| Objective:Homeobox genes, a superfamily of evolutionarily conserved developmental genes, function as critical master regulatory factors in embryonic development and the process of hematopoietic cell proliferation, differentiation, apoptosis. As a member of homeobox gene family, HOXA5 is located in chromosome 7, coding a transcription factor that plays key roles in controlling the expression of genes, cell differentiation and regulator of the morphogenesis. This study used shRNA aims to create the expression vector pGPU6/GFP/Neo-shHOXA5 and screen the most effective plasmid expressing HOXA5 short-hairpin RNA (shRNA), investigating the effect of knocking down HOXA5 gene expression by RNA interference on the proliferation, apoptosis of leukemia U937 cells.Methods:1. To design three pairs of siRNA targeting HOXA5, then create three kinds of plasmid expressing HOXA5 short-hairpin RNA (shRNA) named pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3 and transfect them respectively into U937 cells after enzyme digestion and sequence identification successfully. The effect of each shRNA on down-expression of HOXA5 was observed by Real-time RT-PCR and Western blot analysis. The shRNA was screened out of best silience effect for the following experiments.2. U937 cells were transfected with the specific plasmid expression vectors pGPU6/GFP/Neo-shHOXA5. Downregulation of HOXA5 expression was confirmed by Real-time RT-PCR and Western blot analysis. The effect of HOXA5 silence-expression on growth, cell cycle distribution and apoptosis of U937 cells were measured by CCK-8 and flow cytometry. The expression of Survivin,Caspase-3 was examined by Real-time RT-PCR and Western blot analysis.Results:1. The expression vector targeting HOXA5 (pGPU6/GFP/Neo-shHOXA5) was created successfully detected by enzyme digestion and transfected them into U937 cells for 48 h. The plasmid expression vectors pGPU6/GFP/Neo-shHOXA5-3 was screened out of best silience effect and could inhibit HOXA5 expression effectively, compared to the other two groups (P<0.05).2. U937 cells were transfected with the specific plasmid expression vectors pGPU6/GFP/Neo-shHOXA5-3 for 48 h. Real-time RT-PCR and Western blot analysis confirmed that pGPU6/GFP/Neo-HOXA5 shRNA-3 could inhibit HOXA5 expression effectively, compared to cells transfected with Control shRNA and parental U937, and the silence rates was 70%(P<0.05). CCK-8 assay indicated that after 0,24,48,72 h transfection, the growth of HOXA5 shRNA-3 transfected cells had a slower rate of cell proliferation (P<0.05). After 48 h transfection, Flow Cytometry analysis showed an increase in the proportion of cells in G0/G1 phase and a decrease in S phase in HOXA5 shRNA groups, compared with controls. Cell apoptosis was determined by Annexin V FITC-PI staining using flow cytometry after 48 h of transfection. U937 cells after HOXA5 shRNA transfection significantly increased the apoptosis rate of U937 cells, compared with controls. The apoptosis rates were (7.36±1.63)%, (8.10±1.39)%, (26.52±2.35)% respectively. Western blot analysis showed that compared with control cells, the protein expression level of surviving was significantly reduced, whereas the expression level of caspase-3 was remarkably increased in HOXA5 shRNA-3 transfected U937 cells (P<0.05).Conclusions:1. The plasmid expressing HOXA5 short-hairpin RNA (shRNA) named pGPU6/GFP/Neo-HOXA5 shRNA-1,-2,-3 was created successfully and then screened out the specific plasmid expression vectors which had the best silience effect that could inhibit HOXA5 expression effectively.2. The expression vector targeting HOXA5 (pGPU6/GFP/Neo-shHOXA5) was transfected into U937 cells, and the HOXA5 mRNA and protein levels of the U937 cells were decreased obviously; HOXA5 knockdown by shRNA inhibited proliferation, blocked cell cycle progression and induced apoptosis, which were related to the significantly decreased expression of survivin, elevated levels of caspase-3. |
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