Effect Of RNA Interference Targeting HOXA7 On Proliferation, Apoptosis And Multidrug Resistance Of Leukemia Cell Line U937

Objective To Create the expression vector pGPU6/GFP/Neo-shHOXA7 on the basis of designing and screening small interefere RNA (siRNA) targeting of HOXA7, investigate the effect of knocking down HOXA7 gene expression by RNA interference on the proliferation, apoptosis and multi-drug resistance of leukemia U937 cells, find the new target of gene therapy and MDR for leukemiaMethods1. To design three pairs of siRNA targeting of HOXA7, then transfect them respectively into LETP-a-2 cells, the effect of each siRNA on down-expression of HOXA7 was observed by RT-PCR and Western blot analysis. The siRNA was screened out of best silience effect for the following experiments.2. Design and create the expression vector targeting of HOXA7 (pGPU6/GFP/Neo-shHOXA7), then transfect them into U937 cells after enzyme digestion and sequence identification successfully, The effect of HOXA7 silence-expression on growth and apoptosis of U937 cells were measured by MTT and flow cytometry.3. U937 cells were transfected with the specific expression vectors pGPU6/GFP/Neo-shHOXA7, The stable transfected cells were selected by G418 treatment. Downregulation of HOXA7 expression was confirmed by RT-PCR and Western blot analysis. MTT method was used to detect the sensibility to cytarabine, homoharringtonine and methotrexate of U937 cells. FITC-PI stained flow cytometry was used to detect the promotion on apoptosis of HOXA7 silence-expression.Results1. The siRNA2 was screened out of best silience effect and could inhibit HOXA7 expression effectively, the silence rates were(52.219±0.550)% and(57.344±4.743)% on protein and mRNA levals, significantly lower than the siRNAl and siRNA3 groups(P<0.05).2. The expression vector targeting of HOXA7(pGPU6/GFP/Neo-shHOXA7) was create successfully detected by enzyme digestion and transfect them into U937 cells, The inhibitory rate of 72h was (52.617±9.292)% and the apoptosis rate of 48h was (24.677±4.161)%, significantly lower than the control groups(P<0.05).3. The transcription and expression of HOXA7 were significantly lower in U937 cells transfected with pGPU6/GFP/Neo-shHOXA7 than the control groups, the relative expression level were(23.910±1.662)% and (25.980±1.651)% on mRNA and protein levals(P<0.05). The IC50 of Ara-C and HHT in U937 transfected cells were decreased by 3.5- and 8.5-fold compare to those in the control cells. The apoptosis rates were (78.636±5.993)%, significantly higher than the control group(P<0.05). The cell proliferation inhibition and apoptosis rates of RNA interference targeting of HOXA7 combined with MTX were obviously higher than that of RNAi or only MTX group(P<0.05).Conclusions1. The best silience effect siRNA2 of HOXA7 expression was screened out for the following experiments.2. The expression vector targeting of HOXA7 (pGPU6/GFP/Neo-shHOXA7) was created successfully and could inhibit proliferation and promote apoptosis of U937 cells effectively.3. The inhibition of HOXA7 gene expression by pGPU6/GFP/Neo-shHOXA7 could enhance the sensibility of Ara-C, HHT and MTX to U937 cells, induce apoptosis of U937 cells and could reverse the multidrug resistance of leukemia cells.