Background
Noncoding RNAs (ncRNAs) are usually transcribed by RNA polymerase II and have intrinsic functions without being translated into polypeptides [
1-
4]. MicroRNAs (miRNAs), are important ncRNAs of approximately 20 bp that silence specific target genes [
5-
9], while long noncoding RNAs (lncRNAs) are between 200 bp and several kb and have numerous roles in cellular functions and gene regulation [
10]. However, apart from a few examples, the molecular mechanisms by which most lncRNAs function remain to be fully elucidated [
11,
12]. Accumulating evidence suggests that some lncRNAs are involved in diseases, such as cancer [
13,
14]; therefore, it is important to understand their roles to facilitate the search for new therapeutic targets and to design new diagnostic methods.
Acute myeloid leukemia (AML) is a disease characterized by mutations in a set of genes [
15-
17]. These genes can be divided into two classes. One class includes genes related to cell differentiation,
HOXA9,
AML1,
MLL and
RARα, and the other consists of genes related to proliferation or survival of cells and includes
FLT3,
ABL,
RAS and
KIT. Mutations in genes of both classes need to occur to give rise to AML [
15,
18]. Fusion of
BCR and
ABL (
BCR/
ABL) is well known to be sufficient to cause chronic myeloid leukemia (CML) [
19]. However, acute blastic crisis of CML is often accompanied by mutation of genes that are also mutated in
AML, such as
HOXA9 or
AML1 [
20,
21]. Thus, hematopoietic tyrosine kinases, including
FLT3,
ABL,
RAS and
KIT, seem to have a similar role in AML and CML [
22]. Considering the importance of imatinib, an ABL tyrosine kinase inhibitor used in the treatment of
BCR/ABL-positive CML, inhibitors against other tyrosine kinases are likely to become increasingly important for the treatment of AML [
19,
23]. KIT is a receptor tyrosine kinase that is considered to have a role in AML because of its frequent up-regulation in patients. Expression of
KIT in leukemia stem cells (LSCs) from pediatric AML patients who relapsed after chemotherapy was increased compared with that in patients who did not relapse [
24].
The function of the lncRNA,
CCDC26, is not known. There is no evidence for a functional CCDC26 protein; moreover, the hypothetical 109 amino acid protein encoded in its mRNA has no homology with known proteins [
25]. Despite the ambiguous nature of
CCDC26, several lines of evidence support a relationship of
CCDC26 with tumors, including AML. Radtke and colleagues investigated copy number alterations (CNAs) in pediatric AML genomes and found that the most common CNA is a low burden increase of a region within the
CCDC26 locus [
26]. Studies by ourselves and others have shown that part of or the entire
CCDC26 gene is often amplified in AML cells harboring aberrant double minute chromosomes [
27-
29]. Others suggest that sensitivity of AML cells to anticancer drugs, including retinoic acid, is lost with integration of retroviral DNA into the
CCDC26 locus [
30]. Linkage of
CCDC26 with tumors, including low-grade glioma, was also suggested by genome wide association studies [
31].
In this paper, we demonstrate that knockdown of CCDC26 in CML-derived K562 cells results in transcriptionally-altered expression of several genes, including activation of KIT, and prolonged cell survival under low or no serum conditions. ISCK03, a KIT inhibitor, abolished this prolonged survival. These results provide evidence for a new role of CCDC26 in myeloid leukemia through the regulation of a set of genes that includes KIT.
Discussion
We found several regions in the
CCDC26 locus from which transcripts are produced. These transcripts may be independently transcribed or processed from
CCDC26 introns that have been spliced out of its precursor mRNA. The transcripts accumulate in K562 cells but, at present, it is difficult to determine which products are functional among the mature mRNA and intronic
CCDC26 transcripts [
35]. However, accumulation of mature
CCDC26 products in the nucleus suggests a functional role [
36,
37].
An shRNA usually suppresses its target gene by post-transcriptional gene suppression (PTGS), which includes RNA degradation via the RNA-induced silencing complex (RISC). But in some cases, transcriptional gene suppression (TGS) can take place via the RNA-induced transcriptional silencing complex (RITS) [
38,
39]. In this study, shRNA-mediated PTGS does not account for the suppression of the majority of
CCDC26 transcripts in KD strains (22B11, 32H8, 3–4 and 3D1); partial TGS may have occurred in 32H8 cells because the THS1 transcript was not suppressed in these cells, as shown in Figure
2B. A straightforward explanation in this case is TGS. Moreover, the chromatin range over which TGS occurs is somewhat different among the
CCDC26-KD clones and 1.0 μM TSA and 1.0 μM AzdC partially prevented the silencing (Figure
3C). Release from silencing by TSA and AzdC was observed at genes located on the borders of the suppressed chromosomal region. This suggests that TGS of these genes by epigenetic mechanisms is accompanied by repressive chromatin modification in the
CCDC26-KD clones. In contrast,
CCDC26 itself and the adjacent gene,
LOC728724, remained silent even after treatment with TSA and AzdC (Figure
3C), indicating that TGS of these genes occurs via a different mechanism.
All the KD clones showed lower growth rates than non-KD control K562 cells. It seems likely that CCDC26 acts as an oncogene to control cell proliferation, either directly or indirectly. Paradoxically, however, the KD clones proliferated more rapidly in low serum conditions compared with non-KD cells. Additionally, the KD clones survived for longer under conditions of very low or no serum compared with non-KD control cells, which died relatively rapidly under these conditions. This observation indicates that suppression of CCDC26 enables leukemia cells to survive and proliferate despite a severe shortage of growth factors.
DNA microarray and quantitative PCR analysis revealed that the expression of several genes was changed in KD clones. Among them, activation of
KIT is especially interesting because this gene is over-expressed in many AML patients. Of note, however, activation of KIT protein in KD cells seemed to be stochastic in spite of their monoclonal origin (Figure
6B). It is likely that there are cells that never express
CCDC26 and cells that express more than one molecule, because the number of
CCDC26 transcripts in knockdown cells was calculated at less than one per cell, but the transcript was still detected. This might explain the heterogeneous expression of KIT protein in KD cells, although further investigation is needed to resolve this point. Although the existence of KIT-positive cells
per se is not a prognostic factor, point mutations in
KIT that cause enhanced tyrosine kinase activity are frequently accompanied by chromosomal translocation t(8;21) in adult AML, which results in poor prognosis [
40]. Other genes, including
CD24 (a sialoglycoprotein expressed on mature granulocytes and B cells),
PASD1 (a transcription factor expressed in diffuse large B-cell lymphoma),
MS4A3 (a hematopoietic cell-specific membrane protein),
SAMSN1 (a protein with an SH3 domain and nuclear localization signals) and
MS4A4 (from the same family as
MS4A3) are also interesting because of their involvement with tumors but their relationships to myeloid leukemia are not clear. Nevertheless, we cannot exclude the possibility that
CCDC26 regulates cell proliferation or other properties of cells through some of these genes. Overall, we suggest that this ncRNA has novel, previously unknown roles in cellular function. At present, a regulatory function of
CCDC26 has been shown in only a single cell line, K562. Despite our efforts, we have not been successful in suppressing the
CCDC26 transcripts by RNA interference to sufficient levels in cell lines other than K562 for unknown reasons so far (unpublished results). Further study with different approaches including genome editing might be required to confirm its function.
Although CCDC26 is a low-burden amplified gene expressed in some leukemia cells, amplification occurs partially and does not extend across the whole gene in most cases. This makes it difficult to know whether partial amplification of the gene results in enhancement or loss of its original function. KIT is a well-known oncogene; therefore, if over-expression of the incomplete CCDC26 RNA masks KIT function, our findings are consistent with CCDC26 suppressing some oncogene(s). However, further investigation is required to fully understand this relationship.
LSCs with CD34+ and CD38− surface markers are considered to be a cause of AML recurrence because they have the potential to survive in niche sites that escape the influence of drugs [
41].
CCDC26-KD cells might share some properties with LSCs, including relatively slow growth and the ability to survive under certain conditions, such as a shortage of growth factors. Constitutive activation of KIT might contribute to survival of these cells by an autocrine mechanism involving stem cell factor, a ligand for KIT [
42]. Although LSCs are usually negative for KIT in
de novo AML [
43], the existence of KIT-positive LSCs is related to an increased tendency of pediatric AML recurrence after chemotherapy. This can arise from constitutive activation of KIT protein [
44].
Methods
Cell lines
HL-60 cells are described elsewhere [
47,
48]. K562 and other cells were obtained from ATCC. All cells were maintained in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, 100 U/ml streptomycin at 37°C in a humidified incubator with a 5% CO
2 atmosphere. KD strains derived from K562 were maintained with 0.8–1.0 mg/ml G418 and cultured for at least 12 hours in fresh medium without G418 prior to use in experiments. Cells were counted with a hemocytometer after staining with 0.25% trypan blue. Dead cells were stained blue and live cells excluded the dye. TSA (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 5 mM. The 5 mM TSA solution was diluted 1/5000 in culture medium. AzdC (Sigma-Aldrich) was dissolved in water and added directly to culture medium. Growth of K562 cells was inhibited by 40–60% in media with 1.0 μM TSA and 1.0 μM AzdC. Equivalent concentrations of DMSO were added to control cells without TSA and AzdC.
Plasmids
The shRNA expression vector, pGER, is a derivative of the pGE-1 GeneEraser plasmid purchased from Stratagene (La Jolla, CA, USA). The original SV40 virus early gene promoter of pGE-1 that drives the G418 resistance marker gene was substituted with the Rous sarcoma virus (RSV) promoter, which is more suitable for leukemia cells. In brief, a 580 bp RSV promoter isolated from pRSV.5(neo) (GenBank: M83237.1) by digestion with HindIII and NdeI was blunt-end ligated to a 2.6 kb fragment purified from pGE-1 partially digested with StuI and PvuII.
Isolation of KD clones
Transfection to K562 cells was performed using Dimrie-C (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions using 0.5 μg of plasmid DNA per 105 cells in 500 μl medium. Cells were plated in methylcellulose based-matrix (ClonaCell; Stem Cell Technologies, Vancouver, Canada) on six-well culture plates to form colonies and were selected with 1.6 mg/ml G418 from 48 hours after transfection. Visible colonies were isolated using a Pipetman under microscopic observation after selection for 6–8 days. Cells were transferred to 96-well plates and were maintained to proliferate in culture medium containing 1.6 mg/ml G418. For the first screening, 104 cells were used for RNA preparation. Clones selected in first screening were subcloned by the limiting dilution method and propagated. RNA was then prepared for a second screening. Colonies of empty pGER transformants were pooled and used as control non-KD cells.
Quantification of RNA
RNA for quantification was prepared using RNAiso Plus (Takara Bio, Otsu, Japan) according to the manufacturer’s instructions. Synthesis of cDNA was performed using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) according to the manufacturer’s instructions. Quantification of cDNAs using primer sets specific to each gene or transcript was performed with the LightCycler system (Roche Diagnostics, Indianapolis, IN, USA). To measure the absolute number of cDNA molecules, known concentrations of PCR product were used to generate a calibration curve. Total RNA in a single cell was estimated at 10 pg (1 mg for 10
8 cells). In other quantification, we used
HPRT gene expression as an internal standard. Primer sets used in PCR experiments are listed in Additional file
9: Table S4.
Isolation of nuclei
Isolation of nuclei was performed according to Spector et al. [
49] with slight modification. In brief, 1 × 10
7 cells were washed once with PBS(−) and suspended in 0.5 ml of buffer A (10 mM Hepes-KOH [pH 8.0], 10 mM KCl, 1.5 mM MgCl
2, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.5 U/μl RNase inhibitor). Cells were incubated on ice for 10 min and then lysed in a Dounce homogenizer. Crude nuclei were collected by centrifugation at 1,300 × g for 5 min and resuspended in 0.5 ml buffer B (0.25 M sucrose, 10 mM Tris–HCl [pH 7.9], 5 mM MgCl
2, 1 mM DTT, 0.5 mM PMSF). Suspended crude nuclei were centrifuged on a cushion of 0.4 ml buffer C (1.2 M sucrose, 10 mM Tris–HCl [pH 7.9], 5 mM MgCl
2, 0.1% TritonX-100, 1 mM DTT, 0.5 mM PMSF) at 10,000 × g for 30 min. Precipitated nuclei were resuspended in 0.5 ml buffer B and again centrifuged on a cushion of 0.4 ml buffer C. All procedures were carried out at 0–4°C. The precipitated nuclear fraction was used directly for RNA preparation using RNAiso plus (Takara Bio).
DNA microarray analysis
Total RNA from each clone was prepared using RNAeasy (Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. Using an Ambion WT Expression Kit (Ambion, Foster City, CA, USA) and a WT Terminal Labeling Kit (Affymetrix, Santa Clara, CA, USA), 250 ng of RNA was converted to biotin-labeled single stranded cDNA. Fifteen micrograms of single stranded DNA was hybridized to a Human Gene 1.0 ST Array (Affymetrix), followed by staining and washing. Scanning was carried out using a GeneChip Scanner 3000 7G (Affymetrix).
PI-staining and FACS analysis of dead cells
Cells were washed with PBS and stained in buffer containing 3% FBS, 0.05% NaN3 and 2 μg/ml propidium iodide (Sigma-Aldrich) for 15 min at room temperature and analyzed on a FACSCalibur flow cytometer (Beckton Dickinson, Franklin Lakes, NJ, USA).
Western blot analysis
K562 cells, pGER vector-transformed K562 cells (Vec) and four strains of K562-KD cells were cultured at 37°C in a 5% CO2 atmosphere until the logarithmic growth phase. 2 × 106 cells were lysed by sonication in 0.2 ml Laemmli buffer (125 mM Tris–HCl, pH 6.8, 4% sodium dodecyl sulfate (SDS), 100 mM dithiothreitol). Ten microliters of protein extract, equivalent to 1 × 105 cells, were separated on a SDS polyacrylamide gel electrophoresis pre-made 5–20% gradient gel (Wako Pure Chemical Industries, Osaka, Japan) and then transferred to polyvinylidene fluoride membrane (Immobilon-P Transfer Membrane; Millipore, Billerica, MA, USA). The blot was blocked in 7% BlockAce (DS Pharma Biomedical, Osaka Japan) at 4°C for 2 nights to suppress nonspecific signal and then incubated in CanGetSignal solution 1 (Toyobo) with 1/1500 diluted mouse monoclonal anti-KIT antibody (#Ab-81; Cell Signaling Technology, Danvers, MA, USA) and mouse monoclonal anti-beta-actin antibody (#E1C605; Enogene, New York, NY, USA) (to determine loading). After washing, the blot was incubated in CanGetSignal solution 2 (Toyobo) with anti-mouse IgG secondary antibody conjugated with horseradish peroxidase (KPL, Gaithersburg, MD, USA) and then visualized with Chemi-lumi One Super (Nakalai Tesque, Kyoto, Japan). Images were recorded with a Lumino imaging analyzer FAS-1000 (Toyobo).
Immunocytochemistry
Cells were suspended and fixed in PBS containing 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, cells were suspended in 0.2% Triton/PBS and incubated for 10 min at room temperature. After washing with PBS, cells were spread on glass slides using the Thin-layer cell preparation system Cytospin 4 (Thermo-Fisher Scientific, Waltham, MA, USA). Cells were then incubated with 2% BSA in PBS to block nonspecific signals followed by incubation with 1/100 diluted mouse monoclonal anti-KIT antibody (#Ab-81; Cell Signaling Technology) in CanGetSignal immunostain solution B (Toyobo) overnight at 4°C. Alexa Fluor 546 goat anti-mouse IgG (Invitrogen) was used as a secondary antibody. The specimens were counterstained with 1 μM Hoechst 33428 (AnaSpec, Fremont, CA, USA) and observed under a fluorescence microscope (BA-9000; Keyence, Osaka, Japan).
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
RY carried out the cell fractionation study. HH and YH prepared samples from cell lines. AI and TY planned and supervised execution of the project. All authors read and approved the final manuscript.