Two suspension leukaemic cell lines with t(8;21), Kasumi-1 and SKNO-1, were used in the present study. The former was obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA) while the latter was a kind gift from Professor Olaf Heidenreich (Northern Institute for Cancer Research, Newcastle University, UK). Kasumi-1 and SKNO-1 cells were cultured in RPMI-1640 medium containing 10 and 20% foetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), respectively. SKNO-1 was also supplemented with 10 ng/ml of granulocyte-macrophage colony stimulating factor (GM-CSF). Cell lines were incubated at 37°C in a humidified incubator with 5% CO₂.

Kasum-1 and SKNO-1 cells cultivated to late log growth phase, concentrated to 10⁷ cells/ml in culture medium without serum, were electroporated with siRNA using a Bio-Rad Gene Pulser Xcell™ at 330 V for 10 m. The sequences of siRNAs used are as follows: siRNA-MAPK1 (siMAPK1) sense, 5′-GUUCGAGUAGCUAUCAAGATT-3′ and antisense, 5′-UCUUGAUAGCUACUCGAACTT-3′; siRNA-RUNX1-RUNX1T1 (siRR) sense, 5′-CCUCGAAAUCGUACUGAGAAG-3′ and antisense, 5′-UCUCAGUACGAUUUCGAGGUU-3′; and scrambled mismatch siRNA (siMM) from Qiagen (Hilden, Germany).

For isolated downregulation of RR and MAPK1 gene expression, both cell types were electro-transfected with 200 nM of siRR and 100 nM of annealed siMAPK1 respectively; and for co-transfection a mixture of both siRR and siMAPK1 were used. These concentrations were derived from dose-dependent experiments performed in our laboratory. In experiments requiring extended transfection, cells were sequentially transfected on days 0, 4 and 7. Immediately after electro-transfection, cells were diluted 20-fold in complete culture medium and returned to the incubator. All cell groups were electroporated in triplicates.

Quantitative RT-PCR total RNA was isolated using RNeasy Mini kit (Qiagen) according to the manufacturer's protocol. RNA integrity was analysed on an Agilent 2100 Bioanalyser RNA 6000 NanoLabChip (Agilent Technologies, Palo Alto, CA, USA) to produce an electrophoresis trace of 18S and 28S peaks using 2100 Expert Software (Agilent Technologies). The RNA integrity number (RIN) was calculated directly from the peak areas, a value of 10 corresponded to intact RNA while 1 indicates a total degradation. Our optimised methods showed a 28S:18S ratio of 1.9–2.1:1 and an RIN of 9–10.

Quantitative real-time-polymerase chain reaction (qRT-PCR) was performed on RNA isolated at various time points post-transfection to quantify the mRNA expression levels of MAPK1 and RR genes in cells treated with respective siRNAs.

Gene-specific primers for MAPK1 (forward, 5′-CTGCTGCTCAACACCACCT-3′ and reverse, 5′-GCCACATATTCTGTCAGGAACC-3′); and B2M (forward, 5′-GGCATTCCTGAAGCTGACAG-3′ and reverse, 5′-TCTGCTGGATGACGTGAGTAA-3′) were designed using PrimerPlex software (Premier Biosoft, Palo Alto, CA, USA). Primers to quantify RR expression were: Forward, 5′-AATCACAGTGGATGGGCCC-3′ and reverse, 5′-TGCGTCTTCACATCCACAGG-3′ as described by Heidenreich et al (5).

A mixture of 50 ng of total RNA together with 0.5 µM of MAPK1 and B2M, and 0.2 µM of RR primers was reverse transcribed and amplified simultaneously in the same reaction tube using the QuantiFast SYBR^®-Green RT-PCR One-Step PCR Master Mix (Qiagen) in a final volume of 20 µl on a StepOnePlus Real-Rime thermocycler (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA). The thermocycler was programmed at 50°C for 10 min for RNA reverse transcription into complementary cDNA followed by 95°C for 5 min for the initial DNA polymerase activation step. Quantitative amplification was carried out in 35 cycles of denaturation at 95°C for 10 sec, and annealing and extension at 60°C for 30 sec. All reactions were performed in triplicates.

Relative quantity of MAPK1 and RR mRNA expression was computed using the comparative cycle threshold (2^−∆∆Ct) method normalised with the β₂ microglobulin B2M expression as endogenous reference gene. Mock cells were used as a reference sample for normalisation and AllStars negative siRNA (Qiagen) as a mismatch (siMM) control for validation purposes.

Differentiation ability of RR- and MAPK1-suppressed Kasumi-1 cells was monitored by CD34 expression. Kasumi-1 cells sequentially transfected on days 0, 4 and 7 with either siRR, siMAPK1 or a combination of siRR + siMAPK1 were double stained on day 9 with allophycocyanin conjugated anti-CD34:APC and peridinin-chlorophyll A protein conjugated anti-CD45:PerCP (Becton-Dickinson, Franklin Lakes, NJ, USA).

Briefly, ~10⁵ transfected Kasumi-1 cells were treated with Fc blocking reagent [phosphate-buffered saline (PBS) containing 0.02% sodium azide and 1% BSA] and incubated on ice for 30 min to block non-specific antibody binding. After incubation, the cells were washed and re-suspended in 100 µl of PBS. Cells were subsequently stained with 0.5 µl of allophycocyanin conjugated anti-CD34:APC and 2 µl of peridinin-chlorophyll A protein conjugated anti-CD45:PerCP (Becton-Dickinson). Cells were sorted on a FACSCanto II equipped with BD FACSCanto Clinical Software for acquisition and analyses. Mock cells and siMM-transfected Kasumi-1 cells were used as controls.

To determine the suppressive effects of MAPK1 and RUNX1-RUNX1T1 genes on apoptosis induction, Kasumi-1 cells were sequentially transfected on days 0, 4 and 7 with siMAPK1 and siRR singly, and in combination. Apoptosis was quantified on day 9 using an Annexin V/fluorescein isothiocyanate (FITC) assay kit (AbD Serotec^®; Bio-Rad Laboratories, Raleigh, NC, USA) using a FACSCanto™ II (BD Biosciences; Becton-Dickinson and Company, Franklin Lakes, NJ, USA) flow cytometer. In brief, cells in logarithmic growth phase after treatment with siRNA were washed once in cold 1X PBS and once in cold 1X binding buffer. Cells were suspended to a density of 1×10⁵ cells in 100 µl of 1X binding buffer and treated with 5 µl of Annexin V:FITC for 10 min in the dark at room temperature. On completion, the cells were washed again in 1X binding buffer and re-suspended in 190 µl of 1X binding buffer with 10 µl of propidium iodide (PI) and directly analysed on a flow cytometer. siMM-transfected cells were used as a negative control.

Distribution of Kasumi-1 cells in different phases of the cell cycle were examined using EZCell™ cell cycle analysis kit (BioVision, Milpitas, CA, USA) according to the manufacturer's protocol. Three aliquots of Kasumi-1 cells were eletrotransfected on days 0 and 4 with siRR and siMAPK1, and in combination respectively. Kasumi-1 cells electroporated with siMM and mock-transfected cells were used as controls. Cell cycle analysis was performed on day 6 by FACS analysis. Briefly, cells were washed in PBS, adjusted to a cell density of 1×10⁶ and fixed in 100% ice-cold ethanol for 24 h. On the next day, PBS washed cells were digested with 100 µl of RNase A for 30 min at 37°C followed by staining with 400 µl of PI and subsequently analysed using FACSCanto II flow cytometer at 488 nm wavelength using FL3 filters with wavelength of 670 nm for the proportion cell cycle distribution in the G₀/G₁, S and G₂/M phase.

The rate of viable cells was determined using trypan blue (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) staining method that selectively stains dead cells. Cell counting was carried out using a haemocytometer.

To quantitatively determine the number of viable cells in proliferation on days 0, 4 and 8 following electrotransfection, the siRR, siMAK1 and combined siRR/siMAPK1-treated Kasumi-1 cells were seeded separately, on the day of the experiment, into a 96-well plate at a density of 5×10⁴/well in 100 µl complete growth medium in quadruples. Twenty microliters of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium MTS/PMS solution (as instructed in the Cell Titer 96^® AQueousNon-Radioactive Cell Proliferation Assay kit literature; Promega Corp., Madison, WI, USA) was added and the plate was incubated for 2 h at 37°C in a humidified 5% CO₂ incubator. The absorbance was measured at 490 nm using an ELISA plate reader. Data are represented as mean ± SD of four independent experiments.

Post electrotransfection, genome wide expression (GWE) levels were determined in a series of four experimental conditions set up for each cell line, Kasumi-1 and SNKO-1. They include cells; co-transfected with siRR/siMAPK1; singly transfected with siRR; siMM; and mock-transfected cells. A total of 24 arrays, three replicates for each of the transfection experiment, were processed using Illumina HumanHT-12 v3 Expression BeadChip™ array (Illumina, San Diego, CA, USA) targeting 48,804 human transcripts per array with an average of 15-fold transcript redundancy.

Pre-hybridisation sample preparation used a total of 400–500 ng of RNA from each experiment, which was reverse transcribed to cDNA followed by amplification and in vitro transcription of cDNA to bio-11-dUTP-labelled complementary RNA (cRNA). Quantified (500 ng) and labelled cRNA was then hybridised to BeadChip arrays at 55°C overnight. BeadChips were then wash-cleaned with Illumina high-stringent wash buffer. The hybridisations were scanned after staining with 1 µg/ml streptavidin-Cy3 (Amersham Biosciences, Piscatawy, NJ, USA) on the Illumina BeadArray Reader confocal scanner and BeadScan software to produce bead level data.

Background subtracted bead summary data were exported to GeneSpring™ GX 11.0 software (Agilent Technologies, Santa Clara, CA, USA) for transformation and normalisation, and differential expression analyses. Microarray expression data were normalised to the most stable three reference genes (ACTB, B2M and UBC). Probe sets returning a P-value <0.05 (unpaired t-test) in comparison to the mock cells and experiment classes were considered to be differentially expressed. The differentially expressed genes were then filtered for gene sets that had fold-changes >1.1, and >1.5 together with false discovery rate (FDR) adjusted P-value ≤0.05.

The processed data were used for GO annotation and KEGG pathway enrichment analyses using the Database for Annotation, Visualization and Integrated Discovery [DAVID; (22)].

All statistical analysis was performed using SPSS software version 17 (SPSS, Inc., Chicago, IL, USA). Paired t-test was used to measure mean differences between two variables whereas one-way ANOVA was used to measure mean differences between three variables.

To identify the silencing efficacies of siMAPK1 and siRR, we transfected Kasumi-1 cells with 100 and 200 nM of the respective siRNAs separately in triplicates. Time course suppression of RR and MAPK1 genes in the experimental cells were compared with mock-transfected cells. The results of qRT-PCR showed that siMAPK1 inhibited the expression of MAPK1 by 85% on day one, and lasted for at least three days with 2% recovery (83%) on day two and 6% recovery (77%) on day three (Fig. 1A). siRR-treated Kasumi-1 cells exhibited up to 78% suppression of RR expression at 24 h and continued to further suppress to 82% at 48 h before 16% recovery was observed at 72 h. These observations as measured by qRT-PCR validates that significant knockdown (>80%) of RR and MAPK1 was achieved with respective doses of 200 nM of siRR and 100 nM of siMAPK1 at 48 h post-transfection. Melting curves for RUNX1-RUNX1T1 and MAPK1 are included in Fig. 1B and C, respectively.

We conducted gene expression analysis on mRNA extracts obtained at 48 h post-transfection using Illumina's HumanHT-12 v3 Expression BeadChips. A total of 24 arrays, three replicates for each of the four experimental conditions for both Kasumi-1 and SKNO-1 cells (as described above) were profiled for at least 48,804 transcripts to characterise the genes modulated when RR and MAPK1 were downregulated.

Microarray data for Kasumi-1 cells co-transfected with siMARK1 and siRR showed a total of 4,788 differentially expressed genes (DEG) as compared to the mock cells, accounting for 9.8% of the transcriptome probed in our array. However, when the 4,788 DEGs were conditioned to a fold change (FC) ≥1.5 and P<0.05 (t-test), the gene set condensed to 466 representing 262 upregulations and 204 downregulations. Relative to Kasumi-1 cells, co-knockdown of RR and MAPK1 genes in SKNO-1 cells revealed a more pronounced gene modulation presenting with 6,465 DEGs accounting for 13.2% of the transcriptome studied. Of these 6,465 DEGs, 732 sequences had a FC of ≥1.5 (at P<0.05) representing 415 upregulations and 317 downregulations.

We then examined the 466 and 732 gene sets with FC >1.5 for shared common genes modulated in both Kasumi-1 and SKNO-1 cells using the two-way Venn diagram analysis. In total, there were 148 overlapping genes, which included 109 upregulations and 39 downregulations (Fig. 2A). We also looked at the shared genes between the two cell lines when the fold changes were relaxed to 1.1. At this fold change, we observed 4,855 and 4,974 genes that were modulated in their respective co-transfected Kasumi-1 and SKNO-1 cell lines when compared to their respective mock-transfected cells. When used on two-way Venn diagram, these two enriched gene sets presented with 884 overlapping genes, which included 478 upregulations and 406 downregulations (Fig. 2B).

Whole genome expression studies from isolated RR-depleted Kasumi-1 cells presented with 5,178 DEGs relative to mock-transfected cells, accounting for 10.6% of the transcriptome probed. One-tenth of these, 552 genes, had a fold change expression ≥1.5 including 300 upregulations and 252 downregulations. In turn, RR-depleted SKNO-1 altered the expression of 9,049 genes. Of these, expression of 2,600 genes was significantly changed at a ≥1.5-fold cut-off including 1,267 upregulations and 1,333 downregulations. Two-way Venn diagram analysis enrolling the gene sets observed at fold changes ≥1.5 from Kasumi-1 cells (552) and SKNO-1 cells (2,600) revealed that a subset of 284 DEGs (including 197 upregulations and 87 downregulations) were common between the two cell lines (Fig. 2C), and support their association with the RR knockdown. Moreover, when the absolute fold change cut-off was adjusted to ≥1.1, we observed the number of overlapping genes from the two cell lines rose to 1,891, with 971 upregulations and 920 downregulations (Fig. 2D).

To reveal the potential function of differentially expressed genes common for both Kasumi-1 and SKNO-1 cell lines in terms of their associated biological processes, cellular components and molecular functions, we used the online data-mining tools of DAVID software (23,24). Uploading the 148 overlapping gene set, derived from the siRR + siMAPK experiment as depicted on the Venn diagram Fig. 2A, into DAVID with the default gene ontology (GO) setting (P<0.01) indicated association to 267 biological processes (BP), 49 cellular components (CC) and 52 molecular functions (MF). GO enrichment analysis for the 284 overlapping gene set identified from the Venn diagram analysis (Fig. 2C) from siRR experiments on Kasumi-1 and SKNO-1 cell lines identified 269 BP, 85 CC and 69 MF.

In order to validate the altered gene expression results of the BeadChip experiments, we performed quantitative PCR following reverse transcription (qRT-PCR) on the same RNA isolates used on BeadChips. We chose to quantify the expression levels of MAPK1 and RR, and compared the fold changes.

qRT-PCR assessment of RR and MAPK1 gene expression levels from the co-transfection experiment showed comparable downregulated fold changes validating the microarray data. As illustrated in Fig. 3, RR expression in co-transfected Kasumi-1 and SKNO-1 cells revealed a fold change between 1.75–1.85 and 1.4–1.6, respectively on qRT-PCR, while the same on the BeadChip had fold change downregulations between 2.4–2.6 and 1.9–2.3, respectively. Similar agreements were observed for MAPK1 in the co-transfected experiments. MAPK1 had a downregulated fold changes between 2.4–2.6 and 2.6–3.0 by qRT-PCR and 3.8–3.9 and 2.8–3.6 by BeadChip in Kasumi-1 and SKNO-1 cells, respectively.

The results from the isolated RR knockdown experiment were also consitent with those of BeadChip. On a log2 scale, qRT-PCR revealed that RR expression was downregulated by 1.5–1.7- and 1.6–1.7-fold, while the same on microarray was 2.9–3.3 and 3.2–3.7 in Kasumi-1 and SKNO-1 cells, respectively.

To determine the effect of RUNX1-RUNX1T1 and MAPK1 suppression and their combined influence on the proliferation rate, we transfected Kasumi-1 cells three successive times in a gap of three days with their respective siRNAs individually and in combination. MTS assays were performed on days 0, 4 and 8 to determine the proliferation activity. siMM and mock-transfected Kasumi-1 cells served as controls.

We observed that a single co-transfection with siRR and siMAPK1 or isolated transfection of siRR was not sufficient to inhibit the proliferation of Kasumi-1 cells when compared with siMM (Fig. 4). However, sequential electroporations on every third or fourth day attenuated the proliferation viabilities on days 4 and 8 in both the experiments. Similarly, two successive transfections of siMAPK1 were required to inhibit Kasumi-1 cell proliferation by 18.5% on day 4. The third successive transfection was only sufficient to sustain the proliferation at the same level on day 8. These data suggest that the fusion protein RR plays a more substantial role than MAPK1 in supporting the proliferation of leukaemic cells with t(8;21).

Our GO enrichment analysis revealed that the observed growth suppression upon RR knockdown was accompanied by deregulated expression of classical positive regulator genes that otherwise are known to enhance cell proliferation. They include significant downregulation of FLT3, HIF1A, KIT, MYCN, LYN, UBE2A, TBRG4, RPS9, RPS15A, RPA1, PRDX3, PDF, ODC1, IL13RA1, ITGB1, GRN, FABP1, CDK6, F2R, CDC123, CAPNS1, MARCKSL1, CD40, BCL2L1, AGPAT1, MXI1, CAV2, ENG, EMP3, IL8, MFN2 and TRIM24 genes, and upregulation of CEBPA, S100A11, ING4 and SSTR2 genes. On comparison, GO enrichment analysis for category of proliferation from RR knockdown and RR/MAPK1 co-knockdown experiments revealed several shared differentially expressed genes. On exclusion of the simultaneously altered genes, we observed that downregulation of JUN, NRAS, and STAT1 genes and upregulation of VHL, ZBTB16 and BTG1 genes were unique to MAPK1 depletion. The results of the GO enrichment analysis are summarised in Table I.

Next, we examined the consequences of isolated and co-depletions of RR and MAPK1 on cell apoptosis. Kasumi-1 cells harvested on day 9, after three consecutive transfections, were double stained using Annexin V/FITC and PI.

Three consecutive siRR treatments significantly (P=0.0001) lowered the live cells by 24% when compared with the siMM control indicating that RUNX1-RUNX1T1 suppression has an apoptotic effect on Kasumi-1 cells (Fig. 5). Notably, pronounced apoptosis (30±1.6%) was observed upon isolated MAPK1 deletion, and revealed a significant difference (P<0.0001) when compared with control cells. However, co-depeletion demonstrated no difference when compared with the siMM control cells. FACS results for the apoptosis analysis are shown in Fig. 5B-E.

GO enrichment analysis on shared DEGs from both Kasumi-1 and SKNO-1 cells with fold change of ≥1.5 in response to RR depletion exhibited several changes to genes involved in negative regulation of apoptosis. They included significant upregulation of BCL2, SPHK2, CFLAR, CD24, NOTCH1, ADAM17, BIRC3 and HMGB1, and downregulation of IP6K2 and BECN1 genes. Exclusion of overlapping differentially expressed genes involved in apoptosis between the isolated RR knockdown and the co-knockdown experiments revealed five genes specific for MAPK1 depletion. They include upregulated expression of TP53, TNFSF10 and ADA genes as well as downregulated expression of JUN and NRAS genes. The results of the GO enrichment analysis are summarised in Table I.

To investigate the effect of RUNX1-RUNX1T1 and MAPK1 depletion on the progenitor status of Kasumi-1 cells, we analysed the CD34 expression levels as an indicator of differentiation. Compared with the cells, RR suppression showed statistically significant (P=0.0001) loss of CD34-expressing cells by ~23±1.1% 9 days after the first of the three successive electroporations indicating that the cells were undergoing apparent differentiation (Fig. 6A). The loss of CD34-expressing cells corresponded with an increase in cells expressing CD45 which indicated differentiation. The FACS results are shown in Fig. 6B-E, respectively. Based on the gene ontology analysis on the RR-suppressed microarray data, we identified several genes involved in cell differentiation to be activated or repressed. Upregulated genes included CD24, NOTCH1, DNMT3B, CEBPA, CEBPE, ID2, JMJD6 and IKZF1, while RHOA, CBFB, KIT, CDK6 and FLT3 were downregulated.

When both MAPK1 and RR were co-suppressed, the expression of CD34 in Kasumi-1 cells were relatively less pronounced (13±2.3%) (Fig. 6A). However, CD34 suppression revealed a significant difference (23±1.1%), when compared with the control cell expression in which only RR was suppressed. This implies that MAPK1 abrogated the effect of RR by ~40% suggesting that tandem MAPK1 suppression exerted an anti-differentiation effect. Due to this antagonist effect of MAPK1 and RR co-suppression, we used the same strategy mentioned in the apoptosis section to identify the genes more specific for MAPK1 knockdown. After exclusion, MAPK1 knockdown revealed significant downregulation of c-JUN and upregulation of ADA, both identified to have anti-differentiation effect.

FACS experiment on isolated MAPK1 suppression showed no significant loss of CD34 expression when compared with mock and AllStar siRNA-treated control cells (Fig. 6A). The results of the GO enrichment analysis are summarised in Table I.

Finally, we examined the effect of different siRNAs on cell cycle progression. Kasumi-1 cells were electro-transfected twice o a gap of three days with either siRR, siMAPK1 or co-transfected with siRR/siMAPK1. Cell phase distributions were analysed on day 6 by flow cytometry. In comparison with the mock-transfected cells, RR suppression showed a reduction in the percentage of cells in the S and G2M phases (Fig. 7A). This was correlated with increased cells in the G₀/G₁ phase by ~13%. Gene expression data following RR depletion in the Kasumi-1 and SKNO-1 cell lines showed that several genes involved in positive and negative regulation of the cell cycle were variably expressed. Upregulated genes included FOXO4 and ING4, and downregulated genes were TPX2, CDC2, CKAP5, IL8, MAD2L1, CCNE2, CCNG2, CDK6, TBRG4 and KPNA2. Isolated MAPK1 suppression also caused an increased growth arrest. This was accompanied with reduction in G2M and S phases, while cells in the G₀/G₁ phase were increased (Fig. 7A). FACS results for the cell cycle analysis are included in Fig. 7B-F, respectively.

Combined targeting of RR and MAPK1 yielded further reduction of cells in S and in G2M phase by ~40 and 70%, respectively, when compared to the mock cells. This also led to an increased number of cells in the G₀/G₁ phase by ~25%. Gene ontology for double knockdown of RR and MAPK1, after exclusion of common target genes, demonstrated that several protein coding genes were uniquely responsive to the depletion of MAPK1; they included upregulation of RASSF1, GADD45A, P53 and FBXO6, and downregulation of MAPK11. The results of the GO enrichment analysis are summarised in Table I.