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Expression of WW Domain Containing Oxidoreductase Gene in Cholangiocarcinoma and Its Effect on the Biological Behavior of Cancer Cell Line QBC939

Author: ZhuKai
Tutor: HuangQiang
School: Anhui Medical University,
Course: Surgery
Keywords: Cholangiocarcinoma Immunohistochemistry Gene expression Apoptosis Inbreeding BALB C
CLC: R735.8
Type: Master's thesis
Year: 2011
Downloads: 4
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Abstract


Introduction Cholangiocarcinoma is a devastating malignancy that presents late, is notoriously difficult to diagnose, and is associated with a high mortality. The tumor arises from the ductile epithelium of the biliary tree, either within the liver (intrahepatic cholangiocarcinoma) or more commonly from the extrahepatic bile ducts (extrahepatic cholangiocarcinoma). Studies demonstrated that WWOX expression is lost or reduced in a variety of human malignancies and more recent studies have shown that WWOX protein expression is reduced or lost in tumor cells compared to normal cells. We first detected the expression of WWOX protein by immunohistochemistry in cholangiocarcinoma and normal bile duct tissues, and then stable transfected cholangiocarcinoma cell line QBC939/WWOX was established. We studied the effect of the expression of the exogenous WWOX gene on the cells growth, proliferation and apoptosis in vitro and in vivo.Objective To investigate the effect of transfection of WWOX gene on the apoptosis of human cholangiocarcinoma QBC939 cells in vitro and in vivo and its possible mechanism.Methods the expression of WWOX protein was detected with immunohistochemical method-SP in patients with cholangiocarcinoma and normal bile duct tissues.The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection and positive cell clones were selected and amplified.The mRNA and protein expressions in QBC939 cells stably transfected with WWOX were investigated by Quantitative RT-PCR and Western blotting before and after transfection.Cell proliferation was tested by MTT, Cell apoptosis was assessed by FCM, The alteration of mitochondria membrane potential (△Ψm) was detected by JC-l staining method, Cell invasion was determined by Transwell chamber assay. The expression change of bcl-2、bax、FasL、caspase-3 mRNA and protein was detected by Quantitative RT-PCR and Western blotting. All the experimental subjects were divided into three groups:QBC939 was cultured in natural status;QBC939 was transfected with blank plasmid (QBC939/con group) and QBC939 was transfected with WWOX/pmCherry-N1 (QBC939/WWOX group). The three groups were subcutaneously inoculated into nude mice and the growth of xenografted tumor was observed. TUNEL Was used to evaluate the apoptosis.Results The expression of WWOX protein was significantly lower in cholangiocarcinoma than that in normal bile duct tissues and loss of WWOX protein expression was found in 40.74 % of cholangiocarcinoma specimens (P<0.05) . QBC939 cells with stable transfection of WWOX were established.Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased the proliferation of QBC939 cells (P<0.05), FCM analysis showed that the apoptosis rate after transfection was significantly promoted (P<0.01), JC-1 staining method indicated that the experimental group was loss of△Ψm (P<0.01), transwell chamber assay showed that the number of transfected cells that passed the transwell membrane was significantly less than those of control groups (P<0.01). Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased and the expression of bax、caspase-3 were significantly increased. There was no significant change in the expression of Fasl. The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down (P<0.05). Significant number of apoptotic cells were seen in the QBC939/WWOX group (P<0.01).Conlusion Alterations of WWOX expression may be involved in the tumorigenesis of cholangiocarcinoma, WWOX exerts its antitumor effect against proliferation through inducing cell apoptosis. It may be associated with downregulation of bcl-2 expression, activation of the mitochondrial apoptosis pathway.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Gallbladder, bile duct cancer
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