| RNA interference is the gene silencing of post-transcription induced by small interfering RNA, including the initiation phase and the effect phase. In the initiation phase, the double-stranded RNA(dsRNA) is degraded into small interfering RNAs(siRNA), which consist of 21-23 bp,by an enzyme called Dicer. In the effect phase, RNA-induced silencing complex(RISC) is developed after the siRNA binding to a ribozyme complex, the activated RISC ,located on the homologous mRNA through base-pairing, induce the degradation of mRNA and the subsequent gene silencing of post-transcripition. Lots of research work, which has been done since the phenomenon of RNA interference was founded in 1998, manifested the RNAi technique can not only inhibit the target genes with high specificity and efficiency, but also has a characteristic of high sequence specificity, with less side effects. Till now, the technique has been applied in the field of therapy for many malignant tumors and leukemia with promising gene silencing effect. Therefore, RNAi is a promising method of gene therapy.The Survivn gene is located in the q25 of No.17 chromosome with a length of 14.7kb, encoding a cytoplasmic protein of 142 aa with a molecular weight of 16.5kD which is one of the member of the inhibitor of apoptotic protein (LAP) family. The structural domain of BIR can inhibit the caspase-3 and caspase-7, inducing cell apoptosis. Survivn are non-expressed in the normal adult tissues, but highly expressed in the malignant tumor cells and leukemic cells, and which related the oncogenesis, tumor development, and prognosis. It has been found that the antisense oligonucleotide and RNAi could silence the Survivin gene and induce apoptosis of malignant tumor cells. Therefore, Survivin may be a new potential target of gene therapy for luekemia.Leukemia,which affects the people's quality of life seriously,is a haematological malignancy with very high morbidity rate and malignant degree.Since 1980's,the treatment of the leukemia have made rapid progress,high dose chemotherapy,the hematopoietic stem cell transplantation and symphysis application of the immunization therapy,have raised the survival rate of the leukemic patients.But high dose chemotherapy has serious toxic side effect,as to the hematopoietic stem cell transplantation,high expensive cost,and serious complications such as graft Vs host disease limits its clinical extensive application.Data have showed that RNAi to BCR/ABL gene can induce the apoptosis of K562 cells.However,till now no reasearch has been done in the aera of gene therapy to leukemia by RNAi to survivin.In this study,a siRNA plasmid expression vector against survivin was constructed and transfected into HL-60 cells with Lipofectamine 2000?. The effects of shRNA plasmid expression vector on growth and apoptosis and protein expression of HL-60 cells were assayed in order to provide technique and theories for treatment of leukemia and other tumer,by RNAi targeting survivin gene.Methods:1,To construct eukaryotic expression vectors of siRNA specific for Survivin gene andnon-specific for survivin gene.2,Three groups were classified:transfected with eukaryotic expression vectors ofsiRNA specific for Survivin gene(transfection group); transfected with eukaryoticexpression vectors of siRNA non-specific for Survivin gene(negative group);HL-60cells without transfection(blank group).3,Vector was extracted by Endofree Plasmid Extraction Kit and transfected into HL-60 cells with Lipofectamine 2000TM4,Trypan blue exclusion tests were adopted to investigate the effect of survivinsiRNA on the proliferation of HI-60 cells.5,Cells were collected after transfected 24h, 48h,and72h,total protein was extractedby TRIZOL and the expression of survivin protein was determined by Western-blotassay.6,The apoptosis of HL-60 cells after transfection was evaluated by TUNEL assay.7,SPSS 10.0 statistical software was used to analyse the statistical data . x|-±s andone-way ANOVA were used to compare these data, P value less than 0.05 wasconsidered statistically significant.RESULTS:1,Recombinant plasmid was verified by the restriction map and the sequence analysis, and the results was in agreement with original design.2,After transfection of shRNA plasmid expression vector against survivin gene 24h,48h and 72h,by the trypan blue exclusion test,cells growth in transfection group was inhibited significantly, the inhibitory rate in transfection group was 17.6±8.5%,38.3±4.2%,44.0±1.2% respectively, compared to the blank and negative groups (P<0.01) . the inhibitory rate of HL-60 cells between 24h with 48h and 72h in transfection group were statistically significant (P<0.01) . however,statistically significance was not seen between blank group and negative group or between 48h and 72h in transfection group (P>0.05) .3,After transfected 24h, 48h, 72h, comparing with blank group, the protein expression of transfection group was 0.74±0.01,0.35±0.02,0.34±0.01 respectively; Meanwhile, the result in negative group was 0.99±0.02,1.00±0.02,0.99±01 relatively. Comparing with negative and blank control groups, the protein expression of transfection group was significantly reduced (P<0.01) ; There were statistically significant between 24h with 48h and 72h in transfection group (P<0.01) ,no statistically significance were seen between 48h and 72h in tranfection group (P> 0.05) . 4,After transfected 48h, the apoptotic cells in transfection group, negative group and blank group were 31.0 % ,3-33 % ,3.67 % respectively. The apoptosis rate in transfection group was significantly higher than in negative group or blank group (P<0.01).CONCLUSION:1,The siRNA eukaryotic expression vector against Survivin gene has beensuccessfully conctructed.2,After the siRNA plasmid expression vector against survivin was transfected toHL-60 cells,the growth and proliferation of cells were inhibited.3,After RNAi targeting survivin gene, the protein expression of HL-60 cells wereinhibited significantly,and the was induced.4,After the siRNA plasmid expression vector against survivin was transfected toHL-60 cells,the inhibition of cells growth and proliferation may be related to theexpression of survivin protein and the apoptosis of cells.5,RNAi targeting survivin gene may be used to treat leukemia as a gene therapy. |
