Research On The Expression And Function Of SIRT1 Gene In Cholangiocarcinoma Cells

Background:Cholangiocarcinoma is a kind of digestive tract malignant tumor that arised from biliary epithelial cells with extremely poor outcomes and increasing incidence,it could impair the health seriously and its molecular mechanism of carcinogenesis is still unclear.Molecular targeted therapy has always been a popular topic in tumor research,and SIRT1 has been widely studied in many types of cancer,but its role in tumorigenesis is still controversial.Research of SIRT1 may provide new ideas and means for molecular therapy and clear the pathogenesis of cholangiocarc ino ma.Objective:To compare the expression level of SIRT1 between two kinds of cholangiocarcinoma cells(QBC939 and HuCCT1)and normal bile duct epithelial cells,and to verify the interfering efficiency of SIRT1 small interfering RNA.The effects of downregulationof SIRT1 gene expression level on cholangiocarcinoma cell proliferation,apoptosis and migration ability were also explored.Methods:The difference of SIRT1 expression levels between normal bile duct epithelial cells and two cholangiocarcinoma cell lines was determined by Real-time RT-PCRand Westernblot.The expression of SIRT1 in cholangiocarcinoma cells was downregulated via RNA interference technique,the negative control siRNA(si-Control)and two individual SIRT1 siRNAs(si-SIRT1#1 and si-SIRT1#2)were transfected into human cholangiocarcinoma cell lines including QBC939 and HuCCT1 by using Lipofectamine RNAiMAX Reagent,cells were divided into three groups:negative control group(control)and SIRT1 siRNA transfection group 1(si1),SIRT1 siRNA transfection group 2(si2).The knockdown efficiency of SIRT1 siRNA was validated by Real-time RT-PCR and Western blot.After transfection,cell proliferation was determined by CCK8 and colony formation assay.Cell apoptosis was detected by flow cytometry.Cell migration was assessed by Transwell migration assay.The expression levels of apoptosis related protein Survivin,Bax,Cleaved PARP and epithelial cell marker E-cadherin and mesenchymal cell marker N-cadherin,vimentin were determined by Western blot.Results:Compared with normal bile duct epithelial cells,the expression of SIRT1 mRNA and protein were increased in both of the two kind cholangiocarcinoma cells(P<0.05).Gene silencing by transfecting SIRT1 siRNA efficiently reduced SIRT1 mRNA and protein levels.SIRT1 silence suppressed cell proliferation significantly as cell viability and colony formation of cholangiocarcinoma cells were reduced in CCK8 and colony formation assay obviously(P<0.05);meanwhile,cell apoptosis detected by flow cytometry was increased remarkly(P<0.05);the amount of cells migrated through the micropore membranes to the lower layer of transwell chambers was also reduced significantly in SIRT1 siRNA transfection groups(P<0.05),in other words,the migration ability was weakened obviously;Mechanistically,knockdown of SIRT1 gene downregulated the expressions of Survivin,mesenchymal cell markers N-cadherin and Vimentin,and upregulated Bax,Cleaved PARP and epithelial marker E-cadherin that detected by Western blbt futher(P<0.05).Conclusions:SIRT1 was upregulated in cholangiocarcinoma cells means SIRT1 could promote the tumorigenesis progress of cholangiocarcinoma and act as an oncogene.Knockdown of SIRT1 gene by siRNA could inhibit cancer cell proliferation,suppress cell migration,and induce apoptosis in cholangiocarcinoma cells.The underlying mechanism may be related to the reduction of Survivin,mesenchymal cell marker N-cadherin and Vimentin,whereas upregulation of Bax,Cleaved PARP and epithelial markers E-cadherin.SIRT1 may be a new potential target for molecular therapy in cholangiocarcinoma.