Effect Of NS-398 And Surviving Antisense Oligonucleotide On The Proliferation And Apoptosis Of Human Hepatocellular Carcinoma

Hepatic cell carcinoma(HCC)is one of malignant tumor which prevalence rate and mortality rate are very high in the world.The mortality rate of HCC in our country is the highest in the world.The therapeutic efficiency of HCC were not ideal for its invasive diversion in the earlier period.The HCC recurrence rate were very high.According to the development of molecular biologic technique,gene diagnosis and treatment has been a new diagnosis and therapeutic mode for tumor.Many scientists has been studied to find new tumor marker and new target of gene therapy.Survivin is one of the inhibitor of apoptosis protein which were found recently.Its biological effects includes:①Inhibit apoptosis.②Regulate cell proliferation.③Induce angiogenesis.Survivin mainly express in embryonic tissues,but undetectable in differentiated tissues.It can be detected in many human tumors.Survivin plays a key role in the metastasis of HCC, and it has same correlation with tumorigenesis.It was regarded as "the perfect mark of the antisence targeted therapy".In recently,antisence inhibition and block definite gene expression has been an important method which were used to study gene function and gene therapy.Antisence technology is a good method in biotherapy of tumor for its specificity and controllability.It is an important way in biological therapy of the HCC.How to find the effective target site is crucial in the research of Antisense therapy.The mechanism of survivin in vital movement of cell still need further approach.Many studies showed there are closely relationship between COX-2, surviving and the genesis,development,angiopoiesis of tumor.But the mechanism hasn't been known.COX is rate-limiting enzyme in the process of prostaglandin synthesized by arachidonic.It has two subtype:COX-1 and COX-2.In recently,many studies showed that COX-2 not only is key enzyme which switch on inflammable reaction but also expressed in many human tumors.COX-2 were involved in many tumor's genesis and development by promote cell hyperplasy,inhibit apoptosis and promote new vasifaction. COX-2 has been the new hot spot because its dysexpression in many tumor.In this study,the objective of us is:①To study the expression and its relationship of COX-2 and Survivin in HCC.②To study the effects of NS-398 on cell proliferation and apoptosis of SMMC-7721.③To study the mechanism of action of Survivin-ASODN on SMMC-7721.④To study the effects of combination of NS-398 and Survivin-ASODN on cell proliferation and apoptosis of HCC.Method:①Observe the expression of COX-2 and Survivin in 30 cases HCC tissue,tumor-adjacent tissues,normal tissues and in SMMC-7721 cell respectively by RT-PCR,FCM,western blot technique.②To observe the effects of NS-398 on cell proliferation and apoptosis of SMMC-7721 by MTT and FMC techniques.To study the effects of NS-398 on the expression of COX-2 and Survivin by RT-PCR.③To synthesize the antisense oligodeoxyribonucleotide(ASODN)of Survivin.Observe the effects of Survivin-ASODN on the expression of Survivin in SMMC-7721 by MTT, FMC and RT-PCR.④To abserve the combinated effect of NS-398 and Survivin-ASODN on cell proliferation and apoptosis of SMMC-7721.All data was analyzed by the soft of SPSS 13.0 for windows.The data were analyzed by t-test and chi square test.The analysis of dependablity were by Spearman correlative-analysis.All data were expressed as means±S.E.M.A value of P＜0.05 was considered statistically significant.Results:①The expression of COX-2 and Survivin in HCC were higher than in latero-liver cancer tissue and normal liver tissue.COX-2 was significantly related with Survivin expression in HCC.②NS-398 could inhibit the expression of COX-2 and Survivin in SMMC-7721 in the manner of dose and time dependent.③NS-398 could inhibit the SMMC-7721cells proliferation in a dose and time dependent manner and could change the cell cycle of SMMC-7721.NS-398 could induce apoptosis of SMMC-7721 by dose and time dependent manner.④Survivin-ASODN could induce apoptosis of SMMC-7721 and inhibit the expression of Survivin.⑤NS-398 combined with Survivin-ASODN could reduce the survival rate of SMMC-7721 cell. There are significant difference compared with control group.Conclusion:①NS-398 could inhibit cells proliferation of SMMC-7721 and induce apoptosis of SMMC-7721 by inhibit the expression of COX-2 and Survivin.The exact mechanism needs further study.②Survivin-ASODN could close the expression of Survivin gene in SMMC-7721 and could inhibit the cell proliferation of SMMC-7721 by change cell cycle and increase apoptosis.③Survivin is an ideal antisense marker of hepatoma.The Survivin-ASODN can be a potential anticancer drugs for hepatoma.④Combinated application of NS-398 and Survivin-ASODN could inhibit cell proliferation of SMMC-7721 significantly.