| Objective:To investigate the expression level of metastasis-associated 1 (MTA1)in hepatocellular carcinoma tissue and hepatocellular carcinoma cell lines with varying invasive and metastatic ability, and to explore the correlation of expression level of MTA1 with invasion, metastasis and biological behavior of hepatocellular carcinoma.Methods:1. The expression of MTA1, nm23 and c-myc in hepatocellular carcinoma and their correlations:Paraffin-embedded liver tissue samples and fresh liver tissue samples are obtained from Provincial Hospital Affiliated to Shandong University.The principle of selecting Cases:①the clinical datas of selected cases are complete;②the selected cases without any preoperative antineoplastic therapy;③the pathology of selected cases are confirmed for hepatocellular carcinoma.30 cases of Paraffin-embedded liver tissue samples are selected from the archive paraffin block of surgical resection in this hospital January 2007 to december 2008,each paraffin block contains the hepatocellular carcinoma tissues and corresponding adjacent liver tissues,the selected paraffin-embedded tumor tissue samples are made into 4-5μm thick serial sections,used in immunohistochemical.30 cases of fresh tumor tissue samples taken from the hospital surgical resection cases, January to May in 2009, the hepatocellular carcinoma tissue samples are taken from the center of the fresh liver tumor tissues without haemorrhage or necrosis,and the corresponding adjacent liver tissue samples taken from the edge of 3-5cm adjacent tissues, respectively, part preserved in -80℃refrigerator for RNA extraction; The other part fixed in 10% formalin for 24 hours,paraffin embedded, made into 4-5μm thick serial sections for immunohistochemistry.Immunohistochemistry and semi-quantitative RT-PCR were used to detect the MTAl mRNA and protein in hepatocellular carcinoma tissues and corresponding adjacent liver tissues. The relationship between MTA1 expression and clinical pathological features were analyzed.2. The study of MTA1 in hepatocellular carcinoma cell lines:Hepatocellular carcinoma cell lines with varying invasive and metastatic ability which are used in the experiment are human normal hepatic cell line L02, human hepatocellular carcinoma cell lines MHCC97-H, SMMC7721and HepG2.Real-time PT-PCR and Western blot were used to detect the MTA1 mRNA and protein in human normal hepatic cell line L02, human hepatocellular carcinoma cell lines MHCC97-H, SMMC-7721 and HepG2;Transwell chamber in vitro cell invasion test was used to detect the invasion ability of the cell lines.Results:1. Immunohistochemical results show:MTA1 protein mainly expressed in the nucleus, showing brown granular, MTA1 protein in liver cancer, positive expression rate was 65.0% (39/60), significantly higher than in adjacent tissues (30.0%,18/60), the statistical differences between them were significant(x2=14.737,P=0.000). and the expression of MTAl protein in hepatocellular carcinoma tissue was associated with tumor differentiation, the presence of intrahepatic, extrahepatic metastasis, portal vein tumor thrombus (P<0.05).2. Semi-quantitative RT-PCR analysis show:The average relative expression level of MTA1 mRNA in hepatocellular carcinoma(0.406±0.037), was significantly higher than that in the corresponding adjacent tissues (0.189±0.021), the statistical differences between them were significant (t=4.077, P=0.005<0.05).the expression of MTAl mRNA in hepatocellular carcinoma tissue was associated with tumor differentiation, the presence of intrahepatic, extrahepatic metastasis, portal vein tumor thrombus (P<0.05) consistent with immunohistochemical results.3. Spearman correlation analysis showed that between the expression of MTA1 and nm23 there was negative correlation (rs=-0.625,P<0.01), the expression of MTA1 and c-myc was positive correlation (rs=0.392, P<0.05).4. The real-time quantitative RT-PCR analysis show:with human normal hepatic cell line L02 as control, The average relative expression level of MTA1 protein in the cell line SMMC7721, MHCC97-H, HepG2,respectively0.336,0.665,0.250, ANOVA analysis showed that The expression of MTAl mRNA in cell lines with varying invasive ability different cell lines was the highest in MHCC97-H, the second in SMMC7721, and then in HepG2, the lowest in L02, and the statistical differences between each tow groups were significant (P<0.05)5. The Western blot gel image analysis show:The average relative expression level of MTA1 protein in the cell line L02, SMMC7721, MHCC97-H, HepG2, respectively 0.050,0.392,0.615,0.237, ANOVA analysis showed that The expression of MTA1 protein in cell lines with varying invasive ability different cell lines was the highest in MHCC97-H, the second in SMMC7721, and then in HepG2, the lowest in L02, and the statistical differences between each tow groups were significant (P< 0.05)6. Transwell chamber in vitro cell invasion test show:the average number of penetrating cells in cell line MHCC97-H, SMMC7721, HepG2, L02, were (14.17±1.18)/at high magnification vision, (8.83±0.53)/at high magnification vision, (3.26±1.04)/at high magnification vision, (0.00)/at high magnification vision, and the statistical differences between each tow groups were significant (P<0.05)Conclusions:1. The expression of MTA1 mRNA and protein in hepatocellular carcinoma tissue was significantly higher than that in corresponding adjacent liver tissue, and the expression of MTA1 mRNA and protein in hepatocellular carcinoma tissue was associated with tumor differentiation, the presence of intrahepatic, extrahepatic metastasis, portal vein tumor thrombus. Between the expression of MTA1 and nm23 there was negative correlation, the expression of MTA1 and c-myc was positive correlation.MTA1 may be closely associated with the invasion and metastasis of hepatocellular carcinoma. Combined detection of three indexes can be used to estimate the biological behavior of hepatocellular carcinoma, help to the clinical treatment of hepatocellular carcinoma.2. The expressions of MTA1 mRNA and protein in cell lines with varying invasive ability different cell lines were the highest in MHCC97-H, the second in SMMC7721, and then in HepG2, the lowest in L02. Transwell chamber in vitro cell invasion test showed that the cell line invasive and metastatic ability was the highest in MHCC97-H, the second in SMMC7721, and then in HepG2, the lowest in L02. There may be a relationship between the level of MTA1 and invasive potentiality of human hepatocellular carcinoma cell lines. MTAl may play a role in the molecularmechanism of promoting migration and invasion of hepatocellular carcinoma cell lines and be a potential target for gene therapy of hepatocellular carcinoma. Further studies of MTA1 in human hepatocellular carcinoma are warranted. |
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