Apart from microRNAs, little is known about the regulation of expression of non-coding RNAs in cancer. We investigated whether small nucleolar RNAs (snoRNAs) accumulation displayed specific signatures in acute myeloblastic and acute lymphoblastic leukemias. Using microarrays and high-throughput quantitative PCR (qPCR), we demonstrate here that snoRNA expression patterns are negatively altered in leukemic cells compared with controls. Interestingly, a specific signature was found in acute promyelocytic leukemia (APL) with ectopic expression of SNORD112–114 snoRNAs located at the DLK1-DIO3 locus. In vitro experiments carried out on APL blasts demonstrate that transcription of these snoRNAs was lost under all-trans retinoic acid-mediated differentiation and induced by enforced expression of the PML–RARalpha fusion protein in negative leukemic cell lines. Further experiments revealed that the SNORD114-1 (14q(II-1)) variant promoted cell growth through cell cycle modulation; its expression was implicated in the G0/G1 to S phase transition mediated by the Rb/p16 pathways. This study thus reports three important observations: (1) snoRNA regulation is different in normal cells compared with cancer cells; (2) a relationship exists between a chromosomal translocation and expression of snoRNA loci; and (3) snoRNA expression can affect Rb/p16 cell cycle regulation. Taken together, these data strongly suggest that snoRNAs have a role in cancer development.
In cancer cells much effort has been devoted to the understanding of microRNA expression.1, 2 Other classes of non-coding RNAs (ncRNAs) were originally considered products of constitutively expressed housekeeping genes or representing transcriptional noise so until now have been largely neglected. Small nucleolar RNAs (snoRNAs) are 60–300 nucleotide-long ncRNAs that are excised from the intron regions of pre-mRNAs, usually those encoding fundamental housekeeping proteins.3, 4, 5 There are two structurally distinct classes of snoRNAs, called the box C/D and H/ACA snoRNAs, which function mainly as guide RNAs in the site-specific 2′-O-methylation and pseudouridylation of rRNAs, respectively.3, 6, 7 However, a subgroup of so-called ‘orphan’ snoRNAs exists that lacks any apparent complementarity to cellular RNAs and therefore has unknown function. A few orphan snoRNAs, such as SNORD112 to SNORD116, are encoded by multiple, tandemly arranged intronic genes that display high sequence similarity.8, 9
Recently, new unexpected regulatory functions have been described for human snoRNAs. In terms of pathologies, the human HBII-52 cluster that expresses several variants of the SNORD115 snoRNA was implicated in a neurodevelopmental disorder, the Prader-Willi syndrome.10 Moreover, recent reports suggest that snoRNAs could be relevant to oncogenesis and solid cancer prognosis.11, 12, 13, 14 Thus, we hypothesized that, in cancer, expression of human snoRNAs is regulated and displays specific signatures. We used the acute myeloblastic leukemia (AML) model for two reasons: (1) it represents an interesting model of oncogenesis that is mainly based on blockade of cell differentiation and (2) chromosomal anomalies are frequently observed due to recurrent translocations forming oncogenic fusion proteins. As microRNA signatures can be specific to subsets of chimeric proteins in leukemia,15 we assumed that this was also the case for snoRNA profiles. We tested snoRNAs already present on microarrays together with high-throughput quantitative reverse transcription (RT-qPCR) on microfluidic chips.
Materials and methods
Patients and healthy donors
Fresh and thawed samples from AML or acute lymphoblastic leukemia (ALL) patients (Supplementary Table 1) and from healthy donors (bone marrow, CD3+, CD19+ and CD33+ cells) were obtained as described in Supplementary Methods.
RNA preparation and cDNA synthesis
Total RNA was prepared by extraction with TRIzol (Invitrogen, Grand Island, NY, USA) from mononuclear-sorted cells (CD33+, CD3+ and CD19+) and fresh or thawed samples (from AML and ALL patients). RNA integrity was evaluated using the RNA 6000 Nano Chip kit (Agilent Technologies, Massy, France). Only RNAs with a RIN>7.5 were used. RNAs were reverse transcribed into cDNA using 3 μM of random primer mix (Biolabs, Evry, France) and Superscript III reverse transcriptase (Invitrogen). For high-throughput qPCR, RNAs were polyadenylated by the Poly(A) Polymerase Tailing Kit (EPICENTRE Biotechnologies, Madison, WI, USA) before RT.
GeneChip microRNA arrays
Four hundred nanograms of total RNA were hybridized on GeneChip microRNA array (Affymetrix, High Wycombe, UK). Unlogged data were treated with the dChip software (Boston, MA, USA) with a specific cutoff (filter) to generate dendrograms. The cutoff value of 0.5 applied to microRNAs could not be used for snoRNAs because their expression was lower, thus, we used a cutoff value of 0.25. Rows (corresponding to RNAs of interest) were standardized by respective RNA means of expression and distant metric system applied is correlation. Sample clustering was carried out by the centroid method. Significance analysis of microarrays was used to analyze statistical significance of the results.
The Fluidigm high-throughput qPCR method (Biomark, Fluidigm, South San Francisco, CA, USA) was used as described previously16 and developed in Supplementary Methods. Primers used for amplification are described in Supplementary Table 2. Data were analyzed as for qPCR using 5S rRNA as the housekeeping gene (GAPDH and ACTIN were also tested). Non-supervised clustering was realized using the dChip software as for microarray analysis with a cutoff value of 0.85. The unpaired t-test was used for statistical analyses.
qPCR and data analysis
For real-time PCR, the 25 μl reaction contained 1xSYBR Green PCR Master Mix (Sigma-Aldrich, St Louis, MO, USA), with each primer at 0.3 μM, and 5 μl of cDNA (see above) diluted at 1:5. The primer efficiency (>85%) was checked before the experiments. The 2−ΔCT (threshold cycle) values were used for the analysis (5S rRNA was selected as the housekeeping gene).
In vitro differentiation of acute promyelocytic leukemia (APL) blast cells
APL blasts were cultured (Supplementary Methods) in the presence of 1 μM all-trans retinoic acid (ATRA) (Sigma, St Louis, MO, USA) diluted in ethanol, or in ethanol alone. After 3 and 5 days, the percentage of differentiated cells was assessed by cell morphology with May-Grünwald-Giemsa stained cytocentrifuge slides and by expression of CD11b as determined by flow cytometry. The monoclonal antibodies used for staining were as follows: CD11b-PE-Cy7 (BD Biosciences, Le pont de Claix, France) and CD33-PE-Cy5 (BD Biosciences) and isotype-matched control conjugates. Flow cytometry was performed using a BD-LSRII flow cytometer (BD Biosciences).
PML-RARalpha transient transfection into a KG-1 cell line
A cDNA encoding PML-RARalpha (kindly provided by Valerie Lallemand-Breitenbach; St Louis Hospital, Paris, France) was cloned into an expression vector pCDNA3 (Invitrogen). KG-1 cells (ATCC_CCL-246, Molsheim, France) were transfected with pCDNA3 or pCDNA3-P/R using nucleofection Amaxa Technology (Lonza, Basel, Switzerland) and Ingenio universal solution (Mirus Bio LLC, Madison, WI, USA). SnoRNA expression was monitored by RT-qPCR after 48 and 72 h of transfection. PML-RARalpha expression was monitored by RT-PCR using primer F (5′-IndexTermCAGTGTACGCCTTCTCCATCA-3′) and primer R (5′-IndexTermGCTTGTAGATGCGGGGTAGA-3′).
14q(II-1) overexpression and silencing
Sequences containing snoRNA (Figure 4b) were amplified by a Pfu-Ultra enzyme, sequenced and then cloned. The latter construct or empty vector were transiently transfected by Amaxa technology (Solution V, T16-program) in K562 cell line (ATCC_CCL-243). Cell growth was evaluated by Malassez cell counting or MTS ([3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt) proliferation assay (Promega, Madison, WI, USA). Overexpression of 14q(II-1) snoRNA was checked by qPCR using the primer described in Supplementary Table 2.
14q(II-1) silencing in K562 cell line was realized using siRNA (siRNA-14q(II-1), Sigma; siRNA-negative control, Eurogentec, Angers, France) transfection (25 nM as a final siRNA concentration) using Lipofectamine RNAiMax (Invitrogen). Effective targeting after 48 h was analyzed by qPCR as described above. Total Rb and p16 protein expressions were evaluated by western blotting using a mouse anti-human total Rb monoclonal antibody (Sc-102), mouse anti-human p16 (CDKN2A) antibody (Sc-9968) and anti-β-actin (MAB150L). Rb phosphorylation on Ser780 was analyzed using rabbit anti-human phosphor-ser780-Rb (Sc-9307).
Cell cycle analysis
Cells were washed with phosphate-buffered saline and fixed in cold 70% ethanol for 20 min. Cells were then washed twice with phosphate-buffered saline-0.1% bovine serum albumin and once with phosphate-buffered saline then labeled using propidium iodide staining for 30 min (Invitrogen). Cell cycle distribution was evaluated by fluorescence analysis on a FACScan cytometer (Beckton Dickinson Biosciences). Cell doublets were excluded and 20 000 events per condition were analyzed.
Global downregulation of snoRNAs in ALL and AML
To characterize specific snoRNA expression patterns in AML, we first used GeneChip microRNA arrays that allow the simultaneous screening of almost all snoRNAs and microRNAs that are listed in the ENSEMBL and snoRNABase databases (http://www.ensembl.org and http://www-snorna.biotoul.fr/). SnoRNA expression levels were screened in a cohort of 12 AML samples and compared with 4 CD33+ myeloid-sorted cells from healthy donors. By using non-supervised clustering, we observed a global downregulation of snoRNAs compared with their non-neoplastic counterparts, thus distinguishing leukemic cells from normal cells (Figure 1a). To verify that these observations were robust, we used the same method to analyze ALL samples matched with CD3/CD19-sorted cells. This confirmed a global snoRNA downregulation that was the main characteristic differentiating healthy donors from cancer samples (Figure 1a). SnoRNA profiles from CD3+ and CD19+ control samples were also analyzed and demonstrated that the latter can be regarded as a single-control group (Supplementary Figure 1). To evaluate the accuracy of the snoRNA signatures, we also analyzed microRNA expression profiles and compared them to those reported in the literature. We successfully reproduced the microRNA signatures previously described for AML and ALL, confirming that our experimental design was robust (Figure 1b).17
To further confirm these results we used the Biomark digital PCR system (Fluidigm) for high-throughput expression studies on 62 selected snoRNAs, among which 46 significantly varied on microarrays. The remaining 16 snoRNAs, 5 guide and 11 orphan RNAs did not vary on the microarrays. In addition, we also tested four spliceosomal small nuclear RNAs (U2, U4, U6 and U12) and two snoRNAs (U8 and U3) that are synthesized from independent genes. The 5S ribosomal RNA was used to normalize snoRNA expression due to its stability and length. We extended our RT-qPCR experiments to 26 AML cases. Non-supervised clustering indicated that the snoRNA expression profiles of the AML samples were different from those of CD33+ myeloid cells (Figure 1a), CD34+ progenitors or total bone marrow (Supplementary figure 1). It is worth mentioning, however, that Flt3-ITD-mutated AML cosegregated with controls, suggesting that this group of leukemia is distinct with regard to snoRNA profiles (Figure 1a). To confirm the identity of snoRNA expression patterns obtained in ALL cases by microarray analysis, we also checked the delineation of ALL samples compared with ‘lymphoid controls’ in a larger cohort (25 ALL cases). In leukemic samples, 61% of snoRNAs tested were significantly underexpressed compared with control cells (Figure 1a; Supplementary Table 3). This is in keeping with the global underexpression reported for microRNAs in several types of cancers.1, 18, 19 Thus, RT-qPCR experiments validated the global downregulation of snoRNA expression previously noticed on microarrays (Figure 1a).
Specific snoRNA expression profiles identify APL cases
Although snoRNAs appeared to be globally downregulated in leukemia cells, few snoRNAs located into clusters are overexpressed in some AML cases. Their expression was associated or not to specific chromosomal translocations (Supplementary figure 2). Thus, through microarray analysis we noticed ectopic expression of a set of snoRNAs from the DLK1-DIO3 locus in APL samples carrying identical PML-RARalpha_bcr1 translocations (Figure 1a). DLK1-DIO3 snoRNAs are located within the introns of the maternally expressed gene 8 (Meg8), which carries 1, 9 and 31 highly related copies of SNORD112 (14q(0)), SNORD113 (14q(I)) and SNORD114 (14q(II)) snoRNA genes, respectively (Figure 2a).8, 20 As expected (because of their restricted expression pattern), they were not detected in controls (healthy CD33+ myeloid cells) or in PML/RARα-negative AML samples (Figures 2b and c). Using microarrays, we observed that only eight sequence variants of SNORD114, five variants of SNORD113 and SNORD112 were expressed in APL patients (Figure 2b). Of note, the level of expression of these particular snoRNAs varied significantly within the same cluster.
DLK1-DIO3 snoRNA expression profiles were confirmed by RT-qPCR (Figure 2c). Owing to their high sequence homology, we selected only the snoRNAs for which we could design specific primers: SNORD112 (14q(0)), SNORD113-6 (14q(I-6)), SNORD113-7 (14q(I-7)), SNORD113-8 (14q(I-8)), SNORD113-9 (14q(I-9)) and SNORD114-1 (14q(II-1)). We first tested SNORD113-3 whose expression did not vary when assayed by microarrays. RT-qPCR confirmed the lack of expression indicating that, despite high sequence homology of SNORD113 snoRNAs, the PCR-based quantification was specific.
Several microRNA genes are located around the SNOR112-114 gene cluster in the DLK1-DIO3 locus and seem to have the same expression pattern as their neighboring snoRNAs.21, 22 To validate snoRNA results from microarrays in APL, we examined the expression of the microRNAs from the DLK1-DIO3 locus in APL and PML-RARalpha-negative cells using GeneChip microRNA arrays. When compared with AML samples, we confirmed the significant overexpression of hsa-miR-127, miR-370 and miR-154, as already reported in APL, as well as 11 other microRNAs (Figure 2d).15
PML-RARalpha impacts on DLK1-DIO3 snoRNA expression
To test whether snoRNA expression from the DLK1-DIO3 locus is controlled by PML-RARalpha, we treated leukemic blast cells from three APL patients in vitro with ATRA, which directly targets PML-RARalpha, leading to granulocytic differentiation.23 Blast differentiation was monitored by May-Grünwald-Giemsa staining and CD11b overexpression (Figures 3a and b). RT-qPCR revealed that expression of the SNORD112, SNORD113 and SNORD114 gene cluster in the DLK1-DIO3 locus decreased during differentiation (Figure 3c). Interestingly, levels of the six snoRNAs did not decrease at the same time. After 3 days of treatment, only SNORD113-8 (14q(I-8)) was significantly underexpressed. After 5 days, all snoRNAs were significantly decreased. These findings demonstrate that expression of orphan snoRNAs encoded within the DLK1-DIO3 locus is linked to PML-RARalpha. Moreover, these data corroborate the notion that within the 14q polycistronic snoRNA clusters expression of individual snoRNAs is regulated in a surprisingly independent manner. To further investigate whether the PML-RARα protein chimera influenced snoRNA expression in the DLK1-DIO3 locus, we transfected the human AML M1 (non-APL) cell line KG-1 with a plasmid that expressed PML-RARalpha_bcr1. At both 2 and 3 days after transfection, we verified expression of PML-RARalpha mRNA (Figure 3d) and measured the levels of SNORD112, SNORD113-6, SNORD113-7, SNORD113-8, SNORD113-9 and SNORD114-1 snoRNAs by RT-qPCR. In non-transfected control KG-1 cells, there was a low level of basal expression of all these snoRNAs (<2−12 relative expression compared with 5S rRNA). Transient expression of PML-RARalpha resulted in an upregulation of all the snoRNAs tested at 48 h post-transfection (Figure 3e). Similar data were obtained when we expressed PML-RARalpha in KG1a cells, an AML M0 (non-APL) cell line (data not shown).
The SNORD114-1 (14q(II-1)) variant is implicated in cell growth through cell cycle regulation
To test whether DLK1-DIO3 snoRNAs had a role in APL pathogenesis, we selected the SNORD114-1 (14q(II-1)) variant to analyze its impact on cell growth. This variant was chosen because among the snoRNAs it displayed the highest expression of the locus and because expression of this variant was sustained the longest following treatment with ATRA. The 14q(II-1) variant was cloned and transiently transfected into the K562 cell line, which represents the best model described for studying the DLK1-DIO3 locus (that is, this locus is transcriptionally active in these cells).24 To upregulate 14q(II-1) snoRNA, a vector was constructed that contained the intron hosting the snoRNA sequence and its flanking exons. RT-qPCR quantification of expression of the 14q(II-1) variant revealed a mean fold change of 57 in transfected cells (Figure 4a). We then sought to ensure that snoRNA processing from this construct was effective. RT-PCR was carried out with primers specific to the intronic lariat (the flanking sequences of snoRNA) or primers specific to the mature snoRNA sequence. Thus, we noticed a strong overexpression of the mature snoRNA sequence compared with that of the intron lariat (Figure 4b). Overexpression of the 14q(II-1) variant in the K562 cell line induced a significant increase in cell proliferation as detected with both an MTS cell proliferation assay (Figure 4c) and Malassez counting (Figure 4d). Conversely, snoRNA silencing carried out with 25 nM of siRNA 14q(II-1) in K562 cells caused a 50% reduction in snoRNA expression (Figure 4e) and a significant decrease in cell growth at 24 and 48 h (Figure 4f). We then looked at the cell cycle distribution in siRNA-treated cells and demonstrated that after 48h of treatment 14q(II-1) snoRNA targeting induced a significant increase of cells in G0/G1 phase and, conversely, a significant decrease in S phase (Figure 5a).
The Rb/p16 pathways are involved in snoRNA-mediated cell growth
The effect of 14q(II-1) snoRNA on cell growth and the cell cycle prompted us to focus on what signaling pathways were potentially activated. Indeed, in K562 cells important cell cycle regulators such as p16(CDKN2A) and p53 are known to be inactivated.25, 26 Therefore, we investigated the impact of 14q(II-1) snoRNA silencing on the Rb pathway using western blot analysis and noticed a clear-cut overexpression of the corresponding protein (Figure 5b). This result is in keeping with those described above for cell cycle distribution. To validate these observations, we analyzed the effect of 14q(II-1) overexpression on Rb. As expected, we observed an underexpression of total Rb protein in 14q(II-1) overexpressing cells (Figure 5c). In addition, we noticed that the remaining Rb protein was hyperphosphorylated on serine-780 compared with that under control conditions (Figure 5c), suggesting that cell cycle activity was increased. To test whether this effect on Rb was also observed in APL patients, 14q(II-1) silencing by siRNA was performed using APL blasts from five patients. We confirmed an effective targeting of the 14q(II-1) variant at the RNA level (Figure 6a). Interestingly, a cell growth decrease of 30% was also observed after 48h (Figure 6b). We therefore analyzed the expression of total Rb protein in APL blasts transfected with siRNA-14q(II-1) or an siRNA-negative control. In three of these patients, we confirmed an increase in Rb expression after 14q(II-1) silencing (Figure 6c). In the two remaining patients, Rb expression was almost undetectable whereas p16 expression was detected. It has been previously reported that in cancer cells p16 is often deleted or mutated and Rb/p16 expressions are inversely regulated.27, 28, 29 In APL cells with a basal level of p16, siRNA-14q(II-1) fosters the expression of the protein (Figure 6c). Similar to the K562 cell line, we could not find p53 expression (using western blot analysis with two different antibodies) in APL patients’ cells. Overall, we demonstrated that 14q(II-1) snoRNA modulated cell growth through the Rb pathway in both a K562 cell line and in APL cells ex vivo. Interestingly, in some cases we observed that the p16 pathway could be an alternative regulator of the cell cycle.
SnoRNAs represent an abundant group of non-protein-coding RNAs with their main function being ribosomal RNA maturation.3 Recent new cellular functions have led us to hypothesize that snoRNA accumulation patterns could be modulated in cancer.10, 11, 12, 14, 30, 31, 32, 33, 34, 35 Acute leukemia is a good model to try to answer this question as it is characterized by restrictive oncogenic events leading to a blockade in differentiation.
Using microarrays and a high-throughput qPCR strategy, we showed that neoplastic samples display specific snoRNA accumulation patterns allowing us to differentiate between tumor and control samples. Thus, as already reported for microRNAs,1, 18, 19 global downregulation is a characteristic feature of snoRNAs in cancer cells. Despite a large body of data on microRNAs, there is no clear explanation for this phenomenon. For snoRNAs however, their location within the introns of housekeeping genes implies that downregulation does not result from a transcriptional default but rather from disbranching alterations of the splicing lariat.
Despite global downregulation of snoRNAs in cancer cells, specific signatures were noticed in tumor subtypes that corresponded to ectopic expression of clusters. In particular, a specific signature was noticed in APL cases corresponding to transcriptional activation of restricted chromosomal regions. Thus, DLK1-DIO3 snoRNAs are part of such a chromosomal region containing clustered intronic orphan snoRNAs produced from long nuclear-retained non-coding mRNA-like transcripts such as Meg8.8, 20 The Meg8 transcript has no known functions, and the lack of sequence conservation between species suggests that spliced Meg8 ncRNAs yield only intronic SNORD112, SNORD113 and SNORD114 snoRNAs. We made the interesting observation that only 8 out of 31 sequence variants of SNORD114 and 5 out 9 variants of SNORD113 and SNORD112 were expressed in APL patients. The regulatory mechanism of differential snoRNA expression from the Meg8 region remains unclear, although this region is believed to be transcribed from one promoter (this has not been confirmed experimentally).20 It is possible that the Meg8 region has multiple internal promoters rather than being a single transcription unit. Alternatively, expression of these snoRNAs may be regulated by differential splicing of their host introns, as removal and disbranching of introns are essential for snoRNA production. We sought to determine the impact of PML-RARalpha on DLK1-DIO3 snoRNA expression under ATRA treatment. This corroborated the notion that within these Meg8 polycistronic clusters expression of snoRNAs are either independent from each other or produced in specific subsets. PML-RARalpha seems to be implicated in DLK1-DIO3 snoRNA expression but we could not demonstrate a direct relationship. Regulatory elements that control gene and small ncRNA expression at this locus remain poorly characterized. Nevertheless, consistent with a role for PML-RARalpha in DLK1-DIO3 transcriptional activation, Martens et al.36 recently reported a binding site for PML-RARalpha close to the DLK1 gene using chip sequencing. As AML subtypes display specific methylation profiles, another hypothesis is that a global modification of imprinting by PML-RARalpha leads to a deregulation of the DLK1-DIO3 locus.37 Indeed, apart from DLK1, other elements in this locus display tumor suppressor effects in other models.24, 38, 39, 40 Thus, DLK1-DIO3 snoRNAs may be key players participating in APL leukemic cell proliferation in the context of late differentiation blockade. In line with this, we showed that an individual snoRNA, the SNORD114-1 (14q(II-1)) variant, could impact on cell growth through a negative regulation of the cell cycle and the Rb pathway. Low Rb protein levels have been previously noted in APL cells 41, 42 and the expression of DLK1-DIO3 snoRNAs could negatively regulate the Rb pathway through the control of total Rb protein. In addition, the remaining Rb protein is probably inactivated by direct binding to PML-RARalpha as already shown.43
In conclusion, we report herein a global downregulation of snoRNAs in acute leukemia cells when compared with non-neoplastic counterparts, similar to that reported previously for microRNAs. The hallmark signatures observed for APL support the hypothesis that genes encoding snoRNAs, similar to microRNAs, might be specifically regulated by chimeric transcription factors like PML-RARalpha, in a background of chromosome instability, and thus may have an important role in oncogenesis.
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We are grateful to the patients who participated in this study and their referring physicians. We thank the Genotoul platform, Toulouse, France (Naïs Prade and Jean-José Maoret). We also thank Valerie Lallemand-Breitenbach, José Ángel Martínez Climent and Pauline Gravelle for their helpful comments on this work. This study was supported by grants from the Institut Universitaire de France, the Association pour la Recherche sur le Cancer (ARC) and the CITTIL program (Cooperación de Investigación Transpirenaica en la Terapia Innovadora de la Leucemia).
The authors declare no conflict of interest.
Supplementary Information accompanies the paper on the Leukemia website
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Valleron, W., Laprevotte, E., Gautier, EF. et al. Specific small nucleolar RNA expression profiles in acute leukemia. Leukemia 26, 2052–2060 (2012). https://doi.org/10.1038/leu.2012.111
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