Effect Of RNAi-mediated Gene Silencing Of Livin On Apoptosis Of Human Pancreatic Cancer Cell Line PANC1

Objiective Livin is a member of the inhibitors of apoptosis protein (IAP) gene family, which encodes negative regulatory proteins that prevent cell apoptosis. Livin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to chemotherapeutic agents. Several studies in vitro and in vivo have demonstrated that down-regulation of Livin expression increases the apoptotic rate, reduces tumor growth potential and sensitized tumor cells to chemotherapeutic drugs. There is evidence that Livin gene high expression was typically detected in pancreatic cancer tissues,but not,or to substantially lower levels in the adjacent normal tissues. The aim of present study was to employ the sequence-specific siRNA silencing the Livin gene and investigate its effects on apoptosis of human pancreatic cancer cell line PANC1.Methods 1 structure of Livin mRNA was analysed by software to select targeted interference sites . Three siRNA and one negative control siRNA were constructed and chemically synthesized ,then be transfected into pancreatic cancer cell line PANC1 by positive ion liposome Lipofectamine2000.Transfect efficiency was assessed by flow cytometry. Use RT-PCR to detect the expression of Livin mRNA,then estimate the interference efficieny, screening the most effective siRNA. 2 After transfected by the most effective site,Use RT-PCR to detect the expression of Caspase-3 mRNA,Expressionof Caspase-3 protein was detected by spectrophotometric method,cell proliferation capacity was established by MTT,The apoptic rate were analysed by flow cytometry.Results 1 Negative control FAM-siRNA was successfully transfected into human pancreatic cancer cell line PANC1,The transfection efficiency was about 81.0% detected by FCM with cell density at 2×105/well and siRNA concentration with 80 nmol/L in 24 well play. 2 livin-siRNA were successfully transfected into PANC1 after 12h detected by RT-PCR,Livin mRNA levels in PANC1 cells was significantly inhibited,siRNA3 resulted in the highest inhibiting rate.3 Contrasted with negative control and blank,the expression of Caspase-3 mRNA and protein were obviously increased 24h,48h and 72h after si-RNA3 transfection,the cells proliferation was also strongly inhibited,the same results was found that the cells apoptosis rate was strongly increasedConculusions Livin-siRNA is successfully constructed and efficiently transfected into PANC1 cells.Livin-siRNA can efficiently inhibit the expression of Livin mRNA levels and on the contrary the expression of Caspase-3 mRNA and protein obviously increased .cells proliferation greatly decreased and cells apoptosis rate increased accordingly. All these suggested that livin gene could be a potential target for gene therapy of pancreatic carcinoma, RNAi is expected to be an effective measure for tumor gene therapy.