The Influence Of HOXA5-specific SiRNA On The Expression Of Livin And Smac Proteins

Objectives: To explore the relationship of the development between leukemia and HOXA5 genes,Smac and Livin,screen specific small interfering RNA sequence to restrain the expression of HOXA5 gene and identify its function,thus constructed the HOXA5 sh RNA eukaryotic expression vector p RNAT-GFP-Neo-HOXA5 C,transfected with human acute T lymphoblastic leukemia cell line(Jurkat),observe the effects on Livin and Smac proteins expression in acute T cell leukemia Jurkat cell,to study the mechanism of leukemia and provide theoretical basis for targeted treatment.Methods :1.Short hairpin RNA(sh RNA)design,screening and synthesis: For the Jurkat cell experiments,three specific sequences of HOXA5 si RNA were chemically designed and synthesized by Adicon.2.Group of Experiments: The cells were divided into group A,the blank control group(only the same amount of cells and culture media);group B,the negative control group(liposomal transfection with negative control si RNA);and group C,the experimental group(liposomal transfection with HOXA5 targeting si RNA).3.The HOXA5-specific si RNA was ransfected to Jurkat cells using lipofectamine TM2000.Western blotting and quantitative fluorescent polymerase chain reaction(QF ? PCR)were used to detect the relative HOXA5 m RNA expression,HOXA5 protein,Livin protein and Smac protein expression in each cell group.Results: 1 ? ThereHOXA5 gene targeted sh RNA plasmid expression vector were designed and constructed,HOXA5 gene expression in experimental group was significantly decreased,after transfection experiment group Jurkat cell gene expression were inhibited,with inhibition ratio being p RNAT-GFP-Neo-HOXA5A(24.62±2.34)%,p RNAT-GFP-Neo-HOXA5B(35.07±3.21)%,and p RNAT-GFP-Neo-HOXA5C(70.89±6.41)% respectively,p RNAT-GFP-Neo-HOXA5 C is the most significant.3Effects of the recombinant vector on the expression of HOXA5 m RNA in Jurkat cells.2 ? Effects of the recombinant vector on the expression of HOXA5 m RNA in Jurkat cells.HOXA5 m RNA expression quantity in experimental group(p RNAT-GFP-Neo-HOXA5C)(0.39± 0.01)% higher than its in control groups.The expression of HOXA5 protein levels in Jurkatc cells in experimental group(p RNAT-GFP-Neo-HOXA5C)(0.18±0.01)expression quantity was higher than in control groups(0.84±0.03)(P<0.05).3 ? Livin protein expression in Group C(0.20 ± 0.02)was significantly lower compared to negative control group(1.45±0.04)and blank control group(1.33 ±0.02)(P<0.05).However,differences between negative control group and blank control group had no statistical significance(P>0.05).4?Smac protein expression in Group C(1.26±0.04)was significantly higher compared to negative control group(0.87±0.03)and blank control group(0.86 ± 0.02)(P<0.05).Difference between negative control group and blank control group had no statistical significance(P>0.05).Conclusion:1?HOXA5-specific si RNA effectively silences HOXA5gene expression and downregulation of HOXA5 induced the downregulation of Livin protein expression and upregulation of Smac protein in Jurkat cells,thus inhibiting cell proliferation.2?We suggest the HOXA5 gene to be considered as the new target for acute leukemia gene therapy.Therefore,this eukaryotic expression carrier has the potentialto become an effective gene therapy to treat acute lymphoblastic leukemia.