Effect of silent information regulator of transcription 1 on biological features of cholangiocarcinoma cells

Objective To explore the expression of silent information regulator of transcription 1 (SIRT1) in cholangiocarcinoma cells, and to investigate its effects on proliferation, apoptosis and migration of cholangiocarcinoma cells. Methods The expression of SIRT1 in human cholangiocarcinoma cell lines QBC939 and HuCCT1 were compared with that in normal bile duct epithelial cell line HIBEpiC at mRNA and protein level by real-time polymerase chain reaction (PCR) and Western blot. The QBC939 and HuCCT1 cells were divided into negative control group, the first sequence transfected group and the second sequence transfected group, transfected with negative control small interfering RNA(siRNA), siRNA sequence 1 for SIRT1 and siRNA sequence 2 for SIRT1, respectively. The interference effects were identified by real-time PCR and Western blot. After transfection, cell proliferation was determined by cell count kit and clone forming test, cell apoptosis was detected by flow cytometry, and cell migration was assessed by Transwell migration assay. The expression of apoptosis related protein survivin, B-cellymphoma-2 associated X protein (Bax), cleaved poly adenosine diphosphate-ribose polymerase (PARP) and epithelial cell marker E-cadherin and mesenchymal cell marker N-cadherin, vimentin were determined by Western blot. The t test was performed for statistical analysis. Results The expressions of SIRT1 at mRNA level in cholangiocarcinoma cell lines QBC939 and HuCCT1 were 3.30±0.43 and 4.55±0.50 times higher than that in normal bile duct epithelial cell line HIBEpiC, and the differences were statistically significant (t=7.29 and 9.79; both P<0.05). The increased expressions of SIRT1 in cholangiocarcinoma cell lines at protein level were similar to that at mRNA level. After transfected with SIRT1 siRNA, the expression of SIRT1 in cholangiocarcinoma cell lines significantly reduced at mRNA and protein levels. At 72 hour after transfecting, the cell viability rate of the first sequence transfected group and the second sequence transfected group in HuCCT1 cells were (19.72±1.39)% and (27.48±2.67)%, which were both lower than that of negative control group ((84.65±3.18)%), and the differences were statistically significant (t=37.42 and 27.54; both P<0.05). In HuCCT1 cells, the portion of early apoptosis cells of the first sequence transfected group and the second sequence transfected group were (19.43±1.72)% and (19.80±0.86)%, which were both significantly higher than that of negative control group ((5.43±0.31)%), and the differences were statistically significant (t=11.30 and 22.23; both P<0.05). In HuCCT1 cells, the number of migrated cells through the micropore membranes of the first sequence transfected group and the second sequence transfected group were 56.00±2.61 and 58.80±3.19, which were both lower than that of negative control group (76.60±5.08), and the differences were statistically significant (t=7.21 and 5.93; both P<0.05). The cell viability rate, the portion of early apoptosis cells and migration ability of QBC939 cells were similar to those of HuCCT1 cells. After the expression of SIRT1 in cholangiocarcinoma cells downregulated, the expressions at protein level of survivin, N-cadherin and vimentin remarkably reduced, and the expressions of Bax, cleaved PARP and E-cadherin upregulated. Conclusions The expression of SIRT1 is higher in cholangiocarcinoma cells. As the expression of SIRT1 in cholangiocarcinoma cells downregulates, cell proliferation and cell migration are inhibited, and cell apoptosis is induced. The mechanism may be related with the downregulation of survivin, upregulation of Bax and the suppression of epithelial-to-mesenchymal transition. Key words: Cholangiocarcinoma; SIRT1; RNA interference; Cell proliferation; Apoptosis; Cell movement