上皮细胞粘附分子在肝癌中的表达及转移中的作用
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摘要
目的：
探讨EpCAM在肝癌组织的表达与临床病理相关性及患者预后的关系；研究EpCAM在多种肝癌细胞系的表达,选出进一步研究EpCAM调节肝癌浸润转移机制的合适细胞系。
方法：
1、收集2005年9月至2006年11月于天津医科大学附属肿瘤医院手术切除的符合试验条件(见表1-1)的106例肝细胞癌患者的临床病理资料,采用免疫组织化学方法检测标本中EpCAM的表达状况,分析EpCAM的表达情况与临床病理因素之间的关系,进一步进行生存分析和Logistic回归研究,分析各因素对肝癌预后预测的价值,尤其是EpCAM的表达情况与肝癌复发转移的相关性。为进一步研究EpCAM调节肝癌浸润转移提供前期工作。
2、RT-PCR及Western blot研究EpCAM在多种肝癌细胞系的表达,筛选EpCAM调节肝癌浸润转移机制的合适细胞系。
结果：
1、EpCAM在肝癌组织中呈过表达,采用免疫组化技术检测106例肝癌中EpCAM的表达,发现其主要定位于胞膜,部分弥散到胞浆。
我们比较了随访后转移状态不同肝癌间EpCAM的表达状况。106例肝癌密切随访,3年复发率50.9%(54/106)。EpCAM在有转移复发肝癌中表达的阳性率70.4%(38／54),无转移复发肝癌中EpCAM表达的阳性率19.2%(10／52),有转移复发肝癌的EpCAM免疫组化染色评分为7.11±1.32,无转移复发肝癌的免疫组化染色评分4.63±2.72 (P<0.05)。EpCAM的表达情况在患者的性别、年龄、HBsAg、AFP、Edmonson分级、肝硬化程度、肿瘤侵犯血管、术前肝功能Child-Pugh分级方面的差异没有统计学意义(P>0.05),而在HCV、肿瘤直径、肿瘤形态和有无包膜方面的差异有统计学意义(P<0.05)。单因素分析显示：HBsAg、HCV、AFP、肝硬化程度、Edmonson分级、肿瘤直径、肿瘤形态、包膜有无、肿瘤血管侵犯及EpCAM是影响患者无瘤生存时间的危险因素,而患者性别、年龄、和术前Child-Pugh分级为混杂因素(P>0.05)。COX回归显示患者HCV、HBV、AFP、肿瘤直径、肝硬化程度、肿瘤侵犯血管及EpCAM表达情况是影响患者无瘤生存时间的独立危险因素(P<0.05)。Logistic回归显示EpCAM表达情况、肿瘤直径大小、患者肝硬化程度、肿瘤侵犯血管、HCV、HBsAg及AFP是影响患者复发／转移的独立危险因素。
2、RT-PCR和Western blot结果显示,Hep3B和HepG2细胞系的EpCAM mRNA和蛋白水平表达均较高,以Hep3B为最高。
结论：
肝细胞肝癌中存在不同程度的EpCAM的表达,其表达与否是预测肝癌术后转移复发的独立危险因素。EpCAM的表达与肝癌的转移状态呈正相关；研究结果显示EpCAM在肝癌进展中有着促癌基因的作用,为EpCAM在肝癌复发转移的分子诊断和有针对性的研发药物提供了理论依据。
Objectives
(1)To investigate the expression of EpCAM in hepatocellular carcinoma and the relationship of expression intensity with the clinicopathologic characteristics of hepatocellular carcinoma, especially the relationship with metastasis.(2)To examine the expression of EpCAM in hepatocellular carcinoma cells, and to choose the suitabale cell lines for further analysis of metastatic mechanisms of hepatocellular carcinoma.
Methods
(1)The subcellular location and expression of EpCAM in hepatocellular carcinoma, including metastatic and non-metastatic cases were determined by immunohistochemistry analysis.
(2)Semiquantitative RT-PCR and Western blot were applied to detect the EpCAM mRNA and protein level in hepatocellular carcinoma cell lines.
Results
1. EpCAM was over-expressed in hepatocellular carcinoma tissues and cell lines, especially in metastatic hepatocellular carcinoma. Immunohistochemistry carried out on 106 paraffin-embedded hepatocellular carcinoma sections showed that EpCAM was predominantly expressed in the membrane of liver cells and occasionally diffused into cytoplasm. In hepatocellular carcinoma tissues,45.3% (48of 106 cases) was overexpressed. Recurrences were found in 54 patients during 3 years follow-up, Positive expression rate 70.4% (38/54) of EpCAM in hepatocellular carcinoma with recurrence or metastasis is higher than those without metastasis hepatocellular, expression of EpCAM positive rate of 19.2%(10/52). Liver metastasis EpCAM immunohistochemistry score was 7.11±1.32, also significantly higher than those without metastasis of liver cancer immunohistochemical staining score 4.63±2.72 (P <0.05.) Further analysis of EpCAM expression and clinicopathological features of the patients revealed a negative association of its expression with sex, age, HBsAg, AFP, liver cirrhosis,Edmonson stage,tumor vascular invasion, preoperative Child-Pugh classification P>0.05). However,significant difference was found in tumor size, shape of tumor and Tumor capsule formation (present/absent) among the two groups(P<0.05). Kaplan-Meier analysis demonstrated HBsAg、HCV、AFP,liver cirrhosis, tumor diameter, shape of tumor, Edmonson classification, Tumor capsule formation (present/absent), tumor vascular invasion and EpCAM expression were significant factors influencing the disease-free survival of HCC patients(P<0.05). COX regrssion showed HBsAg, HCV, AFP, liver cirrhosis, tumor diameter, tumor vascular invasion and EpCAM expression were significant risk factors influencing the disease-free survival of HCC patients(P<0.05). Logistic regression indicated that HBsAg, HCV, AFP, liver cirrhosis, tumor diameter, tumor vascular invasion and EpCAM experssion were risk factors of tumor metastasis and recurrence (P<0.05).
2、Semi-quantitative RT-PCR and Western blot analysis on hepatocellular carcinoma cell lines, MHCC 97L,SMMC-7721,QGY-7703,Hep3B and HepG2 revealed that EpCAM was highly-expressed in both of mRNA and protein levels in Hep3B and HepG2 cell lines.
Conclusions
EpCAM is highly expressed in some hepatocellular carcinoma tissues, which is an independent risk factor in predicting the metastasis and recurrence of HCC. The present work gives the convincing evidence of EpCAM as the oncogene in HCC and paves the way for EpCAM as a novel candidate of diagnosis and treatment of HCC metastasis.
探讨EpCAM在肝癌组织的表达与临床病理相关性及患者预后的关系；研究EpCAM在多种肝癌细胞系的表达,选出进一步研究EpCAM调节肝癌浸润转移机制的合适细胞系。
方法：
1、收集2005年9月至2006年11月于天津医科大学附属肿瘤医院手术切除的符合试验条件(见表1-1)的106例肝细胞癌患者的临床病理资料,采用免疫组织化学方法检测标本中EpCAM的表达状况,分析EpCAM的表达情况与临床病理因素之间的关系,进一步进行生存分析和Logistic回归研究,分析各因素对肝癌预后预测的价值,尤其是EpCAM的表达情况与肝癌复发转移的相关性。为进一步研究EpCAM调节肝癌浸润转移提供前期工作。
2、RT-PCR及Western blot研究EpCAM在多种肝癌细胞系的表达,筛选EpCAM调节肝癌浸润转移机制的合适细胞系。
结果：
1、EpCAM在肝癌组织中呈过表达,采用免疫组化技术检测106例肝癌中EpCAM的表达,发现其主要定位于胞膜,部分弥散到胞浆。
我们比较了随访后转移状态不同肝癌间EpCAM的表达状况。106例肝癌密切随访,3年复发率50.9%(54/106)。EpCAM在有转移复发肝癌中表达的阳性率70.4%(38／54),无转移复发肝癌中EpCAM表达的阳性率19.2%(10／52),有转移复发肝癌的EpCAM免疫组化染色评分为7.11±1.32,无转移复发肝癌的免疫组化染色评分4.63±2.72 (P<0.05)。EpCAM的表达情况在患者的性别、年龄、HBsAg、AFP、Edmonson分级、肝硬化程度、肿瘤侵犯血管、术前肝功能Child-Pugh分级方面的差异没有统计学意义(P>0.05),而在HCV、肿瘤直径、肿瘤形态和有无包膜方面的差异有统计学意义(P<0.05)。单因素分析显示：HBsAg、HCV、AFP、肝硬化程度、Edmonson分级、肿瘤直径、肿瘤形态、包膜有无、肿瘤血管侵犯及EpCAM是影响患者无瘤生存时间的危险因素,而患者性别、年龄、和术前Child-Pugh分级为混杂因素(P>0.05)。COX回归显示患者HCV、HBV、AFP、肿瘤直径、肝硬化程度、肿瘤侵犯血管及EpCAM表达情况是影响患者无瘤生存时间的独立危险因素(P<0.05)。Logistic回归显示EpCAM表达情况、肿瘤直径大小、患者肝硬化程度、肿瘤侵犯血管、HCV、HBsAg及AFP是影响患者复发／转移的独立危险因素。
2、RT-PCR和Western blot结果显示,Hep3B和HepG2细胞系的EpCAM mRNA和蛋白水平表达均较高,以Hep3B为最高。
结论：
肝细胞肝癌中存在不同程度的EpCAM的表达,其表达与否是预测肝癌术后转移复发的独立危险因素。EpCAM的表达与肝癌的转移状态呈正相关；研究结果显示EpCAM在肝癌进展中有着促癌基因的作用,为EpCAM在肝癌复发转移的分子诊断和有针对性的研发药物提供了理论依据。
Objectives
(1)To investigate the expression of EpCAM in hepatocellular carcinoma and the relationship of expression intensity with the clinicopathologic characteristics of hepatocellular carcinoma, especially the relationship with metastasis.(2)To examine the expression of EpCAM in hepatocellular carcinoma cells, and to choose the suitabale cell lines for further analysis of metastatic mechanisms of hepatocellular carcinoma.
Methods
(1)The subcellular location and expression of EpCAM in hepatocellular carcinoma, including metastatic and non-metastatic cases were determined by immunohistochemistry analysis.
(2)Semiquantitative RT-PCR and Western blot were applied to detect the EpCAM mRNA and protein level in hepatocellular carcinoma cell lines.
Results
1. EpCAM was over-expressed in hepatocellular carcinoma tissues and cell lines, especially in metastatic hepatocellular carcinoma. Immunohistochemistry carried out on 106 paraffin-embedded hepatocellular carcinoma sections showed that EpCAM was predominantly expressed in the membrane of liver cells and occasionally diffused into cytoplasm. In hepatocellular carcinoma tissues,45.3% (48of 106 cases) was overexpressed. Recurrences were found in 54 patients during 3 years follow-up, Positive expression rate 70.4% (38/54) of EpCAM in hepatocellular carcinoma with recurrence or metastasis is higher than those without metastasis hepatocellular, expression of EpCAM positive rate of 19.2%(10/52). Liver metastasis EpCAM immunohistochemistry score was 7.11±1.32, also significantly higher than those without metastasis of liver cancer immunohistochemical staining score 4.63±2.72 (P <0.05.) Further analysis of EpCAM expression and clinicopathological features of the patients revealed a negative association of its expression with sex, age, HBsAg, AFP, liver cirrhosis,Edmonson stage,tumor vascular invasion, preoperative Child-Pugh classification P>0.05). However,significant difference was found in tumor size, shape of tumor and Tumor capsule formation (present/absent) among the two groups(P<0.05). Kaplan-Meier analysis demonstrated HBsAg、HCV、AFP,liver cirrhosis, tumor diameter, shape of tumor, Edmonson classification, Tumor capsule formation (present/absent), tumor vascular invasion and EpCAM expression were significant factors influencing the disease-free survival of HCC patients(P<0.05). COX regrssion showed HBsAg, HCV, AFP, liver cirrhosis, tumor diameter, tumor vascular invasion and EpCAM expression were significant risk factors influencing the disease-free survival of HCC patients(P<0.05). Logistic regression indicated that HBsAg, HCV, AFP, liver cirrhosis, tumor diameter, tumor vascular invasion and EpCAM experssion were risk factors of tumor metastasis and recurrence (P<0.05).
2、Semi-quantitative RT-PCR and Western blot analysis on hepatocellular carcinoma cell lines, MHCC 97L,SMMC-7721,QGY-7703,Hep3B and HepG2 revealed that EpCAM was highly-expressed in both of mRNA and protein levels in Hep3B and HepG2 cell lines.
Conclusions
EpCAM is highly expressed in some hepatocellular carcinoma tissues, which is an independent risk factor in predicting the metastasis and recurrence of HCC. The present work gives the convincing evidence of EpCAM as the oncogene in HCC and paves the way for EpCAM as a novel candidate of diagnosis and treatment of HCC metastasis.
引文
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