WWOX在胆管癌组织中的表达及其对胆管癌细胞生物学行为的影响

Expression of WW domain containing oxidoreductase gene in cholangiocarcinoma and its effect onthe biological behavior of cancer cell line RBE

目的研究抑癌基因WWOX表达对胆管癌RBE细胞生长的影响。方法采用免疫组化方法检测2005年7月至2010年5月54例胆管癌组织、12例正常胆管组织中WWOX蛋白表达水平。将携有WWOX基因的真核表达载体转染胆管癌细胞系RBE细胞（RBE／WWOX组），筛选稳定转染的细胞并扩增培养，以转染空载质粒（RBE／con组）及未经转染（自然生长组）的RBE细胞作为对照。荧光定量RT－PCR和Western Blot法检测WWOX在RBE细胞中的表达情况；噻唑蓝实验检测转染前后各组细胞增殖活性；FCM法检测各组细胞的凋亡；JC－1染色法检测细胞线粒体膜电位；Transwell小室侵袭实验检测各组肿瘤细胞侵袭力；荧光定量RT—PCR和Western Blot法检测胆管癌细胞bcl-2、bax、FasL、caspase-3表达的变化。结果WWOX在胆管癌中的表达低于正常胆管组织（P〈0．05），蛋白表达的缺失频率为40．7％。建立稳定表达WWOX基因的RBE／WWOX细胞株，mRNA及蛋白表达明显增加。转染后的RBE细胞噻唑蓝吸光度明显下降（P〈0．05）。与自然生长组和RBE/con组比较，FCM显示RBE／WWOX组细胞的凋亡率明显增高（P〈0．01），JC－1显示转染后的线粒体膜电位下降（P〈0．01），侵袭实验显示转移至下室滤膜的细胞数明显减少（P〈0．01）。荧光定量RT-PCR结果显示bcl－2mRNA表达是自然生长组的0．12倍，bax、caspase-3mRNA分别是自然生长组的4．72和2．57倍，FasLmRNA的表达无明显变化；Western Blot法检测发现bcl－2蛋白的表达降低，bax、caspase-3蛋白表达升高，FasL无明显变化。结论抑癌基因WWOX通过诱导胆管癌细胞凋亡发挥抗肿瘤增殖的作用。

Objective To study the effects of anti-oncogene WWOX on cell growth of cholangiocarcinoma. Methods The expression of WWOX protein was detected with immunohistochemical method-SP in 54 patients with cholangiocarcinoma from July 2005 to May 2010 and 12 samples of normal bile duct tissues. The recombinant WWOX eukaryotic expression plasmid was introduced into RBE cells by liposome-mediated transfection and positive cell clones were selected and amplified. The mRNA and protein expressions in RBE cells stably transfected with WWOX were investigated by quantitative RT-PCR and Western Blot before and after transfection. Cell proliferation was tested by MTT, cell apoptosis was assessed by FCM, the alteration of mitochondria membrane potential （Aψm） was detected by JC-1 staining method, cell invasion was determined by Transwell chamber assay. The expression change of bcl-2, bax, FasL, easpase-3 mRNA and protein was detected by quantitative RT-PCR and Western Blot. 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. 7% of cholangiocarcinoma specimens （ P 〈 0. 05 ）. RBE cells with stable transfection of WWOX were established. Quantitative RT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western Blot demonstrated that WWOX protein expression was markedly increased. MTT showed that WWOX gene transfection significantly decreased theproliferation of RBE cells （ P 〈 0.05 ）. FCM analysis showed that the apoptosis rate after transfection was significantly promoted [ （1.1±0. 6）% vs. （1.7±0. 5）% vs. （35.2 ±4. 4）% ,P 〈0. 01 ], JC-1 staining method indicated that the experimental group was loss of Aψm [ （ 12. 6± 1.9 ） % vs. （ 13.6 ± 1.8 ） % vs. （48. 7 ±2.9）%, P 〈 0. 01 ], transwell chamber assay showed that the number of transfected ceils that passed the transwell membrane was significantly less than those of control groups （77 ± 6 vs. 72 ± 8 vs. 48 ± 6, 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. Conclusion WWOX exerts its antitumor effect against proliferation through inducing cell apoptosis in cholangiocarcinoma.