A total of 46 patients with acute leukemia from Shanghai Jiaotong University Affiliated Shanghai First People’s Hospital (Shanghai, China) were enrolled in this study between May 2011 and November 2012. All of the patients were primary or no-remission patients. Patient exclusion criteria were concomitant or previous cancer and acute promyelocytic leukemia. The characteristics of the patients included in this study are listed in Table I. Bone marrow was collected from patients by bone marrow puncture at diagnosis or at follow-up subsequent to therapy. All patients were followed for at least four months or until recurrence. Event-free survival (EFS) and overall survival (OS) were the end-points used for the analysis of the treatment results. EFS (20) was measured from the date of entry into the present study to the date of induction failure, relapse from complete remission (CR), CR with incomplete hematologic recovery or death from any cause. OS was calculated from the date of entry into the study to the date of death from any cause. Data were censored if patient had no event at the last follow-up. Healthy samples were collected from three healthy adults. Written informed consent was obtained from the patients for the biological studies. This study was approved by the Ethics Committee of Shanghai Jiaotong University Affiliated Shanghai First People’s Hospital.

To evaluate miR-125b expression in bone marrow mononuclear cells, qPCR for miRNA was performed. Mononuclear cells were separated using a Ficoll-Hypaque centrifugation gradient from 2 ml bone marrow samples. Total RNA was isolated with TRIzol^® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. To detect miR-125b expression, 10 ng total RNA was reverse transcribed with miRNA-specific primers using a TaqMan^® MicroRNA Reverse Transcription kit (Applied Biosystems, Foster City, CA, USA). qPCR was then performed using TaqMan^® MicroRNA Assays (Applied Biosystems) according to the manufacturer’s instructions, using an StepOnePlus™ Real-Time PCR System (Applied Biosystems). The 20-μl PCR reaction contained the following: 1.3 μl reverse transcription (RT) product, 10 μl TaqMan^® Universal PCR Master Mix and 1 μl primer and probe mix from the TaqMan miRNA assays. The reactions were incubated in optical plates at 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 10 min. U6 small nuclear RNA was used as a control. miR-125b expression relative to that in a healthy sample was calculated using the 2^−ΔΔCT method (21). All of the qPCR assays were performed in triplicate.

The K562, THP-1, Jurkat and REH cell lines (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; Gibco-BRL, Carlsbad, CA, USA), 100 U/ml penicillin and 100 μg/ml streptomycin. HEK293T cells (The Cell Bank of Type Culture Collection of Chinese Academy of Sciences, Shanghai, China) were grown in Dulbecco’s Modified Eagle Medium (HyClone) containing 10% FBS, 100 g/ml L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

In order to overexpress miR-125b in K562, THP-1 and Jurkat cells, 10 μg expression vector or empty vector with 10 μg packing plasmid (gag/pol and vesicular stomatitis virus G) was incubated with Fugene 6^® (Roche, Mannheim, Germany) for 15 min at room temperature and subsequently added to the HEK293T cells. The viral supernatant was harvested after 48 h, and the cells were treated by a spin infection with retroviral supernatant (1 ml supernatant per 1×10⁶ cells plus polybrene) in six-well plates that were pre-coated with retronectin (Takara Bio, Inc., Shiga, Japan). The percentage of green fluorescent protein-positive cells was 70–90%.

In accordance with a previous study (22), miR-125 ‘sponge’ sequences (8–9 tandem repeats, each complementary to miR-125) were designed. An imperfect base-pairing between the miRNA and the miR-125 ‘sponge’ was designed to impair endonucleolytic cleavage by Argonaute-2. The miR-125 ‘sponge’ sequence was 5′-TCACAGGTTACTCAGGGA-3′ and was cloned into the pRetroQ-mCherry-C1 retroviral vector (Clontech Laboratories, Inc., Palo Alto, CA, USA). The miR-125 ‘sponge’ vector was then infected into REH cells using a retrovirus, in order to generate miR-125b-knockdown REH cells. Subsequent to virus infection, REH cells were screened using puromycin (Sigma-Aldrich, St. Louis, MO, USA) at an final concentration of 2 μg/ml. The percentage of red fluorescent protein-positive cells was ~100%.

G protein-coupled receptor kinase (GRK) 2 and p53-upregulated modulator of apoptosis (PUMA) 3′ untranslated region (3′UTR) luciferase reporters were individually generated by inserting full-length human GRK2 and PUMA 3′UTR into the XhoI and NotI sites in the psiCHECK-2 vector (Promega, Madison, WI, USA) downstream from the Renilla luciferase coding sequence. The GRK2 3′UTR was PCR amplified from human genomic DNA (Roche) using the following primer sequences: Forward, 5′-TCGCTCGAGCCC GCCCACCCGCCTTTTA-3′ and reverse, 5′-TCGGCGGCC GCAATCAGGCACCATTTT-3′. The PUMA 3′UTR was PCR amplified using the following primer sequences: Forward, 5′-TCGCTCGAGGACTT TCTCTGCACCAT-3′ and reverse, 5′-TAAGCGGCC GCGGCAAGCAGAAAGAGT-3′. The sequences and cloning direction of the PCR products were validated by DNA sequencing. The HEK293T cells were plated in 96-well plates at 5,000 cells/well the day prior to transfection. Transfection was performed in triplicate with Fugene 6 (Roche) and 150 ng plasmid mixture (135 ng miR-125b expression vector and 15 ng reporter vector). Luciferase assays for firefly and Renilla luciferase were performed 48 h after transfection with a Dual-Glo^® Luciferase Assay kit (Promega). Luminescence was quantified using a NOVOstar machine (BMG Labtech, Ortenberg, Germany). The Renilla luciferase readings were normalized to the firefly luciferase activity in the corresponding well.

Cells were seeded into 96-well plates in RPMI-1640 medium containing 10% FBS and were treated with serial dilutions of DNR (Pfizer, Groton, CT, USA). Cell viability was determined 48 h after DNR treatment using a luminescent cell viability assay (Promega). Luminescence was quantified using a NOVOstar machine (BMG Labtech).

K562 cells were transfected with GRK2 or PUMA siRNA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) using Fugene 6 (Roche) according to the manufacturer’s instructions. The transfection efficiency was ~80% and was calculated as the percentage of fluorescein-labeled cells using fluorescence microscopy. Forty-eight hours after transfection, the cells were harvested for further analysis.

Cells were washed twice with phosphate-buffered saline and then lysed in lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing PhosSTOP (Roche). Cell lysates (~40 μg protein) were loaded on a 10% sodium dodecyl sulfate-polyacrylamide gel and subsequently transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Membranes were blocked in 5% non-fat dry milk in Tris-buffered saline (TBS) for 2 h at room temperature and then incubated with rabbit polyclonal anti-GRK2, anti-PUMA (Santa Cruz Biotechnology, Inc.), anti-caspase-3 (Epitomics, Burlingame, CA, USA) and anti-cleaved caspase-3 (Cell Signaling Technology, Inc., Danvers, MA, USA) antibodies overnight at 4°C. Following three washes with TBS supplemented with Tween 20 (TBST), the membranes were incubated for 2 h with rabbit anti-human immunoglobulin G antibody (Cell Signaling Technology, Inc.) conjugated to horseradish peroxidase. Subsequent to washing with TBST, the membranes were exposed to enhanced chemiluminescent reagent (Millipore) for 1 min and then to Kodak X-ray film (Eastman Kodak, Rochester, NY, USA). Anti-β-actin antibody (Cell Signaling Technology, Inc.) was used to detect β-actin.

Data are presented as the mean ± standard error. EFS was calculated using Kaplan-Meier analysis. The Student’s t-test was performed for comparisons between two groups. Statistical analysis was conducted using SPSS 13.0 software (SPSS, Inc., Chicago, IL, USA). A value of P<0.05 was considered to indicate a statistically significant difference.

To determine the differential expression pattern of miR-125b, qPCR was performed in bone marrow samples from patients with acute leukemia (n=46), including those with AML and ALL. miR-125b expression was observed to be markedly higher in patients with acute leukemia compared with that in the healthy controls, demonstrating an average 4,375-fold increase (data not shown). Patients were divided into two risk groups, low and high, using median miR-125b expression. The median miR-125b expression value in the patients with acute leukemia was 85.24-fold higher than that in the samples from the healthy controls. Patients with an miR-125b expression value below the median were assigned to the low group and those with an expression value above the median were assigned to the high group. Among the 20 patients in the low group, two relapsed, two succumbed during therapy and nine did not achieve remission. A total of 13 patients had an event, and the remaining 35% showed an EFS. The average EFS was 6.2 months [95% confidence interval (CI), 3.5–8.9]. Among the 26 patients in the high group, eight relapsed, three succumbed during therapy, one succumbed during bone marrow transplantation subsequent to achieving complete remission and 12 did not achieve remission. A total of 25 of the patients had an event, and the remaining 3.8% showed an EFS. The average EFS was 3.3 months (95% CI, 1.8–4.8). A significant difference was observed in the EFS between patients in the high group and those in the low group (P<0.05). However, the OS was not observed to be significantly different between the two groups (P=0.336) (Fig. 1).

To investigate the association between miR-125b and DNR chemoresistance in leukemia cells, miR-125b was overexpressed in K562, THP-1 and Jurkat cells and knocked down in REH cells. K562, THP-1 and Jurkat cells were stably transduced with a murine stem cell virus (MSCV)-miR-125b vector to overexpress miR-125b in the cells (23). qPCR was used to assess the expression of miR-125b. In K562 cells, miR-125b expression was observed to be 3.2-fold higher in the cells transduced with MSCV-miR-125b compared with the cells transduced with an empty vector, and 4.9- and 4-fold higher in the THP-1 and Jurkat cells, respectively. The K562, THP-1 and Jurkat cells overexpressing miR-125b were treated with various doses of DNR. Overexpression of miR-125b was associated with a significant increase in the survival of K562, THP-1 and Jurkat cells (Fig. 2A–C). At DNR concentrations ≥0.1 μM, the survival of K562 and Jurkat cells was significantly higher in the miR-125b overexpression group than that in the control group (P<0.05). At DNR concentrations ≥0.01 μM, the survival of THP-1 cells was significantly higher in the miR-125b overexpression group than that in the control group (P<0.05).

The effects of miR-125b-knockdown on REH cells were investigated using transduction with an miR-125b ‘sponge’ vector. The miR-125b ‘sponge’ vector effectively reduced the expression of miR-125b in REH cells, which was verified using qPCR. The expression of miR-125b in the miR-125b-knockdown REH cells was reduced by 75%, compared with that in the control REH cells. The expression of miR-125b was significantly lower in the knockdown cells than that in the control cells (P<0.001) (Fig. 3A). Furthermore, knockdown of miR-125b was associated with a significantly decreased survival rate in the REH cells (Fig. 3B). At DNR concentrations ≥0.01 μM, miR-125b-knockdown REH cells exhibited a significantly lower survival rate than that of the control cells (P<0.05). This suggests that overexpression of miR-125b may contribute to DNR resistance in K562, THP-1 and Jurkat cells and that knockdown of miR-125b may contribute to DNR sensitivity in REH cells. Therefore, these findings suggest that a correlation exists between miR-125b expression and sensitivity to DNR in leukemia cells.

In order to determine whether overexpression of miR-125b affects apoptosis, the release of activated caspase-3 was assessed. Caspase-3 pro and cleaved caspase-3 protein levels were analyzed using western blot analysis. Upon treatment with DNR, K562, THP-1 and Jurkat cells overexpressing miR-125b exhibited higher expression of caspase-3 pro than the control group, and lower expression of cleaved caspase-3 than the control group (Fig. 2D–F). Knockdown of miR-125b was observed to reduce the expression of caspase-3 pro and increase that of cleaved caspase-3 in REH cells compared with the expression in the control cells (Fig. 3C). These results show that overexpression of miR-125b induced DNR resistance through the inhibition of apoptosis and that the inhibition of miR-125b increased the sensitivity of REH cells to DNR by increasing apoptosis. These findings suggest that miR-125b increases the resistance of leukemia cells to DNR through inhibiting apoptosis.

To investigate the effect of miRNA on mRNA expression, miR-125b target mRNAs were obtained from the miRBase (http://www.mirbase.org) and the TargetScan (http://www.targetscan.org) databases (24). The correlation between miR-125b expression and the expression of its predicted target mRNAs, including the number of binding sites in the respective mRNAs and the variation in the correlation coefficients, were statistically analyzed. Among these potential gene targets, the present study focused on GRK 2 and PUMA, which may have effects on cell apoptosis (Fig. 3). It was hypothesized that GRK2 and PUMA are direct targets of miR-125b.

To investigate this hypothesis, HEK293T cells were separately cotransfected with miR-125b and either GRK2 or PUMA 3′UTR luciferase reporters. Transfections with control vector were performed in parallel. Cotransfection resulted in a 55 and 56.6% reduction in the reporter activity for GRK2 and PUMA, respectively (P<0.01; Fig. 4A). These results demonstrate that GRK2 and PUMA 3′UTR are targets of miR-125b. The role of miR-125b in the regulation of GRK2 and PUMA expression in K562, THP-1 and Jurkat cells was then assessed. In the present study, it was hypothesized that overexpression of miR-125b was likely to reduce the GRK2 and PUMA protein expression. GRK2 and PUMA protein levels were observed to be lower in K562, THP-1 and Jurkat cells overexpressing miR-125b, compared with those in the corresponding control cells (Fig. 4). In the miR-125b-knockdown REH cells, GRK2 and PUMA protein levels were found to be significantly higher than those in the control cells (Fig. 3C–E). These findings show that GRK2 and PUMA are targets of miR-125b in K562, THP-1, Jurkat and REH cells.

GRK2 and PUMA siRNA was used to silence the expression of GRK2 and PUMA in K562 cells, respectively. GRK2 expression was observed to be significantly decreased upon transfection with GRK2 siRNA. Of note, K562 cells transfected with GRK2 siRNA exhibited a similar survival pattern to cells overexpressing miR-125b. The level of cleaved caspase-3 protein was also decreased upon DNR treatment in the cells transfected with GRK2 siRNA. Similarly, the expression of PUMA was significantly decreased following transfection of PUMA siRNA, and these cells exhibited a similar survival pattern to cells overexpressing miR-125b. The level of cleaved caspase-3 protein was also observed to decrease upon DNR treatment in PUMA siRNA-transfected cells. These findings show that GRK2 and PUMA have a key role in DNR resistance (Fig. 5), and indicate that miR-125b contributes to DNR resistance through reducing the expression of GRK2 and PUMA in leukemia cells.