TFCP2/YAP转录复合体调控蛋白CPE对肝细胞肝癌的诊断价值

doi:10.3969/j.issn.1673-8640.2019.02.003

摘要/Abstract

摘要：

目的探讨转录因子CP2（TFCP2）/Yes相关蛋白（YAP）转录复合体调控蛋白——羧肽酶E（CPE）在肝细胞肝癌（HCC）中的诊断价值。方法采用酶联免疫吸附试验测定147例HCC患者、42例乙型肝炎患者、29例胃癌患者及100名健康志愿者（正常对照组）血清CPE水平。采用实时荧光定量聚合酶链反应（PCR）检测CPE mRNA水平,免疫印迹法检测CPE、TFCP2及YAP蛋白表达,双荧光报告实验检测CPE启动子基因的活性。CPE与甲胎蛋白（AFP）、丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）的相关性采用Spearman等级相关分析。采用受试者工作特征（ROC）曲线评价CPE和AFP诊断HCC的价值。结果过表达YAP或TFCP2可促进CPE mRNA及蛋白的表达,而YAP和TFCP2共同过表达可明显促进CPE mRNA和蛋白的表达。YAP或TFCP2的敲减可抑制CPE mRNA和蛋白表达,而YAP及TFCP2共同敲减可明显抑制CPE mRNA和蛋白的表达。CPE启动子区域存在TFCP2结合基序,突变该基序后,TFCP2和YAP对CPE启动子的调控作用丧失。HCC组血清CPE水平明显高于乙型肝炎组、胃癌组和正常对照组（P<0.01）,而乙型肝炎组、胃癌组和正常对照组之间差异无统计学意义（P>0.05）。CPE与AFP、ALT、AST均呈正相关（r值分别为0.644、0.454、0.351,P值分别为0.000、0.000、0.001）。ROC曲线分析显示,CPE诊断HCC的曲线下面积（AUC）为0.937,明显高于AFP的AUC（0.679）。结论 CPE在HCC的诊断中具有较高的价值,可作为HCC的血清学诊断标志物。

关键词: 羧肽酶E, 转录因子CP2, Yes相关蛋白, 肝细胞肝癌

Abstract:

Objective To investigate the role of transcription factor CP2（TFCP2）/Yes-associated protein（YAP） transcriptional complex co-regulated protein carboxypeptidase E（CPE） for the diagnosis of hepatocellular carcinoma（HCC）. Methods The levels of serum CPE in 147 HCC patients,42 hepatitis B patients,29 gastric cancer patients and 100 healthy subjects（healthy control group） were determined by enzyme-linked immunosorbent assay. Real-time fluorescence quantitation polymerase chain reaction（PCR） was used to determine the expression of CPE mRNA. Western blotting was used to determine the expression of CPE,TFCP2 and YAP. The dual-luciferase reporter test was used to determine the activity of CPE promoter. The correlations between CPE and other indicators such as alpha-fetoprotein（AFP）,alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were analyzed using Spearman correlation analysis. Receiver operating characteristic（ROC） curve was used to compare the efficiency of CPE and AFP for the diagnosis of HCC. Results The overexpression of YAP or TFCP2 stimulated the expression of CPE mRNA and protein,and the overexpression of YAP and TFCP2 stimulated the expression ofCPE mRNA and protein. The knockdown of YAP or TFCP2 inhibited the expression of CPE mRNA and protein,and the knockdown of YAP and TFCP2 inhibited the expression of CPE mRNA and protein. A TFCP2-binding-motif was existed in CPE promoter region. After mutation,TFCP2 and YAP lost the regulation function on CPE promoter. The level of serum CPE was higher in HCC group than those in healthy control,hepatitis B and gastric cancer groups（P<0.01）,and there was no statistical significance among healthy control,hepatitis B and gastric cancer groups（P>0.05）. The level of serum CPE was positively correlated with the levels of AFP,ALT and AST（r=0.644,0.454 and 0.351,P=0.000,0.000 and 0.001,respectively）. The area under ROC curve（AUC） of CPE for the diagnosis of HCC was 0.937,which is better than that of AFP（0.679）. Conclusions CPE plays a role in the diagnosis of HCC,and CPE may be a diagnostic biomarker for HCC.

Key words: Carboxypeptidase E, Transcription factor CP2, Yes-associated protein, Hepatocellular carcinoma

中图分类号:

Q556

方子祥, 张骁, 陈岩, 吴棋, 邾国庆, 刘雅, 王佳谊, 孙奋勇, 俞蕾. TFCP2/YAP转录复合体调控蛋白CPE对肝细胞肝癌的诊断价值[J]. 检验医学, 2019, 34(2): 103-109.

FANG Zixiang, ZHANG Xiao, CHEN Yan, WU Qi, ZHU Guoqing, LIU Ya, WANG Jiayi, SUN Fenyong, YU Lei. Role of TFCP2/YAP transcriptional complex co-regulated protein CPE for the diagnosis of hepatocellular carcinoma[J]. Laboratory Medicine, 2019, 34(2): 103-109.

图/表 5

图1 TFCP2和YAP对CPE表达的影响注：（a）过表达YAP及TFCP2后CPE mRNA的相对表达量;与空白组比较,*P<0.01;与YAP-HA+TFCP2-Myc组比较,#P<0.01;（b）YAP及TFCP2敲减后CPE mRNA的相对表达量;与GFP-sh组比较,*P<0.01;与YAP-sh+TFCP2-sh组比较,#P<0.01;（c）过表达YAP及TFCP2后CPE蛋白的表达量;（d）敲减YAP及TFCP2后CPE蛋白的表达量;Bel-7402细胞;SMMC-7721细胞

图2 CPE启动子荧光载体示意图

图3 TFCP2及YAP对CPE启动子活性的调控情况注：（a）过表达YAP及TFCP2后野生型CPE启动子的相对荧光强度,与空白组比较,*P<0.01;与YAP-HA+TFCP2-Myc组比较,#P<0.01;（b）敲减YAP及TFCP2后野生型CPE启动子的相对荧光强度,与GFP-sh组比较,*P<0.01;与YAP-sh+TFCP2-sh组比较,#P<0.01;（c）过表达YAP及TFCP2后突变型CPE启动子的相对荧光强度;（d）敲减YAP及TFCP2后突变型CPE启动子的相对荧光强度;Bel-7402细胞;SMMC-7721细胞

图4 CPE与AFP、ALT、AST的相关性注：（a）CPE与AFP的相关性（r=0.644,P=0.000）;（b）CPE与ALT的相关性（r=0.454,P=0.000）;（c）CPE与AST的相关性（r=0.351,P=0.001）

图5 CPE、AFP诊断HCC的ROC曲线注：CPE;AFP; 参考线

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