外被体蛋白复合物亚基β’在胆管癌组织的表达及其与受体酪氨酸激酶信号间的关系

Expression of coatomer protein complexβ′in cholangiocellular carcinoma and its relationship with receptor tyrosine kinase signaling pathway

目的检测外被体蛋白复合物亚基β’(COPB2)在胆管癌中的表达水平及其临床意义,探讨COPB2在胆管癌细胞增殖中的分子机制。方法免疫组织化学法检测27例胆管癌和癌旁组织COPB2的表达,采用Mann-WhitneyU检验分析COPB2表达的临床意义。通过慢病毒为载体构建COPB2基因敲减稳定转染的胆管癌RBE细胞株(shCOPB2),实时定量聚合酶链反应(Real-time PCR)检测COPB2的敲减率,利用PathScan? RTK Signaling Antibody Array Kit试剂盒筛选受体酪氨酸激酶(RTK)信号差异分子,采用t检验比较两组间差异。结果 COPB2在胆管癌组织中的表达高于癌旁组织(P<0.01)。胆管癌组织中COPB2表达的高低与与肿瘤的病理分级、肿瘤大小、T分期、淋巴结转移、远端转移、TNM分期明显相关(P<0.05),与性别和年龄无明显相关(P>0.05)。与对照组比较,shCOPB2组RBE细胞中,COPB2 mRNA的表达均显著下降(P<0.01)其敲减效率为85.0%;RTK信号分子表达有差异的共有13个,7个磷酸化水平上调,其中上调最显著的为细胞外信号调节激酶1/2(ERK1/2)分子,上调了约39.4%;6个磷酸化水平下调,其中下调最明显的为Src,下调了约为25.5%(P<0.05)。结论 COPB2在胆管癌中高表达,可能是胆管癌预后的危险性指标,COPB2敲减后抑制胆管癌增殖,可能通过激活ERK1/2并抑制Src途径实现的。

Objective To explore the expression and clinical significance of the coatomer protein complexβ′(COPB2)in cholangiocellular carcinoma and cancer-adjacent normal tissues,and investigate the possible molecular mechanism of COPB2 on proliferation of RBE cells.Methods The expression of COPB2 in 27 cases of cholangiocarcinoma and corresponding cancer-adjacent normal tissues was detected by immunohistochemistry,and the clinical significance of its expression was analyzed by Mann-Whitney U test.Cholangiocarcinoma RBE cell line(shCOPB2)stably transfected with COPB2 gene knockdown was constructed by lentivirus vector.The knockdown rate of COPB2 expression was detected by real-time quantitative polymerase chain reaction(Real-time PCR).PathScan®RTK Signaling Antibody Array Kit was used to analyze differentially expressed molecules in receptor tyrosine kinase(RTK)signaling pathway.The differences between the two groups were compared by t-test.Results Compared with cancer-adjacent normal tissues,the expression of COPB2 in cholangiocarcinoma tissues was significantly increased(P<0.01).The correlation analysis revealed that COPB2 was statistically related to TNM stage,tumor size,lymph node metastasis and pathological grade(P<0.05),but not related to sex,age(P>0.05).Compared with the control group,the expression of COPB2 mRNA in RBE cells of shCOPB2 group was significantly decreased(P<0.01),and the knockdown efficiency was 85.0%.Analysis of antibody array showed that compared to shCtrl group,13 molecules in shCOPB2 group were expressed differentially,including 7 up-regulated phosphorylation levels and 6 down-regulated phosphorylation levels,in which the most significantly up-regulated molecule was extracellular signal-regulated kinase 1/2(ERK1/2,up to about 39.4%)and the most significantly down-regulated molecule was Src(down about 25.5%)(P<0.05).Conclusion The high expression of COPB2 in cholangiocarcinoma may be a risk indicator for prognosis of cholangiocarcinoma.Inhibition of cholangiocarcinoma cells induced by COPB2 knockdown may due to the activation of ERK1/2 and supression of Src pathway.