| Objective: To explore the relationship between the development,proliferation and apoptosis of leukemia and HOXA5 genes,this article first observation of children with acute lymphoblastic leukemia children with bone marrow HOXA5 expression.On this basis,screening effectively knockdown the expression of HOXA5 gene specific small interfering RNA sequence and identify its function,thus constructed the HOXA5 shRNA eukaryotic expression vector pRNAT-GFP-Neo-HOXA5 C,transfected with human acute T lymphoblastic leukemia cell line(Jurkat),observe the influence on Jurkat cell cycle and apoptosis,to study the mechanism of leukemia and provide theoretical basis for targeted treatment.Methods:1.Isolation of mononuclear cells from bone marrow.Mononuclear cells from bone marrow samples were isolated by lymphocyte separation.2.Detection of HOXA5 mRNA expression and protein distribution in mononuclear cells in the ALL acute phase group,the ALL remission groups and the control group.3.Short hairpin RNA(shRNA)design,screening and synthesis: For the Jurkat cell experiments,three specific sequences ofHOXA5 siRNA were chemically designed and synthesized by Adicon.4.Group of Experiments: The cells were divided into group A,blank control group(plus an equal amount of cells and culture media only);group B,the negative control group(liposomaltransfection with negative control siRNA);and group C,the experimental group(liposomal transfection with HOXA5 targeting siRNA).5.The HOXA5-specific siRNA 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 mRNA expression and protein distribution in each cell group.6.Cell distribution in the cell cycle and the rate of cells undergoing apoptosis were determined usingflow cytometry.Results:1.As the result ofFQ-PCR for the HOXA5 mRNA relative expression analysis ineach group were:HOXA5 mRNA expression in ALL acute phase(0.76 ± 0.05)% was significantly higher than remission group(0.48± 0.07)%and control group(0.47± 0.08)%(P<0.05).The expression of HOXA5 protein levels in the acute phase of ALL(0.70 ± 0.02)was significantlyhigher than that in ALL in the remission(0.39±0.03)and control groups(0.42±0.02)(P<0.05).2.Design and construction of the three HOXA5 gene targeted shRNA plasmid expression vector,experimental group HOXA5 gene expression was significantly decreased,after transfection experiment group Jurkat cell gene expression were inhibited,through FQ-PCR and Western blot detection on sequence pRNAT-GFP-Neo-HOXA5 C is the most significant.3Effects of the recombinant vector on the expression of HOXA5 mRNA in Jurkat cells.HOXA5 mRNA expression quantity in experimental group(pRNAT-GFP-Neo-HOXA5C)(0.39 ± 0.01)% higher than its in control groups.The expression of HOXA5 protein levels in Jurkatccells in experimental group(pRNAT-GFP-Neo-HOXA5C)(0.17 ±0.01)expression quantity was higher than in control groups(0.73±0.003)(P<0.05).4Effects of siRNA on HOXA5 protein expression levels in Jurkat cells:The experimental group(pRNAT ? GFP ? Neo ? siHOXA5C)affected by siRNA compared withcontrol groups,the proportion of G0/G1 cells increased(56.70%±6.4 %)and the proportion of S phase cells decreased(29.00%±5.5%)(P<0.05),(P<0.05).The experimental group(pRNAT-GFP ? Neo ? siHOXA5C)(24.99±5.16)% under the influence of siRNA showed a flow apoptotic rate was higher as compared to that of the control groups(P<0.05).Conclusion:1 HOXA5 gene is highlyexpressed in ALL and closely associated with the occurrenceof ALL in children.2HOXA5-specific siRNA effectively silences HOXA5 gene expression and induces apoptosis and cell-cyclearrest in Jurkat cells,thus inhibiting cell proliferation.3Therefore,this eukaryotic expression carrier has the potentialto become an effective gene therapy to treat acute lymphoblastic leukemia. |
PDF Full Text Request