Stable Expression Of Livin Specific SiRNA Vector In Gastric Cancinoma Cells And Its Significance

Objective:Gastric cancer is one of the most common malignancies in our country.Most of patients with this disease are diagnosed in advanced stages and they lose the chance of radical surgical intervention.Systemic chemotherapy,the main alleviative treatment for advanced gastric carcinomas(AGC)is still unsatisfactory.With the basic research development,it is realized that the failure of treatment can be attributed to multi-drug resistance,while the resistance to apoptosis may be one of the important reasons of drug resistance.To investigate the mechanism of the resistance to apoptosis,and to induce tumor apoptosis become one of the hot spot of the research fields.Livin is a new member of inhibitor of apoptosis protein(IAP)family.It may play an important role in apoptosis resistance and has become the focus of research increasingly.Some studies have suggested that the relationship exist between livin up-regulation and the development of malignancies.However the outcome of livin up-regulation in gastric carcinomas remains uncertain. In the present study we investigated the expression of livin in gastric cancer cells,and constructed small interfering RNA eukaryotic expression vector specific to livin and transfected it into SGC-7901 cells.After the transfection livin expression was determined and the growth and apoptosis ability as well as the sensibility to chemotherapy agents of SGC-7901 cells were also analyzed.Methods:(1)Livin mRNA expression was detected in normal gastric mucosa and three gastric cancer cell lines(SGC-7901,AGS,BGC) respectively by reverse transcription polymerase chain reaction(RT-PCR). (2)The protein expression of livin was detected by Western blot technique. (3)The small interfering RNA eukaryotic expression vector specific to Livin was constructed by gene recombination and was transfected into SGC-7901 cells by Lipofectamin 2000.RT-PCR and Western blot were used to validate gene silencing efficiency of livin in SGC-7901 cells.The stable clones were obtained by G418 screening.(4)Flow cytometry was used to analyze cell apoptosis.Cell growth and 50%inhibition concentration(IC50)of 5-Fu and cisplatin on cell were determined by MTT assay.Results:(1)The expression of livin gene can be detected in all the three gastric cancer cell lines,but not in the normal gastric mucosa.The expression of livin protein was coherent with the expression of livin mRNA in those gastric cancer cells.(2)Four small interfering RNA eukaryotic expression vectors specific to livin were constructed by gene recombination. One of them could efficiently silenced livin expression.The inhibition of the gene was not less than 70%(P＜0.01).(3)The recombinated plasmids were extracted and transfected gastric cancer cells.The stable clones were obtained by G418 screening.(4)When livin gene was silenced,the proliferating activity of the gastric cancer cells was significantly lower than that of the control groups(P＜0.05).The IC50 of 5-Fu and cisplatin on gastric cancer cells treated by siRNA was decreased and the cells were more susceptible to proapoptotic stimuli(5-Fu and cisplatin)(P＜0.01).(5) The apoptosis of gastric cancer cells treated by siRNA was increased(P＜0.05)and when 5-Fu and cisplatin was added the increased cell apoptosis was further increased compared to the control groups(P＜0.01).Conclusions:(1)The expression of livin isoforms were detected in three gastric cancer cell lines but not in normal gastric mucosa;(2)SiRNA expression vector targeting livin gene can efficiently silence livin expression of SGC-7901 cells.(3)SGC-7901 cells with silenced livin gene exhibited low growth and high apoptosis ability,as well as increased susceptibility to chemotheraputic agents.Livin might act as a new target for apoptosis-inducing therapy of gastric cancinoma.