Construction Of Targeting Livin SiRNA Recombinant Adenovirus Vector And Its Effects On Proliferation,Apoptosis And Resistance In K562 Cells

Objective: To construct recombinant adenovirus vector targeting Livin siRNA, inhibit the expression of Livin, and study the influence of recombinant adenovirus vector on proliferation,apoptosis and resistance in K562 cells, which will offer experimental basis for exploring the gene therapy of leukemia following.Methods: 1. Constructed recombinant adenovirus vector of targeting Livin siRNA. Cut shuttle plasmid vector pSES-HUS with restricted enzyme, linked vector and Livin siRNA with DNA Ligation Kit, selected and validated the recombinant plasmid pSES-HUS-Livin siRNA containing Livin siRNA. Transducted the plasmid which has been validated to be correct by gene sequencing into adenoviral homologous skeleton plasmid BJ-Adeasy prepared to produce adenoviral plasmid Adeasy-Livin siRNA, and verified with enzyme digestion following. Transfected the product into virus-producing incasing cells with liposome mediated, and gained recombined adenoviral Ad5-Livin with high titer using ping-pong infection. 2. Ad5-Livin siRNA was transfected influenced on expression of Livin in K562 cells. In experiments, cells were classed into four groups: K562 cells group, K562/Ad5-Livin siRNA-1 group, K562/Ad5-Livin siRNA-2 group and K562/Ad5 group. The gene silencing efficiency of Ad5-Livin siRNA was monitored by Real-time PCR and Western Blot in K562 cells. 3. Transfected Ad5-Livin siRNA influenced on proliferation and apoptosis in K562 cells. The proliferation and apoptosis change of being transfected Ad5-Livin siRNA was detected by MTT and AnnexinV-FITC/PI. 4. Transfected Ad5-Livin siRNA influenced on drug-resistant in K562 cells. In experiments, chose different concentrations of ADM,VP16,VCR and DDP. Drug-resistant change of being gene silence of Livin was detected through anticancer drugs sensitive experiment with MTT.Result: 1. Recombined adenovirus plasmid Adeasy-Livin siRNA was constructed successfully, transfected virus-producing incasing cells. Then recombined adenovirus with high titer was gained, and the titer of adenovirus solution obtained reached to 1.1×1011pfu/ml. 2. Thansfection with recombined adenovirus to K562 cells, when MOI was 100, both infected cells'survival rate and infection rate were fairly well. The infection rate can reach to 15.91%. 3. The mRNA and protein are over expressed in K562 cells. After transfection of Ad5-Livin siRNA, the expression of Livin in K562 cells was decreased significantly in mRNA and protein level, and the transfected blank vector cells were no significant change (p<0.01). 4. Inhibiting Livin of recombinant adenovirus can inhibit proliferation and enhance spontaneous apoptosis in K562 cells. Compared with two control groups, there is statistical significance (p<0.05). 5. Transfected Ad5-Livin siRNA can increased the sensibility of K562 cells to anticancer drugs (for example, ADM,VP16 and VCR). The IC50 of drugs were lower the two control groups (p<0.01). However, recombinant adenovirus has no influence on DDP (p>0.05).Conclusions: 1. Recombined adenovirus containing Livin siRNA with high titer was constructed and gained using molecular biological method. 2. Livin siRNA was transfected into K562 cells by recombinant adenovirus vector,and can down regulate the mRNA and protein expression of Livin. 3. RNA interference with Livin can inhibit the proliferation and apoptosis in K562 cells. 4. RNA interference with Livin can increase the sensitivity of K562 cells to ADM,VP16 and VCR.