Upregulation of microRNA-125b contributes to leukemogenesis and increases drug resistance in pediatric acute promyelocytic leukemia - PubMed (original) (raw)

Upregulation of microRNA-125b contributes to leukemogenesis and increases drug resistance in pediatric acute promyelocytic leukemia

Hua Zhang et al. Mol Cancer. 2011.

Abstract

Background: 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.

Methods: 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.

Results: 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,

Conclusions: 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.

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Figures

Figure 1

Differential expression of miR-125b in pediatric AML patients. (A) Differential expression of miR-125b in normal samples and different subtypes of pediatric AML determined by miRNA microarray analysis (the same part of different microarray chips is shown). Bright green dots indicate highly expressed miRNAs. (B) Expression of miR-125b in the normal pool (N); different subtype pools of pediatric AML and M3 complete remission pool (M3 CR) were validated by northern blot. (C) Expression levels of miR-125b in subtypes of 131 pediatric AML were analyzed with qRT-PCR. Data are presented as the fold change of miR-125b expression in patient samples with respect to expression in bone marrow mononuclear cells (MNC) from 13 healthy donors. The average miR-125b expression of each subtype was statistically compared with the average normal value. *p < 0.05; **p < 0.01.

Figure 2

Expression of miR-125b varied in different therapeutic response groups of pediatric APL. The qRT-PCR assay was repeated three times, and similar results were obtained. Representative results are shown as means ± standard deviation (M ± SD). Data presented are the fold changes of expression with respect to the bone marrow MNC from five healthy donors. (A) Expression level of miR-125b in different age groups of pediatric APL patients. (B) The expression levels of miR-125b in 33 pediatric APL patients before and after therapy are presented as fold changes of expression with respect to expression in bone marrow mononuclear cells (MNC) from healthy donors. MiR-125b levels for the same patient before and after therapy were paired. Data were sorted from lowest to highest levels of miR-125b in non-treated patients. (C) Expression level of miR-125b in pediatric APL patients before and after therapy. *p < 0.05, **p < 0.01.

Figure 3

Exogenous miR-125b deregulates Bak1 protein. (A) An outline of luciferase reporter assay for validating the interaction of miR-125b with the 3' UTR of BAK1 is shown. Red text indicates the ''seed'' regions. In mutant reporter constructs, the MRE was deleted. (B) Repression of luciferase activity due to the interaction between miR-125b and the predicted MREs in the luciferase-Bak1-3' UTR constructs. The values represent the average ± SD (n = 3). *p < 0.05. (C) Western blot analysis of BAK1 expression in NB4 cells (left panel) or HL60 cells (right panel) after transfection with 100 nM miR-125b duplex or scrambled duplex. (D) The effects of suppression of miR-125b on Bak1 in mouse model. Upper: the overexpression of miR-125b was examined using qRT-PCR; lower: western blot analysis showed the Bak1 protein was repressed by miR-125b. lv-miR-125b: lentivirus vectors that expressed miR-125b; lv-NC: lentivirus vectors that expressed miR-NC, miRNA negative control. (E) Bak1 protein expression was inversely correlated with miR-125b levels in 33 of 47 pediatric APL patient samples. Red triangle: miR-125b expression (fold change vs. normal average); light blue diamond: Bak1 protein expression (fold change vs. normal average). Both miR-125b and Bak1 average expression levels of healthy donors were set at 1. (F) Western blotting images showing typical high and low Bak1 protein expression levels. The Bak1 protein level was quantified from western blot bands normalized to the β-actin level. The average expressions of Bak1 and miR-125b, presented as fold change compared with healthy samples, are listed in the table. (G) Bak1 protein expression in three normal donors using western blot (left), selected typical Bak1 protein expression in normal donor and pediatric patients with different ATRA responses was analyzed by western blot (right). N, normal donor; P, samples collected in primary diagnosis without treatment; CR, complete remission after therapy.

Figure 4

miR-125b promotes cell proliferation and inhibits apoptosis in leukemic cell lines NB4 and HL60. (A) Time-dependent effects of miR-125b on NB4 cell proliferation were confirmed using an MTT assay. (B) Time-dependent effects of miR-125b on HL60 cell proliferation were confirmed using an MTT assay. At least three independent experiments were performed and similar results were obtained. Data are represented as means ± SD from 3 independent experiments. P < 0.05, compared with mock, scrambled duplex and miR-195 at 48, 72 and 96 hrs. (C) Apoptosis in a leukemic cell line transfected with miR-125b duplex, as detected by flow cytometry. Upper panel: The NB4 cells were transfected with 100 nM scrambled duplex, miR-195 or miR-125b duplex, respectively. Lower panel: The NB4 cells were transfected with 100 nM antisense control, miR-195 or miR-125b antisense, respectively. Forty-four hours following transfection, camptothecin was added to induce cells for four hours and then cells were labeled with Annexin V/PI and analyzed by flow cytometry. Three independent experiments were performed and similar results were obtained.

Figure 5

Apoptosis after knockdown of Bak1 and the involvement of miR-125b in drug-resistant leukemic cells. (A) Western blotting images showing low Bak1 protein expression when NB4 cells were transfected with three different strands of si-RNA of Bak1. (B), (C) and (D) The NB4 cells were transfected with 100 nM si-RNA control, a strand of si-Bak1-3 or a mixture of these three strands of si-Bak. Forty-eight hours following transfection, camptothecin was added and cells were labeled with Annexin V/PI and analyzed by flow cytometry. Three independent experiments were performed and similar results were obtained. (E) miR-125b expression was up-regulated in drug-resistant cell lines compared to their drug-sensitive parental cells. *p < 0.05 compared with corresponding parents cells. (F) Transfection of HL60/DOX cells with miR-125b duplex increases 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 miRNA duplex control and miR-195. Three independent experiments were performed.

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Upregulation of microRNA-125b contributes to leukemogenesis and increases drug resistance in pediatric acute promyelocytic leukemia - PubMed (original) (raw)