Hippo/YAP信号通路调控蛋白ACAN作为新型肝细胞肝癌血清肿瘤标志物的研究

doi:10.3969/j.issn.1673-8640.2019.01.013

摘要/Abstract

摘要：

目的探讨Hippo/Yes相关蛋白（YAP）信号通路调控蛋白——聚蛋白聚糖（ACAN）诊断肝细胞肝癌（HCC）的价值。方法检测156例HCC患者、33例乙型肝炎患者、31例丙型肝炎患者、30例肝硬化患者、26例结肠癌患者、19例肺癌患者、22例胃癌患者、20例乳腺癌患者及100名体检健康者（正常对照组）的ACAN水平。将过表达YAP载体、YAP-shRNA慢病毒质粒转染肝癌细胞系Bel-7402和SMMC-7721。将过表达环磷腺苷效应元件结合蛋白（CREB）、TEA域家族成员（TEAD）、α珠蛋白转录因子CP2（TFCP2）、Runt相关转录因子2（RUNX2）载体分别单独转染及与YAP共转染Bel-7402和SMMC-7721细胞,检测ACAN mRNA的表达,评价不同载体对ACAN野生型及突变型启动子的影响。同时评价ACAN和甲胎蛋白（AFP）对肝癌的诊断效能。结果 HCC组ACAN水平明显高于胃癌组、结肠癌组、乳腺癌组、肺癌组、乙型肝炎组、丙型肝炎组及正常对照组（P=0.000）,而其他各组之间ACAN水平差异均无统计学意义（P>0.05）。过表达YAP后ACAN mRNA表达量明显升高（P<0.01）,而YAP敲除后ACAN mRNA表达量明显降低（P<0.01）。与其他转录因子（TEAD、TFCP2及RUNX2）比较,过表达CREB及共转染CREB和YAP可促进ACAN mRNA表达（P<0.01）。野生型ACAN启动子受CREB、YAP影响,而突变型ACAN启动子则不受影响。ACAN与AFP、丙氨酸氨基转移酶（ALT）及天门冬氨酸氨基转移酶（AST）呈明显正相关（r值分别为0.722、0.517、0.443,P=0.000）。受试者工作特征（ROC）曲线分析显示,ACAN和AFP诊断HCC的曲线下面积（AUC）分别为0.978、0.661;ACAN诊断Ⅰ~Ⅱ期HCC、Ⅲ期HCC的AUC分别为0.903和0.993。结论 ACAN对HCC的诊断效能略优于AFP,且对晚期HCC的诊断效能优于早期HCC。ACAN通过Hippo/YAP信号通路发挥重要作用,或可作为一种新的HCC血清肿瘤标志物。

关键词: 聚蛋白聚糖, 肝细胞肝癌, Hippo/Yes相关蛋白信号通路

Abstract:

Objective To investigate the role of Hippo/Yes related protein（YAP） signaling pathway regulation protein aggrecan（ACAN）in the diagnosis of hepatocellular carcinoma （HCC）. Methods A total of 156 HCC patients,33 hepatitis B patients,31 hepatitis C patients,30 liver cirrhosis patients,26 colon cancer patients,19 lung cancer patients,22 gastric cancer patients,20 breast cancer patients and 100 healthy subjects （healthy control group） were enrolled. Their ACAN levels were determined. YAP over-expressive vector and YAP-shRNA lentivirus plasmid were used to transfect hepatoma cell lines Bel-7402 and SMMC-7721. Over-expressive cAMP-response element binding protein （CREB）,TEA domain family member（TEAD）,alpha-globin transcription factor CP2（TFCP2）,Runt-related transcription factor 2 （RUNX2）vectors were individually transfected and co-transfected with YAP into Bel-7402 and SMMC-7721 cells,respectively. The expression of ACAN mRNA and the effects of different vectors on ACAN wild and mutant type promoters were determined. The diagnostic efficiency of ACAN and alpha-fetoprotein（AFP） for the diagnosis of HCC was evaluated. Results The levels of ACAN in HCC group were higher than those in gastric cancer,colon cancer,breast cancer,lung cancer,hepatitis B,hepatitis C and healthy control groups （P=0.000）,but there was no statistical significance among the other groups （P>0.05）. The expression of ACAN mRNA increased after the over-expression of YAP（P<0.01）,but it decreased after YAP knockout （P<0.01）. Compared with other transcription factors （TEAD,TFCP2 and RUNX2）,over-expressive CREB and co-transfected CREB and YAP could promote the expression of ACAN mRNA （P<0.01）. The wild type ACAN promoter can be affected by CREB and YAP,while the mutant type ACAN promoter was not affected. There was a positive correlation between ACAN and AFP,alanine aminotransferase （ALT）and aspartate aminotransferase （AST）（r=0.722,0.517 and 0.443,P=0.000）. Receiver operating characteristic （ROC） curve showed that the areas under the curves （AUC）of ACAN and AFP were 0.978 and 0.661. The AUC of ACAN were 0.903 and 0.993 for the diagnosis of stage Ⅰ-Ⅱ and stage Ⅲ HCC,respectively. Conclusions The diagnostic efficiency of ACAN for HCC is better than that of AFP. The diagnostic efficiency for advanced HCC is superior to that of early HCC. ACAN can be used as a new tumor marker for the diagnosis of HCC and plays a role through Hippo/YAP signaling pathway.

Key words: Aggrecan, Hepatocellular carcinoma, Hippo/ Yes related protein signaling pathway

中图分类号:

R446.1

尚璐, 张骁, 陈岩, 吴棋, 邾国庆, 刘雅, 王佳谊, 孙奋勇, 张蕾. Hippo/YAP信号通路调控蛋白ACAN作为新型肝细胞肝癌血清肿瘤标志物的研究[J]. 检验医学, 2019, 34(1): 60-66.

SHANG Lu, ZHAO Xiao, CHEN Yan, WU Qi, ZHU Guoqing, LIU Ya, WANG Jiayi, SUN Fenyong, ZHANG Lei. Hippo/YAP signaling pathway regulation protein ACAN as a new serum tumor marker for hepatocellular carcinoma[J]. Laboratory Medicine, 2019, 34(1): 60-66.

图/表 7

表1 各组血清ACAN水平的比较（ng/mL,$\bar{x}\pm s$）

组别	例数	ACAN
正常对照组	100	17.44±6.59^*
乙型肝炎组	33	21.54±7.74^*
丙型肝炎组	31	24.61±4.89^*
肝硬化组	30	15.42±7.20^*
胃癌组	22	14.76±4.53^*
结肠癌组	26	14.24±5.81^*
乳腺癌组	20	16.29±7.22^*
肺癌组	19	13.89±4.66^*
HCC组	156	308.52±49.28
Ⅰ~Ⅱ期	93	153.94±28.06
Ⅲ期	63	536.71±67.15

图1 ACAN在各肝癌细胞系中的表达注：GAPDHA为甘油醛-3-磷酸脱氢酶（glyceraldehyde 3-phosphate dehydrogenase）

图2 YAP过表达及YAP敲除后ACAN mRNA的表达注：与空白组比较,*P<0.01;与GFP-sh组比较,#P<0.01;Bel-7402;SMMC-7721

图3 CREB、TEAD、TFCP2、RUNX2 过表达及与YAP共转染后ACAN mRNA的表达注：与空白组比较,*P<0.01;与CREB组比较,#P<0.01;与CREB+YAP组比较,△P<0.01;Bel-7402;SMMC-7721

图4 ACAN的启动子中CREB的结合基序及野生型、突变型ACAN启动子相对ACAN mRNA表达量(%)

图5 双荧光报告实验检测CREB过表达、YAP过表达、CREB基因沉默、YAP基因沉默及其联合转染后对野生型及突变型ACAN启动子荧光强度的影响注：（a）CREB过表达、YAP过表达及二者共转染后对野生型ACAN启动子荧光强度的影响;与空白组比较,*P<0.01;（b）CREB基因沉默、YAP基因沉默及二者共转染后对野生型ACAN启动子荧光强度的影响;与GFP-sh组比较,*P<0.01;（c）CREB过表达、YAP过表达、CREB基因沉默、YAP基因沉默及其共转染后对突变型ACAN启动子荧光强度的影响;Bel-7402;SMMC-7721

图6 ACAN、AFP诊断HCC的ROC曲线野生型ACAN启动子相对荧光强度（%）敏感性注：（a）ACAN、AFP诊断HCC的ROC曲线;（b）ACAN诊断Ⅰ~Ⅱ期和Ⅲ期HCC的ROC曲线

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