Down The Wt1 Gene Expression In Human Leukemia K562 Cells The Biological Behavior And Gene Expression Profiles

ObjectiveWT1(Wilms tumor suppressor-1,WT1)gene is abnormally overexpressed in different kinds of leukemia and lead to malignant transformation of normal cells.In this study,we wanted to design and construct eukaryotic expression vector of short hair RNA(shRNA) targetting human WT1 gene,trasfect it into human leukemia cells K562,investigate the inhibitory action of pWT1-shRNA to WT1mRNA and protein expression level in K562,in this way to apply RNAi (RNA interference,RNAi) technique to construct WT1 gene down regulation cell modle.Then to observe the influence of WT1 gene down regulation on K562 biological behaviour such as cell morphou,growth condition,cell cycle,proliferation,apoptosis and cell differentiation,investigate the effects of down regulation of WT1 to the sensitivity of K562/AO2 cells to ADM,and approach the influence of WT1 gene down regulation to K562 cells gene expression profile,to research the biological function of WT1 gene in the genesis and development of leukemia and reveal possible effect of WT1 gene down regulation on reversing K562/AO2 cell drug resistance.Methods1.Construction of eukaryotic expression vector for shRNA targeting WT1gene.Firstly,two sites targeting human WT1 gene in gene bank was selected according to the guidelines for the selection of highly effective siRNA sequence and then BLAST test was performed.Secondly,a pair of oligonucleotides fragments were synthesized and annealed to double strand.Thirdly,the double strand was cloned into plasmid vector pGenesil-1,Finally,the recombinant plasmids were identified by restriction enzyme digestion and sequencing analysis.2.Optimization of the transfecion condition of transfecting pWT1-shRNA to K562 cellsLeukemia cells K562 were cultured conventionally,cell morphous and growth condition were observed by inverted microscope;After transfected with transfection complex comprising different proportion of pWT1-shRNA plasmid and Superfect reagent 12h,24h,48h,72h,96h, transfection efficiency was defected by inverted fluorescence microscope and flow cytometery(FCM),choose the optimal proportion of pWT1-shRNA plasmid and Superfect reagent and time point which lead to maximal transfection efficiency for further research.3.Study on the inhibitory action of pWT1-shRNA to WTlmRNA and protein expression level in K562 cellsAfter 48h of being transfected with pWT1-shRNAI(or pWT1-shRNA2 ),total RNA and protein was extracted from EGFP positive K562 cell(sorted by FCM).WTlmRNA expression level was evaluated by Real-time fluorescent quantitation reverse transcription-polymerase chain reaction(RT-PCR) and WT1 protein expression level was evaluated by Western-blot.Total RNA and protein of pCon group(K562 cells transfected with p-Gensil),pNeg group(K562 cells transfected with negative shRNA vector),and non transfected group were also evaluated as control.4.Influence of down regulation of WT1 gene on the biological behaviour of K562 cellsAfter being transfected with pWT1-shRNA,cell growth condition was observed by inverted phase contrast microscope,cell growth curve was drawn;Cell smear was stained by Wreigt-Gmisa,cell morphous was observed by microscope;cytostasis rates was measured by MTT assay;Cell apoptosis was detected by FCM and DNA ladder strap electrophoresis; Distribution of cell cycle was analysed by FCM;Afle being trasfected by pWT1-shRNA,K562 cells was induced by TPA(12-o-tetradecanoylphorbol-13-acetate ),NBT reduction test and FCM analysis of cell differentiation marker CD9 and CD14 were apllied to study the influence of down regulation of WT1 to K562 diffrentiation induced by TPA.5.Effect of down regulation of WT1 to the sensitivity of K562/AO2 cells to adrimycinpWT1 shRNA was transfected into K562/A02 cells.The 50%inhibitory concentration(IC₅₀)of ADM on K562/A02 cells was measured by MTT assay.Intracellular ADM accumulation and ADM-induced apoptosis of K562/A02 cells were detected by flow cytometry.6.Influence of down regulation of WT1 to gene expression profile of K562 cellsCells were devided into pWT1-shRNA group and non-transfected group,after 48h of being transfected with pWT1-shRNA,positive transfected cells were sorted by FCM,total RNA was extracted and reverse transcripted,cDNA was labeled by cy3 and Cy5 respectively,and hybridized with 22K human gene array.Signals were extracted from the scanned images of chips, followed by background extraction and normalization using LuxScan 3.0 image analysis software, subjected for further analysis.Genes altered 2 fold(Ratio＞2,or＜0.5) with statistically significance(p＜0.05) were included in our Gene tree and pathway and GO analysis.Results1.Construction of eukaryotic expression vector for shRNA targeting WT1gene.Restriction enzyme digestion and sequencing analysis showed that eukaryotic expression vector of shRNA targeting WT1 genan had negative shRNA had been constructed successfully.2.Optimizati on of the transfecion condition of pWT1-shRNA to K562 cellsAfter being transfected with different proportion of plasmids to superfect reagent(Giant molecule balsam tranfection reagent) at different time,transfetion efficiency was evaluated by fluorescence microscope and FCM,it showed that the optimal transfection condition was the proportion of pWTshRNA to Superfect reagent as 1.5μg:6μl at 48h. 3.Study on the inhibitory action of pWT1-shRNA to WTlmRNA and protein expression level in K562 cells48h after transfection,EGFP positive cells were sorted by FCM,purity rates reached 98.0%±1.2%,total RNA and protein were extracted,andWT1mRNA was evaluated by FQ-RT-PCR,it showed:Compared with control groups the expression level of WT1mRNA of pWT1-shRNA1 and pWT1-shRNA2 were significantly suppressed(p＜0.05);Suppression efficiency of pWT1-shRNA1 group(86.4%±5.2%) was higher than that of pWT1-shRNA1 group(53.6%±3.7%)(p＜0.05);There were no significance difference about expression level WT1mRNA of pNeg and pCon group before and after transfection(p＞0.05).WT1 protein was evaluated by western blot,it showed:Compared with control groups the expression level of WT1 protein of pWT1-shRNA1 and pWT1-shRNA2 were significantly suppressed(p＜0.05).Suppression efficiency of pWT1-shRNA1 group(85.1%±9.1%) was higher than pWT1-shRNA1 group(52.4%±4.9%)(p＜0.05).There were no significance difference about expression level WT1 protein of pNeg and pCon group before and after transfection(p＞0.05).4.Influence of down regulation of WT1 gene to the biological behaviour of K562 cells4.1 Influence of down regulation of WT1 gene to the mophorlogy of K562Observed by inverted phase contrast microscope,pWT1-shRNA1 group cells appeared multiformity,cell body abnormity,caryomorphism introcession and increased cell death; After Wreigt-Gimsa staining,morphology showed:Cells of non-trasfeced group:cell volum was larger,karyomegaly,blue,nucleoli were clear and karyoplasmic ratio was large;Cells of pWT1-shRNA1 group:cell volume were uneven,some cell appeared karyopycnosis,broken to pieces,vacuolus emerged in kytoplasm,cellular membrane was un integrity even cell disruption.4.2 Influence of down regulation of WT1 gene to the proliferation of K5624.2.1 cell growth curveAfter trasfection,K562 cells of each group were counted every 24h to draw cell growth curve,it showed:compaired with non-transfected group,cells of pWT1-shRNA group grew slowly,while cells of pNeg and pCon group grow normally.4.2.2 proliferative inhibition rates assayed by MTTCompaired with non-transfected group,proliferative inhibition ratio was higher at each time point(p＜0.05)while proliferation inhibition ratio of pNeg and pCon group had no significance difference(p＞0.05).4.3 Influence of down regulation of WT1 gene to apoptosis of K5624.3.1 AnnexinV/PI analyzed by FCM48h after transfection,apoptosis rates of EGFP positive cells were analyzed applying AnnexinV/PI double parameter method by FCM.It showed:compaired with non-transfected control group(6.6%±0.5%),apoptosis rates of pWT1-shRNA1 group(33.2%±3.1%) were higher (p＜0.05);while apoptosis rates of pNeg group and pCon group had no significance difference(p＞0.05).4.3.2 DNA ladder electrophrosis72h after transfection,cells of pWT1-shRNA1,pCon group and pNeg group,were obtained and EGFP positive cells were sorted(atleast1×10⁶) by FCM,DNA was extracted to ladder strap electrophoresis analysis,It showed:ladder strap was appeared only in pWT1-shRNA group.4.4 Influence of down regulation of WT1 gene to cell cycle distribution of K56248h after transfection,EGFP positive cells were sorted(at least1×10⁶) by FCM,and cell cycle were analyzed by FCM.It showed:compaired with each control group,pWT1-shRNA1 group,cell rates of G2/M phase were increased significantly(P＜0.05),while cells rates of S and G0/G1 phase were decreased significantly(P＜0.05).4.5 Influence of down regulation of WT1 gene to cell differtiation of K5624.5.1 NBT reduction testCompaired with non-transfection group,NBT reduction rates of each transfection group had no significance difference(p＞0.05);When TPA was added,NBT reduction rates of each transfection group were increased gradually(P＜0.05);NBT reduction rates of pWT1-shRNA+TPA group cells was higer than other groups at each time point4.5.2 differtiation antigen detected by FCM72h After transfection megacaryocyte differentiation antigen CD9 and monocyte differentiation antigen CD14 were detected by FCM.It showed:compaired with non-transfection group,CD9 and CD14 positive cell rates of each transfection group had no significance difference(p＞0.05).When TPA was added,CD9 positive cell rates of each transfection group were increased gradually(P＜0.05) while CD14 positive cell rates didn't have this variation tendency;CD9 positive cell rates of pWT1-shRNA+TPA group was higer than other groups at each time point.5.Effect of down regulation of WT1 on the sensitivity of K562/AO2 cells to adrimycinAfter transfeced with pWT1shRNA,The relative reverse rate of sensitivity of K562/A02 cells to ADM was 67.3%.The intracellular accumulation of ADM was greatly increase ADM-induced apoptosis of K562/A02 cells was elevated from 3.1%to65.4%.6.Effect of down regulation of WT1 on the expression profile of K562There are 315 genes differentially expressed after down regulation of WT1 in K562,213 genes were down regulated and 102 genes were up regulated,34 pathways were influenced, most of them invoved in apoptosis,cell proliferation,cell cycle,ttranscription,translation or other cell process and physiology process.There are 23 genes related to apoptosis 13 genes related to proliferation underwent differenttially expressed,differenttially expressed genes related to S phase and Go phase were mostly down-regulatd,and some differenttially expressed genes related to G₂M phase were down regulated.FQ-RT-PCR result showed that PDCD4 was upregulated and VEGF was down regulated, and confirmed the chip results.Conclusion1.Eukryotic expression vector of WT1shRNA was constructed successfully.2.Giant molecule balsam tranfection reagent Superfect can transferpWT1-shRNA into K562 cells efficienly via optimize transfection condition.3.Transfection of pWT1-shRNA into K562 cell could inhibit the expression of WT1mRNA,subsequently inhibit the protein expression efficienly.4.Down regulation ofWT1 gene in K562 cells can inhibit cell proliferation,induce cell apoptosis,and cell cycle and promote K562 cell differentian to trophic nucleus induced by TPA.5.Down regulation of WT1 gene in K562 cells effected the expression profile of 62cells,and influences 34 pathways of cell process.differerntially expressed genes invovles apoptosis,proliferation,cell cycle signal transductor pathways.6.Down regulation of WT1 gene could enhance intracellular ADM accumulation in K562/A02 cells,improve sensitivity of K562/A02 cells to ADM,and induce cell apoptosis,therefore,reverse cell resistant to ADM.