Effect Of Antisense Oligodeoxynucleotide Targeting Survivin On Hilar Cholangiocarcinoma Cell Line FRH-0201

Objective:To investigate the effect of antisense oligodeoxynucleotide targeting survivin(survivin ASODN)on the proliferation and apoptosis of hilar cholangiocarcinoma cell line FRH-0201 by depressing the expression of survivin;To investigate the effect of survivin ASODN on the expression level of Caspase-3,Bax and Bcl-2 proteins.Methods:This experiment was composed of two parts.(1)survivin ASODN induced the apoptosis of hilar cholangiocarcinoma cell line FRH-0201.survivin ASODN was transfected into hilar cholangiocarcinoma cell line FRH-0201 by liposome,meanwhile,sense oligodeoxynucleotide(SODN),liposome and RPM1640 medium served as controls.Morphological changes were observed by inverted phase contrast microscope;RT-PCR and Western blot methods were performed to detect the expressions of survivin mRNA and protein respectively;The changes in cell apoptosis were detected by flow cytometry.(2)The expression levels of Caspase-3,Bax and Bcl-2 proteins were detected by Western blot methods in survivin ASODN group, survivin SODN group and the control group respectively.Results:1.Survivin ASODN induces the apoptosis of hilar cholangiocarcinoma cell line FRH-0201.(1)Morphological apoptotic changes were observed.(2)The expressions of survivin mRNA and protein were significantly decreased in survivin ASODN group than in the control group and survivin SODN group (mRNA 0.51±0.03 vs 0.82±0.02,0.79±0.04,P＜0.05;protein 1.82±0.16 vs 3.08±0.38,2.81±0.11,P＜0.05),no significantly statistical differences were found in the expressions of survivin mRNA and protein betwen survivin SODN group and the control group(mRNA 0.79±0.04 vs 0.82±0.02,P＞0.05;protein 2.81±0.11 vs 3.08±0.38,P＞0.05).(3)The apoptosis rate detected with flow cytometry was increased in survivin ASODN group than in the control group and survivin SODN group(11.50±1.49%vs 0.39±0.08%,0.98±0.07%,P＜0.05),no significantly statistical differences were found in the apoptosis rate betwen survivin SODN group and the control group(0.98±0.07% vs 0.39±0.08%,P＞0.05).2.The expression levels of Caspase-3,Bax and Bcl-2 proteins were detected by Western blot methods in survivin ASODN group,survivin SODN group and the control group respectively.(1)The expression level of Caspase-3 protein was increased in survivin ASODN group than in the control group and survivin SODN group(1.25±0.22 vs 0.51±0.19, 0.59±0.17,P＜0.05),no significantly statistical differences were found in the apoptosis rate between survivin SODN group and the control group(0.59±0.17 vs 0.51±0.19,P＞0.05).(2)The expression level of Bax protein was increased in survivin ASODN group than in the control group and survivin SODN group(0.49±0.15 vs 0.21±0.10, 0.23±0.08,P＜0.05),no significantly statistical differences were found in the apoptosis rate between survivin SODN group and the control group(0.23±0.08 vs 0.21±0.10,P＞0.05).(3)The expression level of Bcl-2 protein was decreased in survivin ASODN group than in the control group and survivin SODN group(0.17±0.05 vs 0.88±0.11, 0.76±0.08,P＜0.05),no significantly statistical differences were found in the apoptosis rate between survivin SODN group and the control group(0.76±0.08 vs 0.88±0.11,P＞0.05).Conclusions:(1)Survivin ASODN can induce hilar cholangiocarcinoma cell line FRH-0201 into apoptosis by decreasing the expression of survivin.(2)Survivin ASODN can up-regulate the expression levels of Caspase-3,Bax and down-regulate the expression of Bcl-2,which may be the mechanism of inducing hilar cholangiocarcinoma cell into apoptosis.