织标本中hZNF23及内参GAPDH mRNA的表达水平,并分析其与HCC临床病理特征的关系;体外培养HepG2细胞后,分为4组(对照组、1.25、2.5和5 μg/ml顺铂组)或6组(对照组、1.25、2.5、5、10和20μg/ml顺铂组).采用四唑盐比色法(MTT)和流式细胞仪膜联蛋白V/碘化丙啶染色法检测与观察HepG2细胞的增殖及凋亡情况;实时荧光定量RT-PCR法检测HepG2细胞在不同浓度的顺铂诱导凋亡后,hZNF23及内参GAPDH mRNA的表达水平.结果 37例HCC患者癌组织及其癌旁组织标本中hZNF23相对表达量分别为8.84(3.59～15.05)和22.20(13.85～42.90),差异有统计学意义(U=259.5,P＜0.01).Edmondson Ⅰ+Ⅱ级HCC组织hZNF23 mRNA相对表达量为12.80(4.80～19.50),高于Ⅲ+Ⅳ级的5.01(2.88～11.68),且差异有统计学意义(U=99.00,P＜0.05);合并肝硬化组为9.92(3.80～15.25),未合并肝硬化组为3.21(2.78～3.60)、合并HBV感染组为9.09(3.72～15.25),无HBV感染组为2.48(1.79～12.10),合并肝硬化组与未合并肝硬化组、合并HBV感染组与未合并HBV感染组比较,其hZNF23 mRNA表达量差异均无统计学意义(U值分别为16.00和24.00,P均＞0.05).顺铂作用于HepG2细胞24 h后,用MTT检测,顺铂明显抑制HepG2细胞增殖;膜联蛋白V/碘化丙啶染色法检测细胞凋亡率依次为(0.9±0.2)%、(4.2±0.3)%、(9.8±4.3)%、(23.0±6.0)%,顺铂显著诱导HepG2细胞凋亡,且呈剂量依赖性(F=27.890,P＜0.01).实时荧光定量RT-PCR法检测1.25、2.5、5μg顺铂诱导的HepGR细胞组hZNF23 mRNA相对表达量依次为(10.39±3.08)×10-5、(24.10±2.09)×10-5、(6.90±2.24)×10-4,均显著高于对照组[(9.48±1.80)×10-5,F=6.027,P＜0.01].结论 HCC组织中hZNF23相对表达水平与Edmondson分级密切关联;顺铂对HepG2细胞的凋亡作用可能与hZNF23上调有关.

Abstract：

Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.

Expression and significance of human zinc finger 23 gene in hepatocellular carcinoma

Objective To detect the expression level of human zinc finger 23 (ZNF23) in hepatocellular carcinoma tissue samples and HepG2 cell lines and investigate the relationship between hZNF23 expression and clinicopathological characteristics of HCC and cell apoptosis. Methods The expression levels of hZNF23 and GAPDH mRNA in 37 cases of HCC were measured by real-time RT-PCR. The association between the expression of hZNF23 and the clinicopathological characteristics of HCC was analyzed. Cultured HepG2 cells were divided into 4 groups ( control group, 1.25 μg/ml , 2.5 μg/ml and 5 μg/ml cisplatin)or 6 groups( control group, 1.25 μg/ml, 2.5 μg/ml, 5 μg/ml, 10 μg/ml and 20 μg/ml cisplatin). MTT method was employed to evaluate cell proliferation. Annexin V-FITC assay was used to assess percentage of apoptotic HepG2 cells. The expression levels of hZNF23 and GAPDH mRNA of HepG2 cells after apoptosis induced by cisplatin with a series of concentrations were measured by real-time RT-PCR.Results The median ( quartile1, quartile 3) expression levels of hZNF23 mRNA in 37 HCC tissue samples and adjacent tissue samples were 8.84 (3.59-15.05), 22.20 ( 13.85-42.90 ), respectively. There was significant difference ( U = 259.5, P ＜ 0.01 ). The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in cancer tissue samples with Edmondson stage Ⅰ + Ⅱ [12.80(4.80-19.50)] was much higher than those in stage Ⅲ + Ⅳ [5.01 ( 2.88-11.68 ), U = 99.00, P ＜ 0.05] The median ( quartile1,quartile 3 ) expression levels of hZNF23 mRNA in patients with and without hepatic cirrhosis were 9.92(3.80-15.25) , 3.21 (2.78-3.60), respectively. The median ( quartile1, quartile 3 ) expression levels of hZNF23 mRNA in HBV infection and non-infection patients was 9.09(3.72-15.25 ), 2.48 (1.79-12.10),respectively. There was no significant difference between groups with and without hepatic cirrhosis and between HBV infection and non-infection groups( U = 16. 00 and 24.00, P ＞0.05 ). MTT assay indicated that cisplatin significantly inhibited HepG2 cells proliferation in a dose-dependent manner. Annexin V-FITC/PI assay showed that HepG2 cells apoptosis rates were (0.9 ± 0.2 ) %, ( 4. 2 ± 0.3 ) %, ( 9.8 ± 4. 3 ) %,(23.0 ± 6.0)%, respectively. Cisplatin significantly induced HepG2 cells apoptosis in a dose-dependent manner( F = 27.89, P ＜ 0.01 ). The expression levels of hZNF23 mRNA in cisplatin groups [( 10.39 ±3.08) × 10-5, (24.10 ± 2.09) × 10-5, (6.90 ± 2.24) × 10-4] were significantly lower than that of the controlgroup[(94.80±1.80) ×10-5, F=6.027, P＜0.01]. Conclusions The expression level hZNF23 mRNA is related to Edmondson stage of HCC. The apoptosis effect of cisplatin on HepG2 cells may be associated with the upregulation of hZNF23.