The NB4, HL-60, K562 and U937 cell lines were obtained from the American Type Culture Collection (ATCC, Richmond, MD, USA) and cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (PAA Laboratories Pty Ltd, Morningside, Australia), 2 mM L-glutamine and antibiotics in a humidified incubator containing 5% CO₂ at 37°C. TPA, U0126 and LY294002 were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethylsulfoxide (DMSO) according to the supplier’s instructions. To induce cell differentiation, TPA was added to the medium to a final concentration of 30 nM (13). To inhibit signal transduction, cells were pretreated with specific inhibitors for 30 min prior to TPA treatment (14,15). Antibodies against phospho-ERK1/2, ERK1/2, Dicer, Drosha, Ago1, and Ago2 were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies against phospho-Akt, Akt and β-actin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The horseradish peroxidase (HRP)-conjugated secondary antibodies were also obtained from Santa Cruz Biotechnology, Inc.

Surface antigen expression was measured by flow cytometry according to our previously published protocol (13). Briefly, cells were harvested at the indicated times, washed twice with PBS, and then incubated with 20 μl FITC-labeled anti-CD14 antibody working solution (eBioscience, San Diego, CA, USA) for 30 min at 4°C in the dark. For every sample, separate aliquots of cells were also incubated with isotype antibody controls (eBioscience) to determine non-specific staining. Following incubation, cells were washed twice with PBS and analyzed by flow cytometry with a 488 nm argon laser. For each sample, a total of 10,000 cells were analyzed.

Cell cycle was profiled by flow cytometry as previously described (14). Briefly, cells were harvested and fixed in 70% ethanol at 4°C overnight. After washing with PBS, the fixed cells were re-suspended in PBS containing 100 μg/ml RNase A and incubated for 30 min at 37°C. Finally, the cells were collected by centrifugation and incubated in PBS containing 50 μg/ml of propidium iodide (PI) (Sigma) for 20 min in the dark, and then analyzed using flow cytometry with a 488 nm argon laser. A minimum of 20,000 cells was analyzed for each sample.

For miRNA microarray analysis, total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. CapitalBio Mammalian miRNA Array V3.0 (CapitalBio, Beijing, China) microarrays were then probed, which include 924 probes for human, mouse and rat miRNAs. The microarray analyses were performed according to our previously published protocols (13,16). Significance Analysis of Microarrays (SAM; Stanford University, CA, USA) software was used to determine the differentially expressed miRNAs, and the selection criteria included a fold change ≥1.5 or ≤0.65, a q-value ≤5%, and a SAM score >2 or <−2 (17). The entire dataset described here is available from the Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo) through accession number GSE33537.

To quantify the mature miRNAs, stem-loop RT-PCR assays were performed according to the method of Chen et al (18); snRNA U6 was used as an internal standard. To quantify the pri-miRNAs, total RNA was subjected to DNase I treatment and reverse-transcribed into cDNA with M-MLV (Promega, Wisconsin, WI, USA) (19). Following reverse transcription, PCR reactions were performed using a SuperGreen quantitative PCR kit II (CapitalBio) according to the manufacturer’s instructions. ACTB was used as an internal standard. Relative expression levels were calculated using the formula: Q.rel. = 2^−ΔCT, where ΔCT = average CT test gene - average CT internal standard, and CT is the cycle threshold. All primers used for quantitative RT-PCR are listed in Table I.

The cells were quickly lysed on ice using lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 1 mM Na₃VO₄, and protease inhibitors) and clarified by centrifugation at 14,000 rpm for 10 min; the supernatants represented the cellular protein extracts. Equal quantities of protein extracts were resolved by SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked in TBST (10 mM Tris pH 7.6, 150 mM NaCl, and 1% Tween-20) containing 5% low-fat milk for 1 h. Subsequently, the membranes were incubated with primary antibody dilutions at 4°C overnight, followed by three washes with TBST at room temperature. The membranes were then incubated with secondary antibody dilutions for 1 h at room temperature, followed by four washes with TBST. The enhanced chemiluminescence (ECL) system (Pierce, Rockford, IL, USA) was used to detect reactive proteins.

Experiments were performed in duplicate or triplicate and independently repeated at least three times. Data are presented as the means ± standard deviation (SD). Statistical significance was determined using two-tailed t-tests, and P<0.05 was considered to indicate statistically significant differences.

NB4 (acute promyelocytic leukemia), HL-60 (acute myeloblastic leukemia), U937 (monoblastic leukemia), and K562 (chronic myelogenous leukemia) cell lines are the typical models for studies of human leukemia cell differentiation in vitro. The differentiation of these cells induced by TPA was determined by assessing cell morphology and the expression of a differentiation marker. After TPA treatment, the cells spread and adhered to the culture dishes, and some cells displayed pseudopod-like protrusions (Fig. 1A). A significant increase in CD14 expression was also observed in leukemia cells treated with TPA (Fig. 1B). These results suggest that cellular differentiation was induced.

Using microarrays, the global changes in miRNA expression were analyzed after treatment of the myeloid leukemia cell lines with TPA. The microarray chips contained 924 probes, allowing a survey of 802 mature human, mouse and rat miRNAs after discarding redundant sequences, and a further 122 predicted miRNAs from published data. To reduce individual variability, replicate array analysis used independently treated samples, and a technical replicate was also performed for each sample.

Employing hierarchical cluster analysis, the global expression patterns of miRNAs in the four leukemia cell lines were obtained (Fig. 1C). Through SAM statistics (17), significantly regulated miRNAs in differentiated cells were identified. TPA induction resulted in the upregulation of 21 miRNAs in the NB4 cell line, 17 miRNAs in the HL-60 cell line, 15 miRNAs in the K562 cell line, and 35 miRNAs in the U937 cell line (Table II). To identify specific differentiation-induced miRNAs, the overlaps of upregulated miRNAs were evaluated between these four myeloid leukemia lines (20). The cell lines each exhibited characteristic miRNA profiling due to their different origins. However, 10 unique miRNAs were consistently induced in all four leukemia cell lines after exposure to TPA (Fig. 1D). We support that the differentially regulated miRNAs may represent individual characteristics of each cell line, while the commonly regulated miRNAs may be ‘key players’ in the differentiation of leukemia cells.

To confirm the accuracy of our microarray analysis, stem-loop RT-PCR assays were performed on the identified differentiation-specific miRNAs (except predicted-miR191) using independent samples. The results of qRT-PCR were consistent with those of microarray analysis in all four cell lines (Fig. 2). These nine miRNAs were confirmed to be differentiation-specific in leukemia cells induced by TPA, of which a subset (miR-21, miR-22, miR-146b, miR-23a and miR-24) was selected for further investigation largely based on the magnitude of the detected induction.

To ascertain how the expression of these differentiation-specific miRNAs was induced, cell signaling analysis was performed. Both the MEK/ERK and PI3K/Akt pathways are associated with the induction of differentiation by TPA in leukemia cells (11). To investigate the effects of these pathways on miRNA induction, MEK/ERK and PI3K/Akt signal transduction was blocked using pharmacological inhibitors: U0126 (MEK1/2 inhibitor) and LY294002 (PI3K inhibitor). U0126 blocked the TPA-stimulated phosphorylation of ERK1/2 (Fig. 3A), and inhibited the induction of miR-21, miR-22, miR-146b, miR-23a and miR-24 (Fig. 3B). The changes in the expression of the differentiation marker, growth arrest, and cell morphology were also inhibited by pretreatment with U0126 (Figs. 3C and 4). By contrast, LY294002 failed to suppress the TPA-induced miRNA expression and cellular differentiation (Figs. 3B, C and 4). The reduction of Akt phosphorylation proved the inhibitory effect of LY294002 on PI3K/Akt signaling (Fig. 3A). Thus, MEK/ERK activation contributed to the induction of these differentiation-specific miRNAs.

To investigate the mechanism by which MEK/ERK activation caused the upregulation of differentiation-specific miRNAs, the expression of both miRNA biogenesis machineries and primary transcripts was examined. Using immunoblotting analysis, four proteins that are the key machineries of miRNA biogenesis pathways (Drosha, Dicer, Ago1 and Ago2) were investigated. Drosha and Dicer are involved in successively cleaving primary transcripts into mature miRNAs; Ago1 and Ago2 are the major components of RISC (5,6). The expression of Dicer, Ago1 and Ago2 was not significantly altered in differentiated cells. Only Drosha was downregulated in cells after TPA treatment, and U0126 inhibited the downregulation of Drosha caused by TPA (Fig. 5A). These results suggest that the expression levels of the key miRNA biogenesis machineries in differentiated cells were not responsible for the increased expression of the aforementioned miRNAs induced by TPA.

Next, we assessed if the MEK/ERK activation induced the upregulation of the differentiation-specific miRNAs at the transcriptional level. qRT-PCR was performed to examine the primary transcripts of miR-21, miR-22, miR-146b, miR-23a and miR-24. Since there was a coordinated effect of processing at the miR-23a and miR-24 loci (21), a pair of specific primers was designed to quantify the expression levels of the pri-23a-24-2 transcript loci. The basal levels of these miRNA primary transcripts were increased after TPA induction, and the induction of these transcripts was significantly inhibited by U0126 but not by LY294002 (Fig. 5B). Therefore, the transcription of these differentiation-specific miRNAs was induced by MEK/ERK activation, suggesting a major mechanism for the upregulation of these miRNAs in the differentiation of leukemia cells induced by TPA.