Effection Of Gastric Cabcinoma Growth And Apoptosis By Small Interfering RNA Silencing Of The Livin Gene

Backgroud and ObjectiveGastric cancer is one of the most common malignancies in our country.Most of patients with this disease are diagnosed in advanced stages,and lose the chance of surgical treatment.Despite recent progress in chemotherapy,the overall survival rate of gastric cancer patients in advanced stages is still low,Apoptosis resistance of gastric cancer cell may be one of the "hot spot" of research fields.Inhibitor of apoptosis protein(IAP)family may paly a key role in causing apoptosis resistance and has become the focus of research increasingly.Livin is a novel inhibitor of apoptosis protein(IAP)family member and overexpresses in most cancers.High expression of livin can decrease sensitivity of chemotherapy.So it might elevate the effect of cancer therapy to suppress the expression of livin and increase the sensitivity of cancer cell apoptosis.To construct siRNA vector to aim directly Livin and transfect it into cells of gastric cancer,and then observe the influence about Livin gene on gastric cancer cells growth velocity,cell apoptosis and sensitivity to radioactive rays through regulating the livin gene expression in the gastric cancer cell lines.Methods1.According to the principle of designing siRNA target sequence,we used the Takara,Ambion and promega target siRNA sequence design and analysis software to scan human livin(livin land livin 2)cDNA.19 bases target siRNA sequence was selected and definited by BLAST homology analysis and a contrast sequence was random assorted.Design and construct a specific siRNA expression vector which targeted livin,identify the vector by enzyme digestion and sequeneing to make sure the exact correction of the construction.An unspecific siRNA expression vector was construeted as a control.2.Transfeet livin into the gastric cancer cells line BGC-823.Select the positive cell clone by antibiotic G418.The expression level of BGC-823 mRNA was mesured by semi-quantitative RT-PCR in each group of various siRNAs transfected.3.After siRNA was transfected,we observed the morphous of BGC-823 cells and measured cell proliferation by Microculture Tetrazolium Thiazolylblue(MTT); BGC-823 cells were observed through fluorescence microscope to calculate efficiency of transfection.Results1.After homologization sequence index and optimization,the target siRNA sequence was ensured,which had 19 bases:GCGTCTGGCCTCCTTCTAT(440～458) and GTCTGGCCTCCTTCTATGA(442～460).2.After the hairpin DNA of siRNA annealing,the results of agar gel electrophoresis analysis showed that there was an obvious stripe whose molecular weight was similar to the design,about 100bp.3.Construction of gastric cancer cells BGC-823 expression vector.The results of Restriction map and sequeneing of inserted sequence of pSUPER-EGFP-G were same to anticipation.4.Compared with blank group(0.37±0.10,0.43±0.09) and cells without siRNA transfection(0.11±0.07,0.13±0.04),siRNA control group(0.34±0.08,0.45±0.11) in livinα/βdecreased significantly(P＜0.05).5.Control group with empty siRNA vector group,then switch to 24h,48h,96h and 1 week of cell growth was not affected,siRNA group compared with control group at 24h after transfection and 48h cells growth is not significantly affected,and 96h and 1 week was significantly inhibited(P＜0.01).6.siRNA transfection group(14.85±1.35) cells apoptosis was significantly increased,compared with non-transfected group(4.51±0.36) and empty siRNA vector transfection group(5.13±0.71),the difference was significant(P＜0.05).Conclusion 1.The siRNA vector was constructed successfully.2.The level of livin was obviously decreasing after transfecting with target siRNA vectors in gastric cancer.3.siRNA can silence livin gene expression in BGC-823 cells;thus,livin can serve as a new potential molecular target to gastric cancer in gene therapy by siRNA.