Down-regulation of WT1/+17AA gene expression using RNAi and modulating leukemia cell chemotherapy resistance

Abstract

We have shown that inhibition of WT1/+17AA protein expression following transfection with a vector-based small interfering RNA expression construct in K562 cell lines, leads to a decrease in MDR1 and P-glycoprotein levels, accumulation of Rh123, and enhancement of the doxorubicin cytotoxicity. Our findings suggest that WT1/+17AA exerts its oncogenic function by modulating multidrug resistance in leukemia cells.

The Wilms’ tumor gene (WT1) is now thought to be involved in the pathogenesis of leukemia as well as a wide variety of solid cancers. The WT1 gene is alternatively spliced at two main regions, exon 5 (17 amino acids: 17AA) and the last three codons of exon 9 (KTS), yielding four isoforms: (+17AA/+KTS), (+17AA/−KTS), (−17AA/+KTS), and (−17AA/−KTS).1 All four WT1 isoforms are expressed in primary leukemia cells and each is considered to have a different function in interfering with signaling in cell differentiation, apoptotic and drug resistance pathways. However, the functions of each WT1 isoform in leukemia cells remains controversial. Recently, the co-expression of WT1 and the multidrug resistance related gene (MDR1) has been shown in vitro and in vivo.2 The promoter region of MDR1 has been carefully mapped and an EGR1/SP1/WT1 site at positions −69 to −41 has been identified.3 It is clear that the SP1/EGR1/WT1 site is a key regulatory region for MDR1 transcription, suggesting that WT1 is a potential modulator for leukemia cells’ multidrug resistance.

In this study, a vector-based shRNA expression system was used (Figure 1A). We selected the target region in exon 5 of WT1/+17AA cDNA and designed WT1/+17AA ShRNA and nonspecific control ShRNA oligonucleotides according to Tushul’s principles. Recombinant plasmids were transfected with the LIPOFECTAMINETM2000 transfection reagent into two paired erythroleukemia cell lines: K562 and K562/A02 (sensitive and resistant to doxorubicin, respectively). WT1/+17AA, WT1/−17AA, MDR1 and β2-MG genes were determined by quantitative real-time PCR using the SYBR green I dye. The primer pairs were designed using the Primer Premier software (version 5.0): WT1/+17AA forward primer 5’-AAAGGGAGTTGCTGCTGG-3’, reverse primer 5’-TGC-CGACCGTACAAGAGT-3’(202bp for the product) WT1/−17AA forward primer 5’-CACCTTAAAGGGC-CACAG-3’, reverse primer 5’-TGCCGACCGTACAA-GAGT-3’(157 bp for the product) MDR1 forward primer5’-CCCATCATTGCAATAGCA-3’, reverse primer 5’-GTTCAAACTTCTGCTCCTGA-3’, (157 bp for the product) β2-MG forward primer 5’-CTCGCGC-TACTCTCTCTTTC-3’ reverse primer 5’-CATGTCTC-GATCCCACTTAAC-3’, (330 bp for product).

Figure 1.Down-regulation of WT1 exon 5 transcript expression level following treatment with WT1/+17AA ShRNA. A. Schematic presentation of U6 promoter-based ShRNA expression vector. Sequences encoding ShRNA with 19-nt of homology to the target gene are synthesized as 65 bp double-stranded DNA oligonucleotides and inserted downstream of U6 promoter. B. WT1 isoforms mRNA expression was determined by real-time RT-PCR using SYBR green I dye. It was showed that the expression levels of WT1/+17AAN in K562-+17AAR and K562/A02-+17AAR were statistically lower than that in K562-con (*p<0.005) and K562/A02-con (**p<0.001) controls, respectively. C. WT1 isoform protein expressions were detected by Western blotting using monoclonal anti-WT1 antibody and horseradish peroxidase-conjugated goat anti-mouse secondary antibody. When compared with K562-con and K562/A02-con, there was significant reduction of WT1/+17AA isoform production in both K562-+17AAR (p<0.005) and K562/A02-+17AAR (p<0.001), while there was no significant change in WT1/−17AA expression (p>0.05). There was no difference in actin production among the these groups (p=0.453).

The products of WT1/+17AA, WT1/−17AA, MDR1 and β2-MG were ligated into pUCm-T vectors and serially 10-fold diluted in water with 1 to 10 copies to be a positive template. The normalized WT1 isoform expression levels (WT1/+17AAN and WT1/−17AAN) and the MDR1 expression level (MDR1N) were represented as a ratio of the WT1 isoform’s copy number and MDR1 products to the control β2-MG product, determined by real-time PCR for each RNA sample. It has been suggested that the protein expression of WT1/+17AA and WT1/−17AA was measured using western blotting. The expression of P-gp was detected by flow cytometric analysis using fluorescein isothiocyanate-conjugated anti-P-gp.4 P-gp function was examined for uptake, efflux and accumulation of Rh123. Doxorubicin cytotoxicity was determined by tetrazolium (MTT) assay. Normalized WT1 isoform WT1/+17AAN expression level in K562 and K562/A02 cells reduced rapidly from 182.45±42.32 to 92.77±27.38 (p<0.005, n=9) and from 243.77±56.93 to101.81±37.51 (p<0.001, n=9) respectively. The WT1/−17AAN expression remained relatively constant throughout (p>0.05, n=9). The ratios of WT1/+17AA and WT1/−17AA in paired cell lines were rapidly reduced, dropping from 1.71±0.37 and 1.90±0.41 to 0.77±0.21 and 0.82±0.30 (Figure 1B). Compared with control cells, the WT1/+17AA isoform protein expression significantly decreased in WT1/+17AA ShRNA transfected K562-+17AAR (p<0.005, n=6) and K562/A02-+17AAR (p<0.001, n=6). There was no change in the WT1/−17AA expression in these two cell lines (p>0.05) (Figure 1C). The MDR1 gene expressions were quantified at 8.72±2.93 and 272.54±42.73 in the control ShRNA transfected K562-con and K562/A02-con cells by real-time RT-PCR. In K562-+17AAR and K562/A02-+17AAR cell lines, MDR1 gene expressions decreased to 7.43±2.23 and 105.73±27.73 respectively. The MDR1 transcript expression was markedly down-regulated in the multidrug resistant K562/A02 cell line (p=0.017) following WT1/+17AA ShRNA transfection. The fluorescent intensity of P-gp was 53.13±7.56 in K562/A02-con cells and 4.39±2.32 in K562-con cells (p<0.001). The P-gp expression in K562/A02 cells clearly decreased, obviously, falling to 19.12±4.21, after transfection with WT1/+17AA ShRNA (p<0.005) (Figure 2A). However, the MDR1 gene and P-gp expression levels in K562- +17AAR cells showed qualitatively similar results with those in the K562-con cells (p>0.05). Similarly, Rh123 uptake was dramatically altered after transfection with WT1/+17AA ShRNA in K562/A02 cells. The K562/A02-+17AAR cells displayed rates of Rh123 accumulation in between those of K562-con and K562/A02-con, while the K562/+17AAR cells displayed uptake kinetics similar to that of the parental K562-con cells (Figure 2B). The K562-+17AAR cells also displayed doxorubicin dose-response similar to K562-con parental cells, with an IC₅₀ values of 0.79 and 0.94 μM. After transfection of WT1/+17AA ShRNA, the dose response curve of the K562/A02+17AA cells shifted significantly to the left, reducing the IC₅₀ value from 78.67μM to 22.59 μM. Thus, transfection of WT1/+17AA ShRNA led to a substantial reversal of doxorubicin resistance in K562/A02 cells but had no effect on the doxorubicin dose response profile of the parental K562 cells.

Figure 2.Representative flow cytometric histograms show the effect of WT1/+17AA ShRNA on the levels of cell surface P-glycoprotein expression and P-glycoprotein mediated rhodamine 123 efflux in both K562 and K562/A02 cells. A. P-glycoprotein is present at very high levels in drug-resistance K562/A02-con cells (53.92) when compared with parental drug-sensitive K562-con cells (4.78) (dark lines). The expression rates of P-gp in K562/A02-+17AAR cells (21.47, blue line) decreased obviously compared to K562/A02-con cells, whereas K562-+17AAR cells (4.54, red line) showed similar results with the K562-con cells. B. The uptake processes in K562 cells were unaffected by WT1/+17AA ShRNA transfection, the K562/+17AAR cells (dashed line) displayed uptake kinetics similar to that of the parental K562-con cells (dotted line), whereas Rh123 uptake was dramatically altered upon transfection with WT1/+17AA ShRNA in K562/A02 cells, the K562/A02-+17AAR cells (solid line) displayed rates of Rh123 accumulation between those of K562-con and K562/A02-con (heavy solid line).

It has been suggested that the presence of the exon 5 insert and the maintenance of the correct balance between WT1/+17AA and WT1/−17AA isoforms are essential for the regulation of critical cellular functions including proliferation, differentiation, and resistance to chemotherapeutic drugs.5–7 The changes in exon 5 splicing are also believed to affect the regulation of downstream gene expression profile in the WT1 pathway.8 The MDR1 gene transcription is regulated by the combined actions of several transcription factors that bind to its promoter region.9, 10 We suggest that ShRNA, when directed to WT1 exon 5, might inhibit WT1/+17AA isoform expression and strongly influence expression and function of the MDR1 gene in drug-resistant leukemia cells. To our knowledge, this is the first report of the relationship between the WT1/+17AA isoform and multidrug resistance in leukemia cells. These results provide novel insights into the role of WT1 exon 5 splicing transcript in the regulation of leukemia cells’ survival signaling pathways. This suggests that a designed RNAi targeted WT1 isoform could be a very effective strategy for MDR1 regulation.

Acknowledgments

the authors would like to thank Dr. Bao-An Chen (The Affiliated Zhong-Da Hospital, Southeast University) for providing the leukemia cell lines K562 and K562/A02

Footnotes

• Huiling Shen and Wenlin Xu contributed equally to this study and should be considered as co-first authors.
• Funding: this work was supported by a grant from the Jiangsu Natural Science Foundation of China (grant No. K2005017).