| BackgroundHCC is the third most common cause of mortality due to cancer in the world, its incidence rate is increasing year by year. The incidence of hepatocellular carcinoma in China is second only to lung cancer and gastric cancer, and the mortality is in the top three in all tumors. Suigical resection is the preferred treatment, The low rate of early diagnosis renders the tumors unresectable and results in metastasis to the lymph nodes or distant organs. HCC cells are relatively resistant to chemotherapy; thus, the overall survival rate of chemotherapy is poor. The etiology of hepatocarcinogenesis is still not fully understood, the activation of oncogenes, dysfunction of tumor suppressor genes, and aberrant activation of signal transduction pathways have been described.Therefore, to investigate the role and mechanism of tumor related genes in the development of hepatocellular carcinoma may help us to understand the pathogenesis of hepatoce-llular carcinoma, and provides a new theoretical basis to early diagnosis and targeted cancer therapy research.Ezrin-radixin-moesin-binding phosphoprotein-50(EBP50) is a55-kDa phosphopr-otein, and it belongs to the family of PDZ scaffolding proteins. EBP50contains two PDZ domains and an ERM domain. PDZ domains have been implicated in protein-protein interactions, and the ERM domain binds to ezrin through its ERM-binding region[2].The role of EBP50as an oncogene or a tumor suppressor is controversial. Previous studies suggest that EBP50is a potential tumor suppressor in colorectal, breast, prostate. Shibata et al. found that45%of HCC cases expressed high levels of EBP50mRNA, and55%of HCC cases showed no difference. The cytoplasmic and nuclear accumulation of the EBP50protein was present in55%of HCC tissues compared with the surrounding noncancerous liver tissue. The role of EBP50as an oncogene or a tumor suppressor in HCC remains unclear.In order to understand the role and possible mechanism of EBP50in hepatocellular carcinoma, the the expression level of EBP50in HCC cell lines Hep3B, SMMC7721, and HepG2were examined by Western blot, screening the expression lower levels SMMC7721cell line. We constructed a stable transfected cell lines with EBP50overexpressing by pBK-CMV-HA-EBP50plasmid and anti-EBP50siRNA.To examine the ability of cell growth, apoptosis,migration, invasion after the transfection of EBP50in vivo, the expression level ofβ-catenin, E-cadherin after the transfection also detected. The effect of EBP50overexpressing on tumor growth in vivo was performed with a xenograft tumor model in nude mice.Methods1. Three human HCC cell lines, i.e., SMMC7721, HepG2and Hep3B, were used. The expression level of EBP50in HCC cell lines Hep3B, SMMC7721, and HepG2were examined by Western blot, and the expression levels in three HCC cell lines were compared.2. The SMMC7721cell line which expressed lower levels was chose to be transfected the Pbk-CMV-HA-EBP50plasmid and pBK-CMV-HA vector with Lipofectamine2000. stably transfected cell clones were cultured and amplified in culture medium supplemented with350μg/mL G418. the expression level of EBP50,β-catenin, E-cadherin before and after the transfection also detected by Western blot, to investigate mechanism or signal pathway of EBP50in the development of hepatocellular carcinoma.3. The cell-counting kit-8(CCK-8) colorimetric assay was used to measure cell proliferation and viability12、24、48、72h after transfection. 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 EBP50overexpressing on tumor growth and cell apoptosis in vivo was performed with a xenograft tumor model in nude mice.4. The SMMC7721cell line was transfected the anti-EBP50siRNA with Lipofectamine2000. The cell-counting kit-8(CCK-8) colorimetric assay was used to measure cell proliferation and viability12、24、48、72h after transfection. Changes of the proliferative and migration capacity of EBP50high expression SMMC7721cell lines was observed through the colony forming experimental. Cell apoptosis was demonstrated with Annexin V-FITC.Results:1. Western blot results showed that EBP50were detected in three kinds of liver cancer cell lines Hep3B, SMMC7721, HepG2,of which SMMC7721cell lines express the lowest, thus we selection SMMC7721cell lines to construct the EBP50high expression transfected cell lines.2. The pBK-CMV-HA-EBP50plasmid or the pBK-CMV-HA vector was transfected into SMMC7721cells with LipofectamineTM2000according to the manufacturer’s protocol. The transfection efficiency was determined with western blotting. It showed that compared with normal SMMC7721cell lines, the expression level of EBP50was significantly higher(1.36±0.07vs0.81±0.09, P<0.01). Stably transfected cell clones were cultured and amplified in culture medium supplemented with350μg/mLG418. The expression of EBP50after transfection was significantly higher in the SMMC7721cells was detected by Western blot, and the protein levels of β-catenin was downregulated (0.28±0.07vs0.56±0.12or0.58±0.08, P<0.05) and E-cadherin (0.55±0.08vs0.39±0.07or0.40±0.06, P<0.05). was upregulated in pBK-CMV-HA-EBP50-transfected cells.the interactions of EBP50with P-catenin/E-cadherin enhance the tumor suppressor properties of EBP50in HCC.3. The migrate and proliferative capacity of the SMMC7721cells that expressed high level of EBP50significantly reduced (P<0.05), Compared with the control cells. The percentage of pBK-CMV-HA-EBP50cells in G0/G1phase was increased to61.3%±3.1%from54.0%±2.4%in parental cells and54.5%±1.9%in mock cells (P<0.05). The pBK-CMV-HA-EBP50cells showed an approximately fourfold increase in apoptosis (P<0.05) compared with the parental group and the pBK-CMV-HA vector group(14.8±2.7%vs3.4±1.3%or4.1±1.5%,P<0.05). The result of transwell assay showed that upregulating EBP50expression in SMMC7721cells inhibited tumor cell invasion and migration(5.8±0.8vs21.6±1.3or20.4± 1.1%, P<0.01). tumors formed from pBK-CMV-HA-EBP50were significantly smaller than those from pBK-CMV-HA and control cells in the nude mice model(28.9±7.2mg vs70.1±7.2mg or68.9±7.9mg). The pBK-CMV-HA-EBP50cells showed an increase in apoptosis (P<0.05) compared with the parental group and the pBK-CMV-HA vector group in vivo(P<0.05).4. The migrate and proliferative capacity of the SMMC7721cells that expressed low level of EBP50significantly increased (P<0.05), Compared with the control cells. The anti-EBP50siRNA cells showed an decrease in apoptosis compared with the parental group (4.7±1.36%,2.7v2.11%, P<0.05). The result of colony forming experimental showed that downregulating EBP50expression in SMMC7721cells increased tumor cell invasion and migration(15±2.70'9±1.82, P<0.05).ConclusionThe overexpression of EBP50could inhibit the growth of SMMC7721cells and promote apoptosis by modulating β-catenin, E-cadherin. EBP50may serve as a potential therapeutic target in HCC. |
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