MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia

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Abstract

MicroRNAs (miRNAs) are small non-coding RNAs of 19–25 nucleotides that are involved in the regulation of critical cell processes such as apoptosis, cell proliferation and differentiation. However, little is known about the role of miRNAs in granulopoiesis. Here, we report the expression of miRNAs in acute promyelocytic leukemia patients and cell lines during all-trans-retinoic acid (ATRA) treatment by using a miRNA microarrays platform and quantitative real time–polymerase chain reaction (qRT–PCR). We found upregulation of miR-15a, miR-15b, miR-16-1, let-7a-3, let-7c, let-7d, miR-223, miR-342 and miR-107, whereas miR-181b was downregulated. Among the upregulated miRNAs, miR-107 is predicted to target NFI-A, a gene that has been involved in a regulatory loop involving miR-223 and C/EBPa during granulocytic differentiation. Indeed, we have confirmed that miR-107 targets NF1-A. To get insights about ATRA regulation of miRNAs, we searched for ATRA-modulated transcription factors binding sites in the upstream genomic region of the let-7a-3/let-7b cluster and identified several putative nuclear factor-kappa B (NF-κB) consensus elements. The use of reporter gene assays, chromatin immunoprecipitation and site-directed mutagenesis revealed that one proximal NFB binding site is essential for the transactivation of the let-7a-3/let-7b cluster. Finally, we show that ATRA downregulation of RAS and Bcl2 correlate with the activation of known miRNA regulators of those proteins, let-7a and miR-15a/miR-16-1, respectively.

Introduction

Acute promyelocytic leukemia (APL) is a subtype of acute myelogenous leukemia characterized by maturation arrest at the promyelocytic stage of development and caused by a novel fusion protein resulting from the reciprocal translocation involving the retinoic acid receptor alpha (RARa) on chromosome 17 with the promyelocytic leukemia gene (PML) on chromosome 15 (Melnick and Licht, 1999). This oncogenic protein (PML-RARa) interferes with the process of myeloid differentiation by transcriptional repression of retinoic acid (RA) responsive genes (Melnick and Licht, 1999). In the absence or at physiological levels of RA, PML-RAR fusion protein recruits a corepressor complex that binds to target promoters and represses transcription. Pharmacological doses of all-trans-retinoic acid (ATRA) reverse the dominant-negative effect of PML-RARa fusion protein and induce gene transcription via RA responsive elements that are present in the promoters of RA-induced genes (Melnick and Licht, 1999).

MicroRNAs (miRNAs) are small, non-protein-coding RNAs of 19–25 nucleotides that regulate gene expression by targeting messenger RNAs (mRNA) in a sequence-specific manner inducing translational repression or mRNA degradation (Bartel, 2004). MiRNAs play important roles in the development, differentiation, metabolism, cell growth and death in animals and plants (Poy et al., 2004; Cheng et al., 2005; Dresios et al., 2005). There is also evidence that supports a role for miRNAs in hematopoiesis. In mice, miR-181a is detected in early progenitor cells and dynamically upregulated during B-cell commitment (Chen et al., 2004). Furthermore, the ectopic expression of miR-181a in murine hematopoietic progenitor cells led to a proliferation in the B-cell compartment (Chen et al., 2004). More recently, it has been reported that miR-223 is a key member of a regulatory circuit involving C/EBPa and NFI-A that controls granulocytic differentiation in ATRA-treated APL cell lines (Fazi et al., 2005). Little is known, however, about other miRNAs expression in ATRA-treated APL cells.

In the present study, we report the miRNA gene expression profile of an APL cell line (NB4) upon granulocytic differentiation with ATRA. The results were also confirmed in HL-60 cells, another mycloid leukemia cell line that undergoes granulocytic differentiation upon ATRA treatment and in primary APL patient samples treated in vitro with ATRA.

Results

MiRNAs gene expression in APL cell lines and primary patient blasts treated with ATRA

The treatment of NB4 cells with 100 nM of ATRA for 96 h induced granulocytic differentiation as evidenced by morphological changes and by increased expression of the surface antigen CD11b and CD15 (Figure 1). We initially compared the miRNA expression profiles of ATRA-treated cells with those of untreated cells at the same time point using t-test pair analysis within the significant analysis of microarrays (SAM) (Tusher et al., 2001). Using this method, we identified eight miRNAs upregulated and one miRNAs downregulated (Table 1).

Figure 1
figure1

Morphological and flow cytometry analysis of differentiated NB4 cells upon ATRA treatment. (a) Morphology of differentiated NB4 cells. May-Grunwald Giemsa stain of cytospin preparations of NB4 cells after ATRA treatment for 96 h. (b) Flow cytometry analysis of differentiated NB4 cells. As shown in Figure 1b, a substantial increase in the staining for anti-human CD11b and anti-human CD15 antibodies were observed in ATRA-treated NB4 cells at 96 h.

Table 1 MicroRNAs differentially expressed during ATRA treatment of NB4 cells

We then compared miRNA expression in ATRA-treated vs untreated cells by using quantitative analysis in SAM, which computes the linear regression coefficient of each gene on the outcome. Untreated cells were set to 0 and ATRA-treated cells to the number of days of treatment (i.e., 1, 2, 3 and 4). We thus selected the miRNAs, which would show continuous positive or negative trends upon ATRA treatments. We identified two additional miRNAs that were upregulated, miR-147 and miR-107 (Table 1). Northern blots and miRNA quantitative real time–polymerase chain reaction (qRT–PCR) confirmed the array data (Figure 2). In addition, we found similar miR-223 and let-7a expression in HL-60 cells treated with ATRA (Figure 2e).

Figure 2
figure2

Microarray data confirmation. (a) Northern blots were performed as described previously (Liu et al., 2004) using probes with perfect complementarities to the mature sequence of miR-223 in NB4 cells. Loading control was performed with U6. Immediately below the Northern blotting there is a corresponding normalized miRNA signal intensity (2logged) for each time point obtained by the miRNA microarrays. QRT–PCR of miR-15a (b), let-7a (c) and miR-107 (d) during granulocytic differentiation of NB4 cells induced by ATRA. The miRNA expression is shown in untreated (U) and ATRA-treated cells (A) during the time course after 2(−Δ-Ct) calculations with 18S normalization. Below each bar there is the normalized miRNA signal intensity (2 logged) for each sample obtained by microarrays. (e) Representative Northern blots for miR-223 and let-7a in HL-60 cells untreated or treated with ATRA for 48 h. The treatment of HL-60 was performed as described previously in the text for NB4 cells. Loading control was performed with U6.

We then asked whether the results from cell lines could be reproduced in primary APL patient blasts cultured with or without ATRA in vitro. As shown in Figure 3, we found similar miRNA expression in primary blast cells from three APL patients upon ATRA treatment, except for miR-181b (data not shown).

Figure 3
figure3

MiRNAs expression in APL patient blasts by qRT–PCR. Total RNA was obtained from three APL patient blasts treated with ATRA or DMSO (control) for 48 h. qRT–PCR of let-7d, let-7a-3, miR-223, miR107, miR-15a and miR-16-1 was performed as described. Relative expression was calculated using the comparative Ct method. The results are presented as fold change in the miRNA expression between ATRA-treated cells with respect to the control group (DMSO).

MiR-107 modulates NFI-A

A recent study showed that two transcription factors, NFI-A and C/EBPa compete for binding to the promoter of miR-223. Although NFI-A maintains miR-223 at low levels, its replacement by C/EBPa following RA differentiation, upregulates miR-223 (Fazi et al., 2005). Consistent with these data, we confirmed that miR-223 is induced significantly upon treatment with ATRA. Interestingly, we found that miR-107, which is predicted to target NFI-A, (Figure 4a) was upregulated as well after ATRA treatment (Figure 2d). To confirm a direct interaction between these two genes, we cloned the NFI-A 3′ untranslated region (UTRs) predicted to interact with miR-107 into a pGL3 luciferase reporter vector. When the miR-107 precursor oligonucleotide was transfected into the NB4 cells along with the reporter plasmid containing the NFI-A 3′UTR sequence by nucleoporation, a 40% reduction of normalized luciferase activity was observed compared with the control scrambled oligonucleotide (Figure 4b).We also performed a control experiment using a mutated NFI-A construct lacking four bases in the miR-107 seed match. As expected the deletion completely abolished the interaction between miR-107 and NFI-A (Figure 4b).

Figure 4
figure4

Hsa-miR-107 targets NFI-A. (a) The interaction between miR-107 (green) with the 3′UTR of NFI-A (red) is shown as predicted by PICTAR (Krek et al., 2005). (b) miR-107 interact with NFI-A. The NFI-A 3′-UTR segments containing the target site for miR-107 was cloned into the pGL3 control vector. The results are shown as a relative change of the miR-107+pGL3–NFI-A and mutated pGL3-NFI-A luciferase/renilla activity ratios with respect to that of the negative control (scrambled RNA) transfected cells.

Regulation of the cluster miR-15a/miR-16-1 and Bcl-2 by ATRA

We then asked whether there is a negative correlation between miR-15a and miR-16-1 expression and Bcl-2 during ATRA-induced granulocytic differentiation of NB4 cells. The expression of Bcl-2 mRNA and protein were analysed during ATRA treatment and correlated with qRT–PCR expression of miR-15a and miR-16-1, which are known regulators of this protein (Cimmino et al., 2005). As shown in Figure 5a and b, both mRNA and protein levels were downregulated upon ATRA treatment. In sharp contrast, the expressions of miR-15a and miR-16-1 were increased (Table 1). It has been reported that ATRA induced downmodulation of Bcl-2 mRNA and protein expression in several myeloid cell lines, including APL cell lines (Bruel et al., 1995). However, there is no clear evidence about the mechanism of this downregulation. To confirm the in vivo interaction between the miR-15a/miR-16-1 cluster and Bcl-2 in NB4 cells, we transfected the precursor miR-16-1 (pre-miR-16-1) oligonucleotide or scrambled oligonucleotides into NB4 cells by using nucleoporation and obtained total RNA at 24 h and proteins from whole cell lysates at 48 h. As shown in Figure 5c, there was a significant reduction of the Bcl-2 transcripts by qRT–PCR after transfection with pre-miR-16-1. Likewise, transfection of the pre-miR-16-1, but not the control, resulted in a strong Bcl-2 protein reduction by Western blots at 48 h (Figure 5e).

Figure 5
figure5

RAS and Bcl-2 downmodulation by ATRA. (a) Quantitative expression of Bcl-2 and RAS mRNA levels by TaqMan real-time PCR in untreated and ATRA-treated NB4 cells after 2(−Δ-Ct) calculations normalized with 18S. (b) Representative Western blot analysis of Bcl-2 and Ras proteins during ATRA treatment of NB4 cells. Protein loading control was done with GAPDH (Santa Cruz). (c) Quantitative expression of Bcl-2 mRNA levels in NB4 cells 24 h after transfection with pre-miR-16-1 oligonucleotides (Ambion) or scrambled oligonucleotides (negative control-1). (d) Quantitative expression of RAS mRNA levels in NB4 cells 24 h after transfection with pre-let-7a oligonucleotides (Ambion) or scrambled oligonucleotides (negative control-1). (e) Western blot analysis of Bcl-2 and RAS in whole cell extracts obtained at 48 h from NB4 cells transfected with pre-miR-16-1 or pre-let-7a-3 oligonucleotides. Loading control was performed with β-actin and GAPDH (Santa Cruz).

ATRA modulation of RAS is inversed to let-7 expression

RAS proteins family has been established as the key regulators of diverse cellular processes (Kolch, 2005). Activating mutations have been described in AML (Bowen et al., 2005) and, recently, it has been demonstrated that K-ras cooperates with PML-RARa to induce APL like disease in a mouse model with high penetrance and short latency (Chan et al., 2006). The let-7 miRNA family have been shown to regulate the RAS oncogenes through post-transcriptional repression (Johnson et al., 2005). Our above data indicated that several let-7 members are upregulated by ATRA. Thus, we investigate the expression of RAS mRNA and protein in ATRA-treated NB4 cells. As shown in Figure 5a and b, RAS transcript levels by qRT–PCR were rather increased, whereas there was a decrease in the RAS protein levels at 48 h, indicating a post-transcriptional regulation mechanism, which is typical to that of miRNAs. Moreover, the levels of RAS protein were inversed of that of let-7 family members, suggesting that let-7 could mediate ATRA downmodulation of RAS in NB4 cells (Table 1). Furthermore, we transfected the precursor let-7a-3 or let-7d or scrambled oligonucleotides in NB4 cells by using nucleoporation. The ectopic expression of let-7a-3 or let-7d in NB4 cells resulted in no change in the RAS mRNA levels, but a decrease of the RAS protein at 48 h, compared with the control transfected cells (Figure 5d and e). These results suggest that the modulation of RAS by ATRA could be mediated by let-7 family members.

ATRA treatment recruits nuclear factor-kappa B on the let-7a-3/let-7b cluster promoter

Nuclear factor-kappa B (NFB) proteins are the key regulators of various biological processes, including cell proliferation and differentiation in many systems (Mathieu et al., 2005). Previous studies have shown the activation of NFB upon ATRA-induced maturation of APL (Mathieu et al., 2005) and enrichment for NFB binding sites in RA target genes has been described (Meani et al., 2005). To investigate whether ATRA-induced NFB activation regulates miRNAs, we co-transfected NB4 cells with a potent NFB inhibitor expression plasmid (IkBa-SR) or empty vector and treated the cells with ATRA or dimethylsulphoxide (DMSO) (control). We then measured the mature miRNA expression of all the differentially expressed miRNAs identified by the microarrays analysis. As shown in Figure 6a, the ATRA-induced upregulation of let-7a was blocked by the inhibition of the NFB pathway, suggesting that this miRNA is regulated by NFB. Because let-7a-3 is located at chromosome 22q13 in cluster with let-7b, we measured the mature let-7b transcripts levels by qRT–PCR in NB4 cells after treatment with ATRA and the IkBa-SR and similar results were found (Figure 6a). However, the inhibition of NFB pathway did not block the ATRA-induced upregulation of other miRNAs (data not shown). Successful NFB pathway inhibition was confirmed by decreased NFB reporter luciferase activity (Figure 6b).

Figure 6
figure6

ATRA treatment recruits NFB on the let-7a3/let 7b cluster promoter. (a) Representative qRT–PCR of let-7a, let-7b and miR-15a from three different experiments was performed with total RNA obtained from NB4 cells treated with DMSO (control), ATRA plus empty vector (control 2) and ATRA plus IkBa-SR. The results are shown as relative fold change in the miRNA expression with respect to the control (untreated) NB4 cells miRNA levels, after normalization with 18 s and 2(−Δ-Ct) calculations as described previously (Chen et al., 2004). (b) Confirmation of successful NFB inhibition. To confirm successful inhibition of the NFB pathway by IkBa-SR, we co-transfected 5 × 106 NB4 cells with 5 μg of NFB activity reporter, 0.5 μg of renilla reporter pRL-TK (Promega) and 5 μg of IkBa-SR or empty vector by using nucleoporation. The cells were then treated with ATRA for 24 h. Renilla and luciferase activities were measured consecutively using the dual luciferase assays (Promega). The results are shown as relative change of the dominant-negative IkBa-SR luciferase/Renilla ratios with respect to the empty vector-treated cells (calibrator). (c) NFB consensus element in the promoter of let-7a-3 and let-7b. Putative NFB site in the let-7a-3 and let-7b promoter identified by using a positional weight matrix and MATCH (Kel et al., 2003) program. In this figure, the genomic region that was amplified by the primers (oligos 1) to perform the ChiP experiments is shown. (d) NFB (P50) binds to the promoter of let-7a-3/let-7b. In this ChiP experiment, performed as described in detail in Materials and methods, there is a clear binding of NFB (P50) to the predicted NFB-binding site.

Having shown that the mature forms of let-7a and let-7b are transcriptionally regulated by NFB, we then searched for NFB binding sites, 5000 bp upstream from the precursor sequence of let-7a-3/let-7b cluster by using a positional weight matrix for NFB and MATCH (Kel et al., 2003) program. We identified 31 putative NFB-binding sites (data not shown). To investigate whether NFB is binding to any of these predicted sites, we performed chromatin immunoprecipitation (ChiP) experiments in six of the 31 predicted sites. As shown in Figure 6c and d, we found that the NFB (p50) subunit is associated strongly with one of the predicted sites at −833 nucleotides from the 5′ precursor let-7a-3 upon ATRA treatment. The binding of NFB (p65) subunit was less convincing for this site (data not shown). Further ChiPs experiments using c-Rel and Rel-B antibodies were performed using the oligonucleotides corresponding to this site. However, no binding was detected. Likewise, there was no binding for the p50 or p65 subunits in the other five predicted sites that were tested (data not shown).

NFB activates the transcription of Let-7a-3 and let-7b

To confirm that the p50 binding to the NFB consensus site in the let-7a-3/let 7b promoter is associated with transcriptional activation of this cluster, we cloned the genomic region of the let-7a-3/let-7b promoter containing this site upstream to the luciferase gene in the pGL3 enhancer vector. Next, we co-transfected this reporter with a NFB (p50) mammalian expression vector (pCMV2) or empty vector into K562 cells by nucleoporation. As shown in Figure 7a, the luciferase/renilla ratios were increased sixfold compared with the empty vector. The increased luciferase activity was significantly reduced, when a mutated pGL3-let-7a-3/let-7b reporter vector (lacking three bases in the consensus NFB binding site) was used. Consistent with the ChiP results for this site, co-transfection of the NFB (p65) and the PGL3-let-7a-3/let-7b promoter reporter in K562 cells did not reveal increased luciferase activity. We performed further confirmatory experiments in NB4 cells co-transfected transiently with the PGL3-let-7a-3/let-7b reporter and the IκBα-SR construct (potent and specific inhibitor of NFB) or empty vector. As expected, the increased activity of luciferase induced by ATRA was blocked, when the NF-κB pathway was inhibited by the IκBα-SR, which is a potent and specific NFB inhibitor (Figure 7b). To discover a potential role for NFB (p65) regulation of the let-7a/let-7b cluster, we transfect NB4 cells with antisense oligonucleotides to the NF-κB (p50) and (p65) subunits, each alone and both combined, and treated the cells with ATRA for 48 h. As controls, we used scrambled oligonucleotides. We obtained total RNA at 48 h and performed qRT–PCR for miR-let-7a. As shown in Figure 7c, the inhibition of NF-κB (p65) or/and (p50), resulted in decrease levels of let-7a compared with the controls, suggesting that additional NFB binding sites for p65 may exist.

Figure 7
figure7

NFB (p50) is able to activate the promoter of let-7a-3/let-7b. (a) K562 cells (5 × 106) were co-transfected in 10 ml plates using nucleoporation (AMAXA) with 5 μg of the let-7a-3/let-7b reporter (WT let-7 in the Figure), 0.5 μg of renilla pRL-TK (Promega) and 5 μg of pCMV2-p50 or pCMV2-p65 vectors or empty vector (EV). Firefly and Renilla luciferase activities were measured consecutively using the dual luciferase assays (Promega) 24 h after transfection. The results are expressed as relative luciferase/renilla ratios between p50 and p65 expression plasmids transfected cells with respect to the empty vector (control). We performed an additional control experiment using a mutated NFB let-7a-3/let-7b reporter. K562 cells were nucleoporated as previously described with 5 μg of the mutated let-7a-3/let-7b reporter and 5 μg of pCMV2-p50 or empty vector (EV). As expected the mutation abolished the interaction with NFB (p50). (b) Let-7a-3/let-7b promoter characterization NB4 (5 × 106) cells were transfected with 5 μg of the pGL-3 let-7a-3/let-7b promoter reporter plus 0.5 μg of Renilla pRL-TK by using nucleoporation as described. Half of the cells were treated with ATRA or left untreated (DMSO). In addition, we co-transfected NB4 cells with 5 μg of IκBα-SR or EV plus 5 μg of pGL3-let-7a-3/le-t7b reporter and 0.5 μg of Renilla pRL-TK and treated the cells with ATRA for 24 h. Renilla luciferase activities were measured consecutively using the dual luciferase assays (Promega) 24 h after transfection. The data are presented as relative change of the luciferase/Renilla activity ratio between ATRA-treated cells transfected with pGl-3 let-7a-3/let-7b reporter (pGL3 let-7 ATRA) alone or IκBa-SR or empty vector with respect to pGL-3 let-7a-3/let-7b untreated (DMSO) cells (c). Interfering with p50 or p65 activity block the upregulation of let-7a induced by ATRA. QRT–PCR of let-7a in NB4 cells transfected with NFB (p50) or/and NFB (p65) small interfering RNAs (Santa Cruz) and non-specific Control Pool (negative control) and subsequently treated with ATRA or DMSO. The results are reported as fold change in the miRNA expression from ATRA-treated samples with respect to that of untreated cells.

Discussion

In the present study, we characterized the expression of miRNAs during ATRA-induced granulocytic differentiation of APL patients and cell lines. Using a miRNA microarray platform, we identified a small number of miRNAs upregulated upon ATRA treatment of APL cell lines. More importantly, these results were consistent with the profiling of APL patients blasts after ATRA treatment by using a different platform (qRT–PCR). Among the upregulated miRNAs, let-7a-3 is located at 22q13, about 860 bp upstream from let-7b. Although these two miRNAs are in a cluster and are likely transcribed together, let-7b was not identified by the microarray as differentially expressed after ATRA treatment. One possible explanation is that cross-hybridization with let-7c has occurred owing to the similarities between these two mature miRNAs (they differ by only one nucleotide). Nevertheless, upregulation of let-7b after ATRA treatment of NB4 cells was confirmed by qRT–PCR.

The present data indicate that many miRNAs with strong experimental data supporting a role for tumor suppressors such as miR-15a, miR-16-1 and several let-7 family members are upregulated by ATRA, whereas the expression of their targets (Bcl-2 and RAS) are downregulated. The ectopic expression of miR-15a and miR-16-1 in NB4 cells induced downregulation of both Bcl-2 mRNA and protein. Thus, although we previously described that miR-15a and miR-16-1 target Bcl-2 at the translational level in MEG01 leukemic cells (Cimmino et al., 2005), it is possible that these two miRNAs might affect both transcription and translation of Bcl-2 or could act indirectly through the modulation of other genes that modulates BCL-2 expression in the system we discussed here.

We have demonstrated that ATRA negatively modulates RAS by a post-translational mechanism. This type of regulation is typical for miRNAs. Furthermore, the enforced expression of let-7a-3 recapitulates the ATRA effects on RAS mRNA and protein expression.

We have shown that a small number of miRNAs are induced upon ATRA treatment. However, the mechanism by which ATRA regulates miRNA expression is largely uncharacterized. We hypothesize that miRNAs expression could be regulated in part by ATRA-modulated transcription factors like NFB. In fact, we demonstrated that NFB (p50/p50), but not p65/p50 binds to the upstream sequence of the let-7a-3/let7b cluster and activates its transcription. Previous work by other groups indicates that p50/p50 homodimers are generally repressors of transcription (Mori et al., 1999; Udalova et al., 2000). However, there are several reports that support the ability of p50/p50 homodimers to activate transcription (Dechend et al., 1999; Kurland et al., 2001; Priel et al., 2005). On the basis of our experiments with antisense oligonucleotides against p65 and p50, it is possible that p65/p50 heterodimers may also bind to a yet to be identified site of the let-7a-3/let7-cluster and activate its transcription.

In summary, our results demonstrate that ATRA treatment of APL patients and cell lines modulate a small number of miRNAs, most of which have confirmed targets involved in hematopoietic differentiation and apoptosis. Interestingly, we have confirmed that along with miR-223, miR-107 negatively regulates NFI-A. In addition, we demonstrated that ATRA induced NFB binds to the promoter of let-7a-3 and activates its transcription. Finally, we showed that ATRA downregulation of RAS and Bcl-2 correlated with the activation of two confirmed miRNA regulators of these genes, let-7a- and miR-15a/miR-16-1, respectively.

Materials and methods

Patients, cell lines and ATRA treatment

The human promyelocytic cell line NB4, was a gift of Guido Marcucci. The NB4 cells were maintained in Rosewell's Park Memorial Institute media (RPMI) with 20% fetal bovine serum (FBS) and antibiotics. The cell line HL-60 was obtained from American Type Culture Collection (Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium with 20% FBS and glutamine. NB4 and HL-60 cells were treated with 100 nM of ATRA (SIGMA, St Louis, MO, USA) for 4 days. Bone marrow (2) and peripheral blood (1) leukemic blood samples were collected after informed consent from three patients with newly diagnosed APL, prepared by Ficoll-Hypaque (Nygaard) gradient centrifugation and cryopreserved. After thawing and counting the cells at 37°C, 5 × 105 cells were cultured in AVIM media without serum (Invitrogen, Carlsbad, CA, USA) with ATRA 100 nM or without ATRA. Total RNA was extracted using Trizol (Invitrogen) only after 48 h of culture because of the reduced number of initial cells and the lack of sustained growth over time.

Granulocytic differentiation and characterization

Cytospin preparations of NB4 cells treated with ATRA or untreated were performed and stained with May–Grunwald Giemsa at different time points during the granulocytic differentiation induction. Flow cytometry analysis of differentiated NB4 cells was performed using peridine chlorophyll protein conjugated mouse antihuman CD11b and phycoerythrin conjugated mouse antihuman CD15 antibodies with their respective isotypes (BD Pharmingen, San Jose, CA, USA). For single staining, 1 million of cells were resuspended in 20 μl of the previously indicated antibodies and were incubated for 60 min at 4° in the dark. After two washes with phosphate-buffered saline (PBS) the cells were re-suspended in 0.5 ml of 3% FBS-PBS and analysed by FACScan (BD) using cellquest software (BD).

RNA extraction, Northern blots and microarray experiments

Total RNA for microarray studies was obtained using Trizol reagent (Invitrogen) from NB4 cells after 24, 48, 72 and 96 h of culture with or without ATRA-containing medium. Labeled targets from 5 μg of total RNA were used for hybridization on each miRNA microarray chip-containing 368 probes in triplicate, corresponding to 245 human and mouse miRNA genes (Liu et al., 2004). Northern blots were performed as described in detail elsewhere (Liu et al., 2004).

Data analysis

Expression data were normalized after background subtraction using global median normalization of the bioconductor package (www.bioconductor.org). In two class comparisons (i.e., untreated vs treated), differentially expressed miRNAs were identified by using the t-test procedure within the SAM (http://www.stat.stanford.edu/~tibs/SAM/index.html) (Tusher et al., 2001). SAM identifies genes with statistically significant changes in expression by assimilating a set of gene-specific scores (i.e., paired t-tests). Each gene is assigned a score on the basis of its change in gene expression relative to the standard deviation of repeated measurements for that gene. Genes with scores greater than a threshold are deemed potentially significant. The percentage of such genes identified by chance is the false discovery rate (FDR) (q-value). To estimate the FDR, nonsense genes are identified by anszing permutations of the measurements. The threshold can be adjusted to identify smaller or larger sets of genes, and FDR are calculated for each set. We also used SAM quantitative analysis, which computes the linear regression coefficient of each gene on the outcome.

Western blots

Total cell extracts (50 μg) from untreated or treated NB4 cells after 48 h of culture were extracted using radioimmunoprecipitation assay buffer (Sigma, St Louis, MO, USA) and fractionated by electrophoresis on 10% sodium dodecyl sulfate–polyacrylamide gel, electroblotted to polyvinylidene difluoride membranes (BIORAD, Hercules, CA, USA) and reacted with anti-BCL-2 (Dako, Carpinteria, CA, USA) or RAS (ABCAM, Cambridge, MA, USA). Protein loading control was done with GAPDH (Santa Cruz, Santa Cruz, CA, USA). Appropriate secondary antibodies were used (Santa Cruz).

ChiP experiments

NB4 cells were seeded at 1 × 106 cells/ml and were grown for 48 h with or without 1 μ M RA. After 48 h of treatment, the cells were cross-linked by 1% formaldehyde for 10 min at room temperature. Cell extracts were prepared and sonicated to generate 200–1000 bp DNA fragments. DNA–chromatin complex was immunoprecipitated with anti-p50, -p65, c-Rel, Rel-B antibody (Upstate, Chicago, IL, USA) and with normal rabbit IgG (Santa Cruz) used as internal control. The DNA–protein crosslinks were reversed by heating at 65°C for 4 h. The recovered DNA was PCR-amplified with the oligo 1 (forward (For) 5′-IndexTermTTT CCC CAG GAA GGT GGT AG-3′; reverse (Rev) 5′-IndexTermGTC CAG TGC CTG TCC CTG CG-3′) corresponding at NF-κB consensus binding site at −833 and with the negative control oligo corresponding at KIAA66 (Aviva system negative control primers). Control amplification was carried out on input chromatin before immunoprecipitation (column input) and on mock-immunoprecipitated chromatin (column No-Ab).

Luciferase gene reporter assays

The NFI-A 3′UTR segments containing the target site for miR-107 was amplified by PCR from genomic DNA and inserted into the pGL3 control vector (Promega, WI, USA), using the XbaI site immediately downstream from the stop codon of luciferase. We used the following primers; For: 5′ IndexTermTAC TCT AGA CAA TTC CGA AAA TGG CAA AC 3′ and Rev: 5′ IndexTermCAT TCT AGA CCA ATG AAA TTG CAC TTT ACT CA 3′. We also generated an insert with deletion of four bases from the site of perfect complementarity using the Quiagen XL-site directed mutagenesis kit (Stratagene, La Jolla, CA, USA). Wild-type and mutant inserts were confirmed by sequencing. The human NB4 cell line was grown in 10% FBS in RPMI-1640 at 37°C in a humidified atmosphere of 5% CO2. The cells were co-transfected in 10 ml plates using nucleoporation with kit V (Amaxa, Gaithersburg, MD, USA) according to the manufacturer's protocol using 5 μg of the firefly luciferase report vector and 0.5 μg of the control vector containing Renilla luciferase, pRL–TK (Promega). For each plate, the pre-miR-107 oligonucleotides or the scrambled RNA (Ambion, Austin, TX, USA) were co-transfected. Firefly and Renilla luciferase activities were measured consecutively using the dual luciferase assays (Promega) 24 h after transfection. The upstream sequence of the Let-7a-3/let7-b cluster containing the NFB consensus site (-833) was amplified by PCR from genomic DNA and inserted into the pGL3 enhancer vector (Promega, WI, USA), using the XhoI and BglII sites upstream from luciferase sequence. We used the following primers: For 5′IndexTermCTT TCC CAG GAA GGT AG 3′; Rev 5′ IndexTermCGC AGG GAC AGG CAC TGG AC 3′. A mutated construct, lacking three bases and with a mutation A → T in the NFB consensus site, was also generated by using the Quiagen XL-site directed mutagenesis kit (Stratagene). Wild-type and mutant insert were confirmed by sequencing.

Small interfering RNA experiments

NB4 cells were seeded at 5 × 106 and transfected with 5 μg of NFB (p50) or/and NF-κB (p65) small interfering RNAs purchased from Santa Cruz (Cat# SC_29410 and SC_29407, respectively), and non-specific control pool (negative control Cat# SC 37007) by using nucleoporation (Amaxa) kit V. Total RNA were extracted by TRIZOL (Invitrogen) after 48 h of treatment with ATRA. Mature let-7a expression was analysed by qRT–PCR as described previously. (Chen et al., 2004). Untreated cells transfected with negative control oligonucleotides were used as a calibrator.

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Acknowledgements

This work was supported by National Cancer Institute/National Institutes of Health Grants, P01CA76259, P01CA16058 and P01CA81534 (CMC), P01 CA055164 and the Paul and Mary Haas Chair in Genetics (MA), Lauri Strauss Discovery grants awards (RG), Kimmel Foundation grants awards (GAC and RA) and CLL Global Research Foundation Grant (GAC). We thank Guido Marcucci for kindly providing us with NB4 cells and Dennis Guttridge for his generous support with many reagents used to characterize the NFB regulated miRNAs (The Ohio State University).

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Correspondence to C M Croce.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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Keywords

  • microRNAs
  • promyelocytic leukemia
  • NF-κB

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