MicroRNA-183 promotes cell proliferation via regulating programmed cell death 6 in pediatric acute myeloid leukemia

Abstract

Purpose: The aim of this study was to investigate roles of microRNA (miR)-183 in pediatric acute myeloid leukemia (AML).

Methods: miR-183 expression in bone marrow and patients' sera of childhood AML was detected by real-time quantitative PCR. Functions of miR-183 in malignant phenotypes of two leukemia cell lines were then evaluated. Additionally, putative targets of miR-183 were predicted using three miRNA target prediction algorithms and validated by luciferase reporter assay. Clinical relevance of miR-183 and its target gene were further determined.

Results: miR-183 expression in bone marrow and patients' sera of childhood AML was both significantly higher than those in the corresponding normal controls (both P < 0.001). Enforced expression of miR-183 dramatically enhanced cell proliferation and G1/S transition, but inhibited cell apoptosis of leukemia cells. Bioinformatics prediction and luciferase reporter assay identified programmed cell death 6 (PDCD6) as a direct target gene of miR-183. Moreover, high serum miR-183 combined with low serum PDCD6 mRNA was significantly associated with French-American-British classification subtype M7 (P = 0.01) and unfavorable karyotypes (P = 0.006). Further multivariate analysis identified the combination of serum miR-183 and PDCD6 levels as an independent prognostic factor for both relapse-free and overall survivals. Functionally, re-introduction of PDCD6 markedly reversed the effects of miR-183 in cell cycle, proliferation and apoptosis of two leukemia cell lines.

Conclusion: Combined serum miR-183 and PDCD6 mRNA may serve as a novel prognostic biomarker for pediatric AML. Interestingly, miR-183 may function as an oncogene and may enhance cell proliferation by targeting PDCD6, implying a potential therapeutic target for this malignancy.

Keywords: Pediatric acute myeloid leukemia; Prognosis; Programmed cell death 6; Proliferation; microRNA-183.

Fig. 1

MiR-183 upregulation in bone marrow and patients’ sera of pediatric AML. a Expression levels of miR-183 in bone marrow of patients with pediatric AML and normal controls. b Expression levels of miR-183 in patients’ sera and healthy controls. c Correlations of miR-183 expression levels between the bone marrow and patients’ sera of pediatric AML

Fig. 2

MiR-183 upregulation promotes cell proliferation of human leukemia cells. a, b Expression levels of miR-183 in HL60 and K562 cells transfected with miR-183 mimics were significantly increased compared with those transfected with control mimics (both P = 0.02), while its expression in HL60 and K562 cells transfected with miR-183 inhibitor were significantly decreased (both P < 0.001); c, d MTT assay indicated that the transfection of miR-183 mimics significantly promoted cell proliferation in HL60 and K562 cells compared with normal controls (P < 0.01); in contrast, the loss of miR-183 by the specific inhibitor markedly suppressed cell proliferation of HL60 and K562 cells (P = 0.02 and 0.03, respectively)

Fig. 3

MiR-183 upregulation promotes G1/S transition and inhibits apoptosis of human leukemia cells. a, b HL60 and K562 cells which were transfected with miR-183 mimic, respectively, display the significantly decreased ratio of G0-/G1-phase cells and significantly increased ratio of S-phase and G2-/M-phase cells, compared with the cells in the negative group (both P < 0.05). In contrast, the transfection of miR-183 inhibitor led to the increased cell population of G0-/G1-phase and the decreased cell population of the S-phase and G2-/M-phase of two leukemia cell lines (both P < 0.05). c, d Percentage of apoptotic HL60 and K562 cells, which were transfected with miR-183 mimic, were dramatically decreased (all P < 0.05), but those in miR-183 inhibitor transfection group were significantly increased, compared with the corresponding controls (all P < 0.05)

Fig. 4

MiR-183 downregulates PDCD6 expression by directly targeting its 3′-UTR. a The putative miR-183-binding sequence in the 3′-UTR of PDCD6 mRNA; b luciferase report assay showed that the relative luciferase activities were dramatically reduced in cells co-transfected with the psiCHECK-2-PDCD6-3′-UTR-WT luciferase reporter and miR-183 mimics than those in the negative control cells. However, both inhibitory and enhanced effects were abolished when 3′-UTRs that contained both mutant-binding sites were co-transfected with miR-183; c, d Enforced expression of miR-183 remarkably inhibited the endogenous expression of PDCD6 mRNA and protein in HL60 and K562 cells, respectively. e, f Loss of miR-183 increased the expression of endogenous PDCD6 at both the mRNA and protein levels in HL60 and K562 cells, respectively. The relative expression level of PDCD6 mRNA was obtained by the normalization to the values of GAPDH. Each sample was examined in triplicate. *P < 0.05, comparison with NC-mimics and NC-inhibitor groups

Fig. 5

Serum miR-183 in patients with pediatric AML negatively correlates with serum PDCD6 mRNA. a Expression levels of PDCD6 mRNA in pediatric AML patients’ sera were significantly lower than those in healthy sera (AML vs normal: 1.62 ± 0.88 vs 3.24 ± 1.03, P < 0.001); the relative expression level of PDCD6 mRNA was obtained by the normalization to the values of GAPDH. Each sample was examined in triplicate. b Spearman’s correlation analysis showed the negative correlation between serum miR-183 and PDCD6 mRNA in patients with pediatric AML (Spearman’s correlation: r = −0.556, P < 0.001)

Fig. 6

Kaplan–Meier curves of relapse-free survival (RFS, A) and overall survival (OS, B) of patients with pediatric AML stratified by the combined serum miR-183 and PDCD6 mRNA levels

Fig. 7

MiR-183 promotes cell proliferation in human leukemia cells via regulating PDCD6. a, b Endogenous expression levels of PDCD6 protein were detected by western blot in HL60 and K562 cells transfected with the miR-183 mimic in the presence of PDCD6 or vector control for 24 h. c, d Re-introduction of PDCD6 markedly reversed the enhancing effects of cell proliferation in the miR-183-expressing HL60 and K562 cells

Fig. 8

MiR-183 promotes G1/S transition and inhibits cell apoptosis in human leukemia cells via regulating PDCD6. a, b Re-introduction of PDCD6 markedly reversed the effects of miR-183 overexpression on cell cycle of HL60 and K562 cells, respectively. c, d Re-introduction of PDCD6 markedly reversed the effects of miR-183 overexpression on cell apoptosis of HL60 and K562 cells, respectively