Research On The Correlation Between Epithelial Cell Adhesion Molecule And The Recurrence And Metastasis Of Hepatocellular Carcinoma

Objective In the therapeutic treatment of hepatocellular carcinoma(HCC),the heterogeneity and the lack of suitable biomarker impede the judgement of prognosis and the choice of therapeutic schedule.Epithelial cell adhesion molecule(EpCAM) is considered as an early biomarker of HCC.The aim of this research is to investigate the expression level and pattern of EpCAM in HCC,analyze the correlation between EpCAM and the recurrence,metastasis and prognosis of HCC,and explore the mechanism of EpCAM on biological behavior of HCC.Methods(1) Constructed EpCAM prokaryotic and eukaryotic expression plasmid and studied their expression characteristics.(2) Clinical follow-up data were obtained by a review of medical records of 153 patients who were diagnosed with HBV-related HCC from April 2006 to July 2009.Detected the expression of EpCAM extracellular domain(EpEX) and intracellular domain(EpICD) in HCC tissue sections by immunohistochemistry and immunofluorescence,retrospectively analyzed its correlation with HCC recurrence,metastasis and prognosis.(3) HCC tissues and preoperative plasma samples were collected from 34 HCC patients,who underwent curative resection during August 2013 to January 2014,and the clinical and follow-up materials were collected.Detected EpCAM mRNA expression level in HCC tissues by double-stranded curves method of relative quantification PCR, and tested the EpEX level in plasma by enzyme-linked immunosorbent assay,prospectively analyzed their correlation with clinical characteristics and HCC recurrence.(4) Transfected EpEX and EpICD specific shRNA plasmid into Huh7 cell line, studied the proliferation changes of Huh7 cells by CCK8 test,cell cycle distribution and apoptosis rate by flow cytometry, migration ability by would-healing test,and invasion ability by transwell chamber test.Results(1) We successfully amplfied the whole EpCAM gene sequence in HCC tissues and constructed the prokayotic and eukayotic expression plasmid.(2) In 153 HCC cases,the EpEX cytomembrane expression rate was 16.99%, the EpICD nuclear and the cytoplasmic expression rate were 59.48% and 100%, respectively. The expression rate of cytomembrane EpEX and nuclear EpICD in HCC tissues were higher than those in cancer adjacent tissues (16.99%vs 7.84%, p=0.015; 59.49% vs 15.03%, p<0.001).The expression rate of cytoplasmic EpICD showed no significant difference between HCC tissues and adjacent tissues (100%vs 98.69%, p=0.156).The expression rate of cytomembrane EpEX and nuclear EpICD were independent of HCC staging (p=0.282, p=0.097).A negative correlation was found betwccn the expression rate of cytomembrane EpEX and the age of patients.Both the expression rate of cytomembrane EpEX and nuclear EpICD correlated with the AFP level positively.The median tumor free survival time of the EpEX+EpICD+,EpEX-EpICD-,EpEX+EpICD-and EpEX’EpICD+HCC groups were 19 months,6 months,3 months and 6 months.The median overall survival time of the four groups were 46 months,42 months,28 months and 28 months.There were statistically significant differences between the tumor free survival rate and overall survival rate among the four HCC groups(p=0.008,p=0.041). HCC was prone to relapse earlier, and the rate of tumor free survival and overall survival were lower in EpEX+EpICD" group than in EpEX-EpICD- group (p=0.007,p<0.001) and EpEX+EpICD+group (p<0.001, p=0.004).The initial relapse of HCC mainly characterized by intrahepatic recurrence,which accounted for 62.20% of the total cases of recurrence.The EpEX-EpICD-group was more likely to occure vascular invasion and extrahepatic relapse(50%) than the EpEX+EpICD+(37.5%),EpEX-EpICD-(24%), and EpEX+EpICD-(0%) group.(3) The median normalized value of EpCAM mRNA in HCC tissues and cancer adjacent tissues (13.50×10-4、57.00x 10’4) were higher than those in normal liver tissues (7.00×10-4, p<0.05, p<0.01) and the value in cancer adjacent tissues were higher than in HCC tissues. High level of EpCAM mRNA in HCC tissues were more common in young patients and patients with microscopic tumor thrombs.The follow-up data showed that the tumor free survival rate of patients was lower in group with higher EpCAM mRNA level(one-year recurrence rate 52.94% vs 17.6%,Log-rank test:p=0.039).EpEX were detected in 11.36% plasma of HCC patients by ELISA,and the mean concentration was 5703.92±8846.46pg/ml(ranged from 61.49 to 18723.08 pg/ml).There was no statistical difference between HCC patients and healthy persons(240.87±112.97pg/ml) in the plasma level of EpEX(p=0.34).(4) The expression of EpCAM protein in Huh7 cell line was inhibited by EpEX and EpICD specific shRNA,which resulted in a weakened ability of cell proliferation,migration,invasion in vitro and an increased cell apoptosis rate.Cell cycle analysis revealed a decrease in S phage and an accumulation in G2 phage.Conclusions(1) EpEX+EpICD" in HCC tissues correlated with earlier recurrence and reduced overall survival rate in HCC patients.HCC with EpEX’EpICD+was more likely to occur vascular invasion and extrahepatic recurrence.(2) An increased expression level of EpCAM mRNA in HCC tissues may predict HCC recurrence.Due to the insufficiency of the EpEX detection rate and the sample size,its predictive value in HCC was limited.(3) The inhibition of EpCAM protein expression in Huh7 cell line resulted in a change in cell cycle, apoptosis rate, and weakened ability of cell proliferation,migration and invasion in vitro,which provide a theoretical basis for EpCAM antibody therapy in HCC.