AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used....AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.展开更多
基金Supported by Fundamental Research Funds for the Central Universities of China,No.201130202020005
文摘AIM:To determine the effect and molecular mechanism of ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) in hepatocellular carcinoma(HCC).METHODS:Three human HCC cell lines,i.e.,SMMC7721,HepG2 and Hep3B,were used.We transfected the Pbk-CMV-HA-EBP50 plasmid into SMMC7721 cells with Lipofectamine 2000 to overexpress EBP50.Western blotting were performed to determine the effects of the plasmid on EBP50 expression and to detect the expression of β-catenin and E-cadherin before and after the transfection of the plasmid into SMMC7721 cells.In vitro cell proliferation was assessed with a Cell Counting Kit-8(CCK-8) assay.Cell cycle distribution was assessed with flow cytometry.Invasion and migration ability of before and after the transfection were determined with a transwell assay.Cell apoptosis was demonstrated with Annexin V-FITC.The effect of EBP50 overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.RESULTS:The transfection efficiency was confirmed with Western blotting(1.36 ± 0.07 vs 0.81 ± 0.09,P < 0.01).The CCK8 assay demonstrated that the growth of cells overexpressing EBP50 was significantly lower than control cells(P < 0.01).Cell cycle distribution showed there was a G0/G1 cell cycle arrest in cells overexpressing EBP50(61.3% ± 3.1% vs 54.0% ± 2.4%,P < 0.05).The transwell assay showed that cell invasion and migration were significantly inhibited in cells overexpressing EBP50 compared with control cells(5.8 ± 0.8 vs 21.6 ± 1.3,P < 0.01).Annexin V-FITC revealed that apoptosis was significantly increased in cells overexpressing EBP50 compared with control cells(14.8% ± 2.7% vs 3.4% ± 1.3%,P < 0.05).The expression of β-catenin was downregulated and E-cadherin was upregulated in cells overexpressing EBP50 compared with control cells(0.28 ± 0.07 vs 0.56 ± 0.12,P < 0.05;0.55 ± 0.08 vs 0.39 ± 0.07,P < 0.05).In vivo tumor growth assay confirmed that up-regulation of EBP50 could obviously slow the growth of HCC derived from SMMC7721 cells(28.9 ± 7.2 vs 70.1 ± 7.2,P < 0.01).CONCLUSION:The overexpression of EBP50 could inhibit the growth of SMMC7721 cells and promote apoptosis by modulating β-catenin,E-cadherin.EBP50 may serve asa potential therapeutic target in HCC.
