Effect of transfection of WWOX gene on apoptosis of cholangiocarcinoma QBC939 cells and its mechanism

Objective To investigate the effect of transfection of WWOX gene on apoptosis of human cholangiocarcinoma QBC939 cells in vitro andin vivoand its possible mechanism.Methods The recombinant WWOX eukaryotic expression plasmid was introduced into QBC939 cells by liposome-mediated transfection.The mRNA and protein expression levels in QBC939 cells stably transfected with WWOX were detected by using quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting before and after transfection.Cell apoptosis was assessed by flow cytometry (FCM).The alteration of mitochondria membrane potential ( △ Ψm) was assayed by JC-l staining method.The expression change of bcl-2 mRNA and protein was examined by using quantitative RT-PCR and Western blotting.All the experimental subjects were divided into three groups:QBC939 cells were cultured in natural status (nature control group) ; QBC939 cells were transfected with blank plasmid (blank control group),and QBC939 cells were transfected with WWOX/pmCherry-N1 (experimental 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 QBC939 cells with stable transfection of WWOX were established.QuantitativeRT-PCR showed that the expression of WWOX mRNA was significantly enhanced and Western blotting demonstrated that WWOX protein expression was markedly increased.FCM analysis showed that the apoptosis rate after transfection was significantly promoted [(1.24 ±0.35)％ vs (1.73 ±0.48)％ vs (21.40 ±2.35)％,P＜0.01].JC-1 staining method indicated that the experimental group was loss of △Ψm [ (4.27 ± 0.64) ％ vs (4.96 ± 0.52 ) ％ vs (28.60 ± 3.94 ) ％,P ＜ 0.01 ].Quantitative RT-PCR and Western blotting showed that the expression of bcl-2 mRNA and protein was markedly decreased (P ＜O.05 ).The growth of implanted tumor of WWOX/pmCherry-N1 cells was significantly slowed down (P ＜0.05 ).Number of apoptotic cells was significantly increased in the experimental group [ (4.1 ± 0.6) ％ vs (4.4±0.5)％ vs (13.6 ± 1.5)％,P＜0.01].Conclusion WWOX gene can promote apoptosis of cholangiocarcinoma cells,which may be associated with downregulation of bcl-2 expression,and activation of the mitochondrial apoptosis pathway. Key words: Cholangiocarcinoma;  Gene transfection;  Apoptosis