- Open Access
Upregulation of microRNA-125b contributes to leukemogenesis and increases drug resistance in pediatric acute promyelocytic leukemia
- Hua Zhang†1,
- Xue-Qun Luo†2,
- Dan-Dan Feng1,
- Xing-Ju Zhang1,
- Jun Wu3,
- Yu-Sheng Zheng1,
- Xiao Chen3,
- Ling Xu3Email author and
- Yue-Qin Chen1Email author
© Zhang et al; licensee BioMed Central Ltd. 2011
- Received: 14 April 2011
- Accepted: 1 September 2011
- Published: 1 September 2011
Although current chemotherapy regimens have remarkably improved the cure rate of pediatric acute promyelocytic leukemia (APL) over the past decade, more than 20% of patients still die of the disease, and the 5-year cumulative incidence of relapse is 17%. The precise gene pathways that exert critical control over the determination of cell lineage fate during the development of pediatric APL remain unclear.
In this study, we analyzed miR-125b expression in 169 pediatric acute myelogenous leukemia (AML) samples including 76 APL samples before therapy and 38 APL samples after therapy. The effects of enforced expression of miR-125b were evaluated in leukemic cell and drug-resistant cell lines.
miR-125b is highly expressed in pediatric APL compared with other subtypes of AML and is correlated with treatment response, as well as relapse of pediatric APL. Our results further demonstrated that miR-125b could promote leukemic cell proliferation and inhibit cell apoptosis by regulating the expression of tumor suppressor BCL2-antagonist/killer 1 (Bak1). Remarkably, miR-125b was also found to be up-regulated in leukemic drug-resistant cells, and transfection of a miR-125b duplex into AML cells can increase their resistance to therapeutic drugs,
These findings strongly indicate that miR-125b plays an important role in the development of pediatric APL at least partially mediated by repressing BAK1 protein expression and could be a potential therapeutic target for treating pediatric APL failure.
- pediatric acute promyelocytic leukemia (APL)
- treatment response
- drug resistance
Pediatric acute promyelocytic leukemia (APL), which represents approximately 10% of pediatric AML cases, is a subgroup of acute myelogenous leukemia (AML) characterized by promyelocytic cell morphology (referred to as M3 in the French-American-British classification) [1–3]. APL is characterized by a specific t(15;17) translocation that encodes a fusion of the promyelocytic leukemia (PML) and retinoic acid receptor-α (RARA) proteins [1–5]. Although the outcomes for children and adults with APL have dramatically improved since the successful introduction of all-trans retinoic acid (ATRA) in combination with anthracycline-based chemotherapy, more than 20% of patients presenting with APL will die of the disease, and the 5-year cumulative incidence of relapse is 17% overall and more than 20% in children [6–8]. The combination of ATRA and chemotherapy as initial therapy has become an attractive strategy for all APL patients; unfortunately, approximately 10% of APL patients develop "retinoic acid syndrome (RAS)" [6, 9, 10]. Furthermore, the precise genes and pathways that exert critical control over the lineage fate during APL development remain unclear.
MicroRNAs (miRNAs), a novel class of small noncoding RNAs ranging from 19 to 25 nucleotides in size, regulate specific target genes through translational repression or direct mRNA degradation, thereby regulating many cellular functions, including cell proliferation, differentiation, and apoptosis [11–13]. Recent studies have shown that deregulated expression of specific miRNAs that modulate expression of oncogenes and tumor suppressors is associated with the development of malignancies and that specific miRNA expression signatures can be used to effectively classify human tumors . MiRNA expression signatures associated with specific cytogenetic changes and clinical outcomes of adult CLL, AML, and Hodgkin's lymphoma have been reported [15–20]. Recent data suggest that miRNA inactivation by epigenetic mechanisms plays an important role in myelopoiesis and that modulating specific miRNA levels with drugs can lead to the downregulation of target oncogenes and restoration of cell differentiation . Dysregulated miRNA expression in APL cells following retinoic acid (RA) induction was also reported [22–25]. These studies, however, mainly focused on APL cell lines and included very few clinical APL samples. The role of miRNAs in the clinical progression of APL, especially in pediatric APL, remains to be explained.
Our previous study found that miR-125b was up-regulated in pediatric primary AML using genome-wide miRNA expression profiles in 36 diagnostic acute leukemia bone marrow samples . To further understand the role of miR-125b in pediatric AML, we analyzed miR-125b expression in 169 pediatric AML patients for whom clinical data were available. Interestingly, we found that miR-125b was reduced to normal levels in complete remission (CR) APL patients. Importantly, we found that miR-125b could promote proliferation and inhibit apoptosis of APL cells by targeting BCL2-antagonist/killer 1 (Bak1). The results imply that miR-125b functions as an oncogene in pediatric APL and that it has potential roles as a malignancy biomarker and a predictive marker of response to chemotherapy. In addition, we showed that transfection of the cells with miR-125b could increase cell resistance to chemotherapeutic drugs.
Patients, sample collection, and therapeutic methods
Pediatric AML patients' characteristics
AML Primary (N = 131)
Age at diagnosis, y
WBC count, ×10 9 /L
Less than 10
50 or higher
M3 after therapy (N = 38)
Cell lines and cell cultures
Human leukemia cell line NB4, HL60, K562 and their drug resistant cell lines were maintained in RPMI 1640 medium containing 10% fetal bovine serum (Gibco BRL). Drug resistant cell lines included NB4-R1 (subclones of NB4 with ATRA resistance established by ATRA inducing progressively), NB4-R2 (subclones of NB4 with ATRA resistance established by mutating the E domain of PML/RARA), HL60/DOX (subclone of HL60 with doxorubicin-resistance established by drug inducing progressively) and K562/DOX (subclone of K562 with doxorubicin-resistance established by drug inducing progressively). 293 T cell line was maintained in DMEM medium containing 10% fetal bovine serum (Gibco BRL).
Five-week-old BALB/c (nu/nu) mice were maintained under a specific pathogen-free condition in the Laboratory Animal Center, Sun Yat-sen University. All experiments were performed in accordance with our Institutional Animal Guidelines. The xenografted tumors were established by a single subcutaneous injection at the flank of ALB/c (nu/nu) mice with 2 × 106 HL60 cells (infected with lentivirus vectors that expressed miR-125b or miRNA negative control) in 0.1 ml RPMI-1640 medium. Each group consisted of at least 3 mice. The tumors were harvested, snap-frozen and stored at -80°C.
RNA extraction and quantitative real-time polymerase chain reaction
Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The following two quality criteria were used to assess total RNA quality. The first is purity. Both ratios, OD A260/A280 and OD A260/A230 were in the 1.8-2.1 region. The second is integrity. Total RNA quality has been assessed by denaturing gel electrophoresis electrophoresis and the ratio of 28S and 18S rRNA bands was approximately 2:1. Quantitative real-time RT-PCR (qRT-PCR) was performed as described  and employed a Hairpin-it™ miRNAs Real-Time PCR Quantization Kit containing stem-loop like RT primer, miRNA specific PCR primer and Molecular Beacon probe for miR-125b or the U6 RNA internal control (GenePharma, Shanghai, China). The expression of miR-125b relative to that in healthy samples was calculated using the 2-ΔΔCT method, and the expression of miR-125b relative to internal control U6 RNA in mouse was calculated using the 2-ΔCT method . The efficiencies of PCR amplification per PCR-cycle (by a dilution curve) of both miR-125b and U6 from each patient are higher than 95% (Additional file 2. Figure S1).
Fisher's exact test, paired t-test, and chi-square tests were used to compare baseline characteristics and average miRNA expression between different groups. All reported P values were two-sided and were obtained using SPSS software. P < 0.05 is considered significant.
Target genes prediction
Target genes prediction was performed to meet the following two criteria. First, miRNA targets were analyzed using three algorithms, including TARGETSCAN http://www.targetscan.org, PICTAR http://pictar.mdc-berlin.de and miRBase http://microrna.sanger.ac.uk/sequences/index.shtml. Second, in order to reduce the number of false positives, only putative target genes predicted by at least two of the programs were accepted.
Cells were homogenized in a lysis buffer (25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 1 mM PMSF, 25 μg/ml leupeptin, 1 mM DTT, 0.5% Triton X-100) and centrifuged. Samples of supernatant, each containing 10 μg of total protein, were electrophoresed in a 5-15% gradient gel. Western blotting was performed as following. In brief, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked in 4% non-fat dry milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 hr at room temperature and then probed overnight with rabbit polyclonal anti-Bak1 antibody (Sigma) in the same buffer. After three washes with TBST, the membranes were incubated for 1 hr with goat anti-rabbit IgG antibody (Sigma) conjugated to horseradish peroxidase (HRP). After washing with TBST, the membranes were exposed to a chemiluminescent reagent (ECL Plus, Amersham) for 1 min and then to Kodak X-ray film. Anti-β-actin antibody and anti-tublin antibody (Cell signaling technology) has been used to detect beta-actin and tublin.
MiR-125b expression vector construct
Full-length double-stranded human pre-miR-125b, together with 305 bp of flanking sequence was amplified from human genomic DNA using PCR (primer information is available in Additional file 3. Table S2) and cloned into the pcDNA™6.2-GW/EmGFP-miR control vector (Invitrogen) to generate the miR-125b expression construct pcDNA-miR-125b. The PCR product was also cloned into pIRES2-EGFP (Invitrogen, Carlsbad, CA, USA) and the fragment containing CMV promoter and miR-125b precursor was then cloned into pINCO retroviral vector (Genechem, Shanghai, China) to generate lv-miR-125b. Insertions were verified by DNA sequencing.
pGL3-BAK1 reporter construct and mutagenesis
The 252 bp 3'-untranslated terminal region (UTR) segment of Bak1 was amplified by PCR from human genomic DNA and inserted into the pGL3-control vector (Promega) using the XbaI site immediately downstream from the stop codon of luciferase for miR-125b functional analysis. Similarly, recombinant pGL3 reporter vectors for other selected possible targets were constructed. The Bak1 mutant, pGL3-BAK1-M, which had 8 bp deleted from the site of perfect complementarity to miR-125b, was generated using PCR deletion Wild-type and mutant insertions were confirmed by DNA sequencing. Primer information is available in Additional file 3. Table S2.
0.2 μg of pcDNA-miR-125b or pcDNA-control was cotransfected to 293 T cells with 0.1 μg of pGL3-control or recombinant pGL3 reporter vector and 0.1 μg of the control vector pRL-TK containing Renilla luciferase and TK promoter (Promega) using lipofectamine 2000 (Invitrogen). For NB4 cells, 5 μg of pcDNA-miR-125b or pcDNA-control, 5 μg of pGL3-control or pGL3-BAK1 or pGL3-BAK1-M Firefly luciferase report vector and 2.5 μg of the control vector pRL-TK were cotransfected using electroporation. 293 T cells or NB4 cells were grown in 24-well plates (1 × 105 cells/well) in 10% FBS in DMEM or RPMI-1640 supplemented with sodium pyruvate at 37°C in a humidified atmosphere of 5% CO2. Firefly and Renilla luciferase activities were measured consecutively using dual-luciferase assays (Promega) 24 hrs after transfection, according to the manufacturers' instructions.
Cell proliferation and apoptosis assay
NB4 cells, HL60 cells, NB4-R1 cells, NB4-R2 cells and HL60/DOX cells (1 × 104 per well) were plated in 96-well plates in RPMI medium 1640 and 10% FBS that was supplemented with sodium pyruvate at 37°C in a humidified atmosphere of 5% CO2. Cells were transfected with 100 nM miR-125b duplex (Ambion), scrambled duplex (negative control, Ambion), or 200 nM miR-125b antisense (Ambion) using lipofectamine 2000 (Invitrogen). After culturing for 1, 2 or 3 days, the MTT assay (Promega) or CCK-8 assay (Dojindo Molecular Technologies) was carried out to analyze cell proliferation. For apoptosis, forty-eight hours following the transfection, cells were labeled with Annexin V/PI (MultiSciences Biotech) and analyzed by flow cytometry.
Cell cytotoxicity assay
HL60 or HL60/DOX cells were seeded in 24-well plates at a density of 3 × 105 cells per well and transfected with 100 pmol scrambled duplex, miR-125b duplex or miR-125b antisense, or scrambled duplex (Ambion) in three independent replicates, using Nenon transfection equipment (Invitrogen) according to the manufacturer's protocol with program settings of 1400 V, 10 ms width, and 3 pulses. Forty-eight hours after transfection, cells were reseeded in 96-well plates at a density of 1.5 × 104 per well in the presence of miR-125b and treated with doxorubicin at a range of concentrations (5 to 25 μg/ml) in medium for 72 hrs. Cell survival was analyzed using the Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega).
miR-125b is highly expressed in pediatric APL
Induction therapy suppresses miR-125b expression in pediatric APL
All pediatric APL (M3 subtype) patients received a substantially distinct therapy utilizing the modified PETHEMA protocol (Additional file 1. Table S1) after diagnosis. We then collected 33 matched-pair APL samples at diagnosis and complete remission (CR) with PML-RARα-positive and with three years of clinical follow-up. We also collected 5 relapsed patients for comparison. To establish the relevance of miR-125b expression before and after therapy treatment, we investigated miR-125b expression in pairs of samples with diagnosis-CR or diagnosis-relapse. The results showed that in all CR patients, miR-125b expression decreased sharply after therapy to the same levels as in the normal controls (Figure 2B), which may be due to largely reduced leukemic cells after therapy. However, the expression level in the relapse patients was higher than normal controls, although induction therapy suppressed miR-125b expression in these patients to some degree (Figure 2C). The significantly different expression of miR-125b between pediatric primary, CR and relapse patients suggested that miR-125b could be a biomarker for clinical outcome. This possibility will be further studied using a larger number of patients. This finding raised the possibility that miR-125b, which is involved in the timing of tissue development and cell differentiation , might function as an oncogene in pediatric APL.
miR-125b represses endogenous Bak1 protein in myeloid cell lines and pediatric APL samples and is associated with disease development and treatment outcomes
To further investigate whether overexpression of miR-125b can promote APL myeloid cell progression by targeting tumor suppressor Bak1, we transfected two APL cell lines (NB4 and HL60) with 100 nM miR-125b duplex using the electroporation method (Additional file 4. Figure S2B). Bak1 expression was strongly reduced compared with that transfected with scrambled duplex in both myeloid cell lines NB4 (Figure 3C, left panel) and HL60 (Figure 3C, right panel). To uncover the effects of suppression in vivo, HL60 cells were infected with lentivirus vectors that expressed miR-125b (lv-miR-125b) or miRNA negative control (lv-NC), and then transplanted into nude mice with SC injection. Both groups of mice transplanted with lv-miR-125b-HL60 cells and lv-NC-HL60 cells developed solid tumors three weeks later. The overexpression of miR-125b was examined using qRT-PCR (Figure 3D, upper). The western blot analysis showed the Bak1 protein was repressed in the lv-miR-125b-HL60 tumors compared with the lv-NC-HL60 tumors transplanted with miRNA negative control (Figure 3D, lower).
Next, to examine the possibility that Bak1 was also regulated by miR-125b in clinical samples, we measured the expression levels of these two molecules in 47 pediatric APL clinical samples (only 47 samples have enough proteins to be used for western blot ananlysis) and five normal donors. We found that the levels of Bak1 and miR-125b were inversely correlated in 70.2% (33/47) of the pediatric APL samples (Figure 3E). For comparison, Figure 3F illustrates typical Bak1 protein expression in the 33 samples with the highest (APLH) and lowest (APLL) levels of expressed miR-125b. The levels of miR-125b expression for APLH and APLL were 80,071-fold and 10.24-fold higher than the normal average, respectively, while the levels of Bak1 expression were 50-fold lower in APLH and 2-fold lower in APLL. These results strongly suggest that Bak1 is a target of miR-125b in pediatric APL.
Because induction therapy suppressed miR-125b expression in pediatric APL patients, we would expect that, as a target of miR-125b, Bak1 levels would also be affected by therapeutics. We therefore analyzed the levels of Bak1 and miR-125b in samples from follow-up patients and compared the profiles with the profiles obtained from the same patients before therapy. In addition, three normal marrows were used as controls. Bak1 expression was increased in about 60% of CR patients (19/33) receiving therapeutics (Figure 3G). Thus, a concordant negative correlation existed between miR-125b levels and Bak1 levels with respect to disease development and treatment outcome.
Exogenous miR-125b promotes leukemic cell proliferation and inhibits apoptosis of leukemic cells
We then transfected the myeloid leukemic cell line NB4 with miR-125b duplex (mimics) and miR-125b inhibitor (anti-miR-125b) to assess the possible role of miR-125b in apoptosis in leukemic cells. However, in cancer cells, p53 and Bak1 protein are mainly localized in the cytoplasm, so the endogenous activity of Bak1 is usually insufficient to modulate apoptosis . Additionally, a previous study showed that ectopic expression of miR-125b in cells only suppresses apoptosis when the mitochondrial pathway is fully activated by exposure to the drug . We then treated the cells with 7-ethyl-10-hydrol-camptothecin and performed flow cytometry assays after 48 hrs of transfection. The results showed that ectopic expression of miR-125b significantly suppressed 7-ethyl-10-hydrol-camptothecin-induced apoptosis of NB4 cells (Annexin V-FITC positive) compared with control and miR-195 duplex (Figure 4C, upper panel).
The ability of transfection with miR-125b antisense to control apoptosis was also examined. Interestingly, after being transfected with 100 nM miR-125b (antisense), the percentage of 7-ethyl-10-hydrol-camptothecin-induced apoptotic NB4 cells (ANNEXIN V-FITC positive) at 48 hrs increased compared with control and miR-195 duplex (Figure 4C, lower panel). Similar results were obtained in HL60 cells (Additional file 6. Figure S4A). These results demonstrate that transfection of miR-125b antisense can remarkably increase cell apoptosis in leukemia cells. Furthermore, we inferred that miR-125b directly regulates Bak1 expression, thereby modulating the susceptibility of leukemic cells to apoptosis.
Up-regulation of miR-125b levels was found in resistant cells and reduction of the expression levels increased their sensitivity to therapeutic drugs
We noted above that the expression of miR-125b in all CR patients normalized after therapy, while it remained much higher in relapse patients (Figure 2C). We also found that miR-125b expression was slightly up-regulated in four drug-resistant cell lines, including HL60/DOX, K562/DOX (subclone of K562 with doxorubicin-resistance) and NB4-R1, as well as NB4-R2 (subclones of NB4 with ATRA resistance), when compared with their drug-sensitive parental cells (Figure 5E). These findings imply that miR-125b contributes to the drug-resistant nature of leukemic cells and that up-regulation of miR-125b levels in cells may increase their resistance to therapeutic drugs.
To address this issue, we transfected drug-resistant HL60/DOX cells and NB4-R1 with miR-125b duplex. Transfection of HL60/DOX cells with miR-125b duplex increased their resistance to DOX treatment. The IC50 of HL60/DOX cells transfected with miR-125b duplex was higher (p < 0.05) compared with the IC50 for HL60/DOX cells transfected with the miRNA duplex control and miR-195 duplex (Figure 5F). We also reproduced the same experiment in NB4-R1, and the results are shown in Additional file 6. Figure S4B. Taken together, the above results indicate a link between miR-125b dysregulation and drug resistance. However, the precise pathway and mechanism of action of miR-125b in the acquisition of drug resistance remains elusive.
In our previous investigation, we analyzed genome-wide miRNA expression profiles in pediatric AML and identified miRNA patterns specific to this disease. We also found that unique miRNA expression profiles were present in different subtypes of AML . These results imply that particular miRNAs might have specific roles in carcinogenesis or in the development of certain subtypes of leukemia. We noted that the expression of some miRNAs, including miR-125, was strikingly higher in pediatric APL than other subtypes of AML. This phenomenon led us to consider whether these miRNAs are oncogenic, whether they cause specific subtypes of pediatric leukemia or simply accompany them, or, alternatively, whether they represent a tumor suppressor feedback mechanism that is superseded by malignancy. Elucidating these questions should enhance our understanding of the malignant progression of pediatric APL. Therefore, to address these questions, we focused on the role of highly up-regulated miR-125b in pediatric APL.
The expression of miR-125b has been investigated in many human cancers. It is down-regulated in human breast cancer, thyroid anaplastic carcinomas, ovarian cancer, squamous cell carcinoma of the tongue, and hepatocellular carcinoma, suggesting that it functions as a tumor suppressor [33–38]. Myeloid cell differentiation arrest by miR-125b was also found in leukemia. M. Bousquet et al. demonstrated that miR-125b was able to interfere with primary human CD34+ cell differentiation and overexpression of miR-125b is sufficient both to shorten the latency of BCR-ABL-induced leukemia and to independently induce leukemia in a mouse model [39, 40]. These results reveal that miR-125b is important in cancer development.
The most important finding of this study was the discovery that miR-125b levels are related to the treatment response in pediatric APL patients. In patients with APL and positive PML-RARα gene fusion, miR-125b expression was much higher than in patients with other subtypes of AML, suggesting that miR-125b may be involved in PML/RARA- positive pediatric APL patients. However, the detailed mechanism of its role remains to be characterized. Notably, we found that miR-125b expression is associated with both disease development and treatment response. These results thus suggest a potential that miR-125b may represent a suitable biomarker for predicting the effect of chemotherapy.
Shi et al. showed that in prostate cancer, upregulated miR-125b could promote proliferation and targets Bak1 [40, 41]. Zhou et al. demonstrated that Bak1 is a direct target of miR-125b in breast cancer . However, the role of miR-125b in leukemia, especially in pediatric APL, remains unclear. We also found an inverse correlation between levels of miR-125b and Bak1 and obtained strong evidence that miR-125b could directly inhibit Bak1 expression in pediatric APL. Taken together, these findings suggest that up-regulated miR-125b may be part of a common regulatory pathway in different cancers. Furthermore, exogenous miR-125b is capable of promoting proliferation of malignant cells. This finding indicates that miR-125b may function as an oncogene by inhibiting APL cell apoptosis and promoting APL cell proliferation.
Although conventional therapy, such as the modified PETHEMALPA99 protocol, succeeded in obtaining long term CR in more than 70% of APL patients, early death and relapse in some patients are still a major concern in therapeutics, especially for pediatric APL. However, the mechanism of the patients' resistance to chemotherapeutic treatment is not entirely clear. Therefore, distinguishing the reason behind drug resistance and developing biomarkers are critical.
We have shown that miR-125b differentially expressed between pediatric APL primary, CR and relapse patients, suggesting a potential that high miR-125b expression might be used as a biomarker to indicate the treatment response of pediatric APL patients, although the detailed mechanism is still unknown. More importantly, transfection of NB4 and HL60 cells with miR-125b effectively inhibited apoptosis of these leukemic cells. Previously, M. Bousquet et al. reported that miR-125b was able to block the differentiation of NB4 cells induced by ATRA . Recently, miR-125b was also found associated with drug resistance in breast cancer and pediatric acute lymphoblastic leukemia [42, 43]. In this study, we also demonstrated that up-regulation of miR-125b in pediatric APL cells can increase their resistance to therapeutic drugs. We speculated that increased miR-125b expression might block the differentiation of hematopoietic precursors and respond to induction by ATRA or other chemotherapeutic drugs.
The expression of miR-125b both in pediatric and adult leukemia is currently not well understood. In adult APL, M. Bousquet et al. showed that myelodysplastic syndrome and AML patients carrying the t(2;11)(p21;q23) translocation were associated with miR-125b up-regulation ; however, they only studied 19 adult leukemia patients carrying the t(2;11)(p21;q23) translocation and did not determine whether the expression of miR-125b was up-regulated in adult APL patients. Another miRNA expression profiling assay suggested that miR-125b was up-regulated in seven adult APL patients, but this was not validated . Thus, miR-125b expression should be further examined on a large scale with adult APL patients. Remarkably, in our large-scale qRT-PCR assay using 131 pediatric primary AML samples (M1 to M6), we found that miR-125b was exceptionally highly expressed in the pediatric APL subtype compared with other AML subtypes. Because pediatric AML is a heterogeneous disease made up of various leukemia subtypes that differ markedly in their cytogenetics, the identification of these specific cytogenetic characteristics within each subtype will be helpful for elucidating the mechanism of oncogenesis in particular AML subtypes. Furthermore, this information may benefit the design of chemotherapeutic strategies for patients.
In conclusion, this is the first study of miR-125b expression in pediatric APL patients. Our results suggest that miR-125b might be used as a biomarker of malignancy and as a biomarker to evaluate the effectiveness of chemotherapy in pediatric APL. We also found that miR-125b promots proliferation and inhibits cell apoptosis at least partially mediated by targeting tumor suppressor Bak1 in pediatric APL. More importantly, forcing expression of miR-125b can increase resistance to therapeutic drugs, suggesting that miR-125b is a potential therapeutic target for pediatric APL failure.
The authors would like to thank the following investigators and hospitals that provided samples for the analysis: Dr. Hai-Xia Guo at the Second Affiliated Hospital of Sun Yat-sen University; Li-Bing Huang at the First Affiliated Hospital of Sun Yat-sen University; and Li-Ming Tu Guangdong at the Provincial People's Hospital. We thank Dr. ShaiJuan Chen for kindly providing the NB4-R1, NB4-R2 cell lines. This work was supported by the National Science and Technology Department (2011CB811301, 2011CBA01105 and 2009ZX09103-641) and National Natural Science Foundation of China (No. 30672254, 30872784 and 30772366)
- Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C: Proposals for the classification of the acute leukemias. French-American-British (FAB) co-operative group. Br J Haematol. 1976, 33: 451-458. http://www.ncbi.nlm.nih.gov/pubmed/188440, 10.1111/j.1365-2141.1976.tb03563.xView ArticlePubMedGoogle Scholar
- Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, Sultan C: A variant form of hypergranular promyelocytic leukemia (M3). Ann Intern Med. 1980, 92: 280-288., http://www.ncbi.nlm.nih.gov/pubmed/7352737Google Scholar
- Murthy R, Vemuganti GK, Honavar SG, Naik M, Reddy V: Extramedullary leukemia in children presenting with proptosis. J Hematol Oncol. 2009, 2: 4-, http://www.ncbi.nlm.nih.gov/pubmed/19166619 10.1186/1756-8722-2-4PubMed CentralView ArticlePubMedGoogle Scholar
- de Thé H, Lavau C, Marchio A, Chomienne C, Degos L, Dejean A: The PML-RAR alpha fusion mRNA generated by the t(15;17) translocation in acute promyelocytic leukemia encodes a functionally altered RAR. Cell. 1991, 66: 675-686., http://www.cell.com/retrieve/pii/009286749190113D 10.1016/0092-8674(91)90113-DView ArticlePubMedGoogle Scholar
- Kakizuka A, Miller WH, Umesono K, Warrell RP, Frankel SR, Murty VV, Dmitrovsky E, Evans RM: Chromosomal translocation t(15;17) in human acute promyelocytic leukemia fuses RAR alpha with a novel putative transcription factor, PML. Cell. 1991, 66: 663-674., http://www.cell.com/retrieve/pii/009286749190112C 10.1016/0092-8674(91)90112-CView ArticlePubMedGoogle Scholar
- Tallman MS, Nabhan C, Feusner JH, Rowe JM: Acute promyelocytic leukemia: evolving therapeutic strategies. Blood. 2002, 99: 759-767., http://www.ncbi.nlm.nih.gov/pubmed/11806975 10.1182/blood.V99.3.759View ArticlePubMedGoogle Scholar
- Ortega JJ, Madero L, Martín G, Verdeguer A, García P, Parody R, Fuster J, Molines A, Novo A, Debén G, Rodríguez A, Conde E, de la Serna J, Allegue MJ, Capote FJ, González JD, Bolufer P, González M, Sanz MA, : Treatment with all-trans retinoic acid and anthracycline monochemotherapy for children with acute promyelocytic leukemia: a multicenter study by the PETHEMA Group. J Clin Oncol. 2005, 23: 7632-7640., http://jco.ascopubs.org/content/23/30/7632.long 10.1200/JCO.2005.01.3359View ArticlePubMedGoogle Scholar
- Chow J, Feusner J: Isolated central nervous system recurrence of acute promyelocytic leukemia in children. Pedia Blood Cancer. 2009, 52: 11-13. 10.1002/pbc.21608., http://www.ncbi.nlm.nih.gov/pubmed/18816805 10.1002/pbc.21608View ArticleGoogle Scholar
- Avvisati G, Lo Coco F, Diverio D, Falda M, Ferrara F, Lazzarino M, Russo D, Petti MC, Mandelli F: AIDA (alltrans retinoic acid idarubicin) in newly diagnosed acute promyelocytic leukemia: a Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto (GIMEMA) pilot study. Blood. 1996, 88: 1390-1398., http://www.ncbi.nlm.nih.gov/pubmed/8695858PubMedGoogle Scholar
- Sanz MA, Martín G, Rayón C, Esteve J, González M, Díaz-Mediavilla J, Bolufer P, Barragán E, Terol MJ, González JD, Colomer D, Chillón C, Rivas C, Gómez T, Ribera JM, Bornstein R, Román J, Calasanz MJ, Arias J, Alvarez C, Ramos F, Debén G: A modified AIDA protocol with anthracycline-based consolidation results in high antileukemic efficacy and reduced toxicity in newly diagnosed PML/RARalpha-positive acute promyelocytic leukemia. Blood. 1999, 94: 3015-3021., http://bloodjournal.hematologylibrary.org/content/94/9/3015.longPubMedGoogle Scholar
- Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004, 116: 281-297., http://www.ncbi.nlm.nih.gov/pubmed/14744438 10.1016/S0092-8674(04)00045-5View ArticlePubMedGoogle Scholar
- Ambros V: The functions of animal microRNAs. Nature. 2004, 431: 350-355., http://www.nature.com/nature/journal/v431/n7006/full/nature02871.html 10.1038/nature02871View ArticlePubMedGoogle Scholar
- Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM: Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature. 2005, 433: 769-73., http://www.nature.com/nature/journal/v433/n7027/full/nature03315.html 10.1038/nature03315View ArticlePubMedGoogle Scholar
- Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA, Downing JR, Jacks T, Horvitz HR, Golub TR: MicroRNA expression profiles classify human cancers. Nature. 2005, 435: 834-838., http://www.ncbi.nlm.nih.gov/pubmed/15944708 10.1038/nature03702View ArticlePubMedGoogle Scholar
- Calin GA, Liu CG, Sevignani C, Ferracin M, Felli N, Dumitru CD, Shimizu M, Cimmino A, Zupo S, Dono M, Dell'Aquila ML, Alder H, Rassenti L, Kipps TJ, Bullrich F, Negrini M, Croce CM: MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci USA. 2004, 101: 11755-11760., http://www.pnas.org/content/101/32/11755.long 10.1073/pnas.0404432101PubMed CentralView ArticlePubMedGoogle Scholar
- Calin GA, Ferracin M, Cimmino A, Di Leva G, Shimizu M, Wojcik SE, Iorio MV, Visone R, Sever NI, Fabbri M, Iuliano R, Palumbo T, Pichiorri F, Roldo C, Garzon R, Sevignani C, Rassenti L, Alder H, Volinia S, Liu CG, Kipps TJ, Negrini M, Croce CM: A microRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med. 2005, 353: 1793-1801., http://www.nejm.org/doi/full/10.1056/NEJMoa050995 10.1056/NEJMoa050995View ArticlePubMedGoogle Scholar
- Garzon R, Volinia S, Liu CG, Fernandez-Cymering C, Palumbo T, Pichiorri F, Fabbri M, Coombes K, Alder H, Nakamura T, Flomenberg N, Marcucci G, Calin GA, Kornblau SM, Kantarjian H, Bloomfield CD, Andreeff M, Croce CM: MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia. Blood. 2008, 111: 3183-3189., http://www.ncbi.nlm.nih.gov/pubmed/18187662 10.1182/blood-2007-07-098749PubMed CentralView ArticlePubMedGoogle Scholar
- Garzon R, Garofalo M, Martelli MP, Briesewitz R, Wang L, Fernandez-Cymering C, Volinia S, Liu CG, Schnittger S, Haferlach T, Liso A, Diverio D, Mancini M, Meloni G, Foa R, Martelli MF, Mecucci C, Croce CM, Falini B: Distinctive microRNA signature of acute myeloid leukemia bearing cytoplasmic mutated nucleophosmin. Proc Natl Acad Sci USA. 2008, 105: 3945-3950., http://www.pnas.org/content/105/10/3945.long 10.1073/pnas.0800135105PubMed CentralView ArticlePubMedGoogle Scholar
- Jongen-Lavrencic M, Sun SM, Dijkstra MK, Valk PJ, Löwenberg B: MicroRNA expression profiling in relation to the genetic heterogeneity of acute myeloid leukemia. Blood. 2008, 111: 5078-5085., http://bloodjournal.hematologylibrary.org/content/111/10/5078.long 10.1182/blood-2008-01-133355View ArticlePubMedGoogle Scholar
- Navarro A, Gaya A, Martinez A, Urbano-Ispizua A, Pons A, Balagué O, Gel B, Abrisqueta P, Lopez-Guillermo A, Artells R, Montserrat E, Monzo M: MicroRNA Expression Profiling in Classical Hodgkin Lymphoma. Blood. 2008, 111: 2825-2832., http://bloodjournal.hematologylibrary.org/content/111/5/2825.long 10.1182/blood-2007-06-096784View ArticlePubMedGoogle Scholar
- Fazi F, Racanicchi S, Zardo G, Starnes LM, Mancini M, Travaglini L, Diverio D, Ammatuna E, Cimino G, Lo-Coco F, Grignani F, Nervi C: Epigenetic silencing of the myelopoiesis regulator microRNA-233 by the AML1/ETO oncoprotein. Cancer Cell. 2007, 12: 457-466., http://www.cell.com/cancer-cell/retrieve/pii/S1535610807002681 10.1016/j.ccr.2007.09.020View ArticlePubMedGoogle Scholar
- Fazi F, Rosa A, Fatica A, Gelmetti V, De Marchis ML, Nervi C, Bozzoni I: A mini-circuitry comprising microRNA-223 and transcription factors NFI-A and C/EBPa regulates human granulopoiesis. Cell. 2005, 123: 819-831., http://www.cell.com/retrieve/pii/S0092867405009773 10.1016/j.cell.2005.09.023View ArticlePubMedGoogle Scholar
- Garzon R, Pichiorri F, Palumbo T, Visentini M, Aqeilan R, Cimmino A, Wang H, Sun H, Volinia S, Alder H, Calin GA, Liu CG, Andreeff M, Croce CM: MicroRNA gene expression during retinoic acid-induced differentiation of human acute promyelocytic leukemia. Oncogene. 2007, 26: 4148-4157., http://www.nature.com/onc/journal/v26/n28/full/1210186a.html 10.1038/sj.onc.1210186View ArticlePubMedGoogle Scholar
- Saumet A, Vetter G, Bouttier M, Portales-Casamar E, Wasserman WW, Maurin T, Mari B, Barbry P, Vallar L, Friederich E, Arar K, Cassinat B, Chomienne C, Lecellier CH: Transcriptional repression of microRNA genes by PML-RARA increases expression of key cancer proteins in acute promyelocytic leukemia. Blood. 2009, 113: 412-421., http://bloodjournal.hematologylibrary.org/content/113/2/412.longView ArticlePubMedGoogle Scholar
- De Marchis ML, Ballarino M, Salvatori B, Puzzolo MC, Bozzoni I, Fatica A: A new molecular network comprising PU.1, interferon regulatory factor proteins and miR-342 stimulates ATRA-mediated granulocytic differentiation of acute promyelocytic leukemia cells. Leukemia. 2009, 23: 856-862., http://www.nature.com/leu/journal/v23/n5/full/leu2008372a.html 10.1038/leu.2008.372View ArticlePubMedGoogle Scholar
- Zhang H, Luo XQ, Zhang P, Huang LB, Zheng YS, Wu J, Zhou H, Qu LH, Xu L, Chen YQ: MicroRNA patterns associated with clinical prognostic parameters and CNS relapse prediction in pediatric acute leukemia. PLoS One. 2009, 4: e7826-, http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0007826 10.1371/journal.pone.0007826PubMed CentralView ArticlePubMedGoogle Scholar
- Chen C, Ridzon DA, Broomer AJ, Zhou Z, Lee DH, Nguyen JT, Barbisin M, Xu NL, Mahuvakar VR, Andersen MR, Lao KQ, Livak KJ, Guegler KJ: Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res. 2005, 33: e179-, http://www.ncbi.nlm.nih.gov/pubmed/16314309 10.1093/nar/gni178PubMed CentralView ArticlePubMedGoogle Scholar
- Livak KJ, Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001, 25: 402-408., http://www.sciencedirect.com/science/article/pii/S1046202301912629 10.1006/meth.2001.1262View ArticlePubMedGoogle Scholar
- Lagos-Quintana M, Rauhut R, Yalcin A, Meyer J, Lendeckel W, Tuschl T: Identification of tissue-specific microRNAs from mouse. Curr Biol. 2002, 12: 735-739., http://www.ncbi.nlm.nih.gov/pubmed/12007417 10.1016/S0960-9822(02)00809-6View ArticlePubMedGoogle Scholar
- Galonek HL, Hardwick JM: Upgrading the BCL-2 network. Nat Cell Biol. 2006, 8: 1317-1319., http://www.nature.com/ncb/journal/v8/n12/full/ncb1206-1317.html 10.1038/ncb1206-1317View ArticlePubMedGoogle Scholar
- Leu JI, Dumont P, Hafey M, Murphy ME, George DL: Mitochondrial p53 activates Bak and causes disruption of a Bak-Mcl1 complex. Nat Cell Biol. 2004, 6: 443-450., http://www.nature.com/ncb/journal/v6/n5/full/ncb1123.html 10.1038/ncb1123View ArticlePubMedGoogle Scholar
- Le MT, Teh C, Shyh-Chang N, Xie H, Zhou B, Korzh V, Lodish HF, Lim B: MicroRNA-125b is a novel negative regulator of p53. Genes Dev. 2009, 23: 862-876., http://genesdev.cshlp.org/content/23/7/862.long 10.1101/gad.1767609PubMed CentralView ArticlePubMedGoogle Scholar
- Iorio MV, Ferracin M, Liu CG, Veronese A, Spizzo R, Sabbioni S, Magri E, Pedriali M, Fabbri M, Campiglio M, Ménard S, Palazzo JP, Rosenberg A, Musiani P, Volinia S, Nenci I, Calin GA, Querzoli P, Negrini M, Croce CM: MicroRNA gene expression deregulation in human breast cancer. Cancer Res. 2005, 65: 7065-7070., http://www.ncbi.nlm.nih.gov/pubmed/16103053 10.1158/0008-5472.CAN-05-1783View ArticlePubMedGoogle Scholar
- Visone R, Pallante P, Vecchione A, Cirombella R, Ferracin M, Ferraro A, Volinia S, Coluzzi S, Leone V, Borbone E, Liu CG, Petrocca F, Troncone G, Calin GA, Scarpa A, Colato C, Tallini G, Santoro M, Croce CM, Fusco A: Specific microRNAs are downregulated in human thyroid anaplastic carcinomas. Oncogene. 2007, 26: 7590-7595., http://www.nature.com/onc/journal/v26/n54/full/1210564a.html 10.1038/sj.onc.1210564View ArticlePubMedGoogle Scholar
- Yang H, Kong W, He L, Zhao JJ, O'Donnell JD, Wang J, Wenham RM, Coppola D, Kruk PA, Nicosia SV, Cheng JQ: MicroRNA expression profiling in human ovarian cancer: miR-214 induces cell survival and cisplatin resistance by targeting PTEN. Cancer Res. 2008, 68: 425-433., http://cancerres.aacrjournals.org/content/68/2/425.long 10.1158/0008-5472.CAN-07-2488View ArticlePubMedGoogle Scholar
- Nam EJ, Yoon H, Kim SW, Kim H, Kim YT, Kim JH, Kim JW, Kim S: MicroRNA expression profiles in serous ovarian carcinoma. Clin Cancer Res. 2008, 14: 2690-2695., http://www.ncbi.nlm.nih.gov/pubmed/18451233 10.1158/1078-0432.CCR-07-1731View ArticlePubMedGoogle Scholar
- Wong TS, Liu XB, Wong BY, Ng RW, Yuen AP, Wei WI: Mature miR-184 as Potential Oncogenic microRNA of Squamous Cell Carcinoma of Tongue. Clin Cancer Res. 2008, 14: 2588-2592., http://clincancerres.aacrjournals.org/content/14/9/2588.long 10.1158/1078-0432.CCR-07-0666View ArticlePubMedGoogle Scholar
- Li W, Xie L, He X, Li J, Tu K, Wei L, Wu J, Guo Y, Ma X, Zhang P, Pan Z, Hu X, Zhao Y, Xie H, Jiang G, Chen T, Wang J, Zheng S, Cheng J, Wan D, Yang S, Li Y, Gu J: Diagnostic and prognostic implications of microRNAs in human hepatocellular carcinoma. Int J Cancer. 2008, 123: 1616-1622., http://www.ncbi.nlm.nih.gov/pubmed/18649363 10.1002/ijc.23693View ArticlePubMedGoogle Scholar
- Bousquet M, Quelen C, Rosati R, Mansat-De Mas V, La Starza R, Bastard C, Lippert E, Talmant P, Lafage-Pochitaloff M, Leroux D, Gervais C, Viguié F, Lai JL, Terre C, Beverlo B, Sambani C, Hagemeijer A, Marynen P, Delsol G, Dastugue N, Mecucci C, Brousset P: Myeloid cell differentiation arrest by miR-125b-1 in myelodysplasic syndrome and acute myeloid leukemia with the t(2;11)(p21;q23) translocation. J Exp Med. 2008, 205: 2499-2506., http://jem.rupress.org/content/205/11/2499.long 10.1084/jem.20080285PubMed CentralView ArticlePubMedGoogle Scholar
- Bousquet M, Harris MH, Zhou B, Lodish HF: MicroRNA miR-125b causes leukemia. Proc Natl Acad Sci USA. 2010, 107: 21558-21563. , http://www.ncbi.nlm.nih.gov/pubmed/21118985 10.1073/pnas.1016611107PubMed CentralView ArticlePubMedGoogle Scholar
- Shi XB, Xue L, Yang J, Ma AH, Zhao J, Xu M, Tepper CG, Evans CP, Kung HJ, deVere White RW: An androgen-regulated miRNA suppresses Bak1 expression and induces androgen-independent growth of prostate cancer cells. Proc Natl Acad Sci USA. 2008, 104: 19983-19988., http://www.pnas.org/content/104/50/19983.longView ArticleGoogle Scholar
- Zhou M, Liu Z, Zhao Y, Ding Y, Liu H, Xi Y, Xiong W, Li G, Lu J, Fodstad O, Riker AI, Tan M: MicroRNA-125b confers the resistance of breast cancer cells to paclitaxel through suppression of pro-apoptotic Bcl-2 antagonist killer 1 (Bak1) expression. J Biol Chem. 2010, 285: 21496-21507., http://www.ncbi.nlm.nih.gov/pubmed/20460378 10.1074/jbc.M109.083337PubMed CentralView ArticlePubMedGoogle Scholar
- Schotte D, De Menezes RX, Akbari Moqadam F, Mohammadi Khankahdani L, Lange-Turenhout E, Chen C, Pieters R, Den Boer ML: MicroRNAs characterize genetic diversity and drug resistance in pediatric acute lymphoblastic leukemia. Haematologica. 2011, 96: 703-711., http://www.haematologica.org/cgi/content/full/96/5/703 10.3324/haematol.2010.026138PubMed CentralView ArticlePubMedGoogle Scholar
- Li Z, Lu J, Sun M, Mi S, Zhang H, Luo RT, Chen P, Wang Y, Yan M, Qian Z, Neilly MB, Jin J, Zhang Y, Bohlander SK, Zhang DE, Larson RA, Le Beau MM, Thirman MJ, Golub TR, Rowley JD, Chen J: Distinct microRNA expression profiles in acute myeloid leukemia with common translocations. Proc Natl Acad Sci USA. 2008, 105: 15535-15540., http://www.pnas.org/content/105/40/15535.long 10.1073/pnas.0808266105PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.