Suppression of miR-708 inhibits the Wnt/β-catenin signaling pathway by activating DKK3 in adult B-all

Abstract

Inactivation of Dickkopf-3 (DKK3) is closely associated with a poor prognosis in various solid tumor and hematologic malignancies. Promoter hypermethylation is one potential cause of DKK3 inactivation. However, whether other mechanisms lead to DKK3 inactivation and the subsequent effects of these inactivations on cell proliferation and the Wnt signaling pathway in adult B acute lymphoblastic leukemia (B-ALL) remain unclear. In the present study, we found that low DKK3 expression levels were associated with high miR-708 expression and promoter hypermethylation in adult B-ALL. miR-708 was confirmed to directly decrease DKK3 expression in Nalm-6 and BALL-1 cells. Additionally, a miR-708 inhibitor decreased cell proliferation mainly through apoptosis and cell cycle arrest at the G1 phase, and these effects were eliminated by DKK3 siRNA treatment. Moreover, the demethylating agent 5-aza-2'-deoxycytidine (5-aza) decreased the methylation state of the DKK3 promoter based on methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP), although this demethylation effect was not enhanced by the miR-708 inhibitor. The miR-708 inhibitor or 5-aza significantly increased DKK3 expression and decreased p-GSK3β, cyclin D1 and nuclear and cytoplasmic β-catenin protein expression, indicating that the Wnt/β-catenin signaling pathway was inhibited. These effects became more pronounced when the miR-708 inhibitor and 5-aza were used simultaneously. These findings provide greater insights into the mechanisms that increase DKK3 expression and suggest that a miR-708 inhibitor and 5-aza might be useful as targeted therapies for adult B-ALL.

Keywords: 5-aza-2’-deoxycytidine; DKK3; Wnt/β-catenin; adult B-acute lymphoblastic leukemia; miR-708.

Figure 1. DKK3 mRNA and miR-708 expression levels in acute leukemia patients and cell lines evaluated by PCR

(A and D) Compared with the controls (monocytes (MNCs) from healthy volunteers, n=3), DKK3 mRNA in newly diagnosed AML (n=10), B-ALL (n=20) and T-ALL (n=7) patients was expressed at low levels (A), and miR-708 was highly expressed (D). (B and E) The DKK3 mRNA expression level was low (B) and miR-708 was highly expressed (E) in newly diagnosed and relapsed B-ALL patients (n=6) compared with samples extracted at complete remission (CR). (C and F) Compared with the control (MNCs from healthy volunteers), DKK3 mRNA was expressed at low levels (C) and miR-708 was highly expressed (F) in the B-ALL cell lines. (G-I). The miR-708 and DKK3 mRNA expression levels are negatively correlated in newly diagnosed (G), relapsed (H) and CR (I) B-ALL patients. The data are presented as the means ± standard deviations (SDs) of three different experiments. *P<0.05; ** and^## P<0.01.

Figure 2. DKK3 is a functional target of miR-708 in B-ALL

(A) Predicted binding sites for miR-708 and DKK3. The sequence of the mutant 3′-UTR of DKK3 is also presented. (B) Luciferase assays showing the decrease in relative luciferase activity in Nalm-6 cells co-transfected with miR-708 and DKK3 and the increase in cells co-transfected with a miR-708 inhibitor and DKK3. A mutated 3′-UTR DKK3 plasmid was used as the control. (C-H). The miR-708 (C and F) and DKK3 mRNA levels (D and G) and the DKK3 protein expression levels (E and H) changed after transfection with a miR-708 mimics or a miR-708 inhibitor, respectively. (D and G). The graphs show the fold changes in protein expression relative to the untreated control measured by densitometry. (E and H) The images represent the results of a western blotting analysis of DKK3 protein expression. The data are presented as the means ± SDs of three different experiments. NC indicates the negative control. *P<0.05; **P<0.01.

Figure 3. Influence of miR-708 expression on cell proliferation, the cell cycle and apoptosis

(A, D). A miR-708 inhibitor significantly decreased the proliferation rates of BALL-1 (A) and Nalm-6 (D) cells. (B and E) The miR-708 inhibitor significantly increased the proportions of BALL-1 (B) and Nalm-6 (E) cells in the G1 phase. (C, F) The miR-708 inhibitor increased the numbers of apoptotic BALL-1 (C) and Nalm-6 (F) cells. (G and H). Representative depictions of the cell cycle (G) and apoptosis (H) in the two cell lines. Non-transfected cells were used as controls. Three independent experiments were performed. The data are presented as the means ± SDs. *P<0.05; **P<0.01.

Figure 4. Effects of miR-708 on the expression of proteins related to the cell cycle and apoptosis as determined by western blotting

(A and D). The miR-708 inhibitor decreased cyclin D1 expression in BALL-1 (A) and Nalm-6 (D) cells. (B-F) The miR-708 inhibitor significantly increased Bax expression and strongly decreased Bcl-2 expression in BALL-1 (B and C) and Nalm-6 cells (E and F). Non-transfected cells were used as controls. The fold changes in the relative protein levels were calculated with reference to the control levels. The data are presented as the means ± SDs (n=3, * P<0.05; ** P<0.01). (G and H). The images represent the results of a western blotting analysis of cyclin D1, Bax and Bcl-2 protein expression.

Figure 5. Depletion of DKK3 by siRNA eliminates the effect of a miR-708 inhibitor on Nalm-6 cells

(A-C). DKK3 mRNA (A) and protein (B and C) expression was significantly inhibited by the DKK3 siRNA, as determined by qRT-PCR and western blotting. The fold changes are relative to the untreated controls. (D) The DKK3 siRNA significantly increased cell proliferation in Nalm-6 cells, even in the presence of the miR-708 inhibitor. (E) The DKK3 siRNA strongly decreased the proportion of Nalm-6 cells in the G1 phase and increased the proportion of cells in the S phase, even in the presence of the miR-708 inhibitor. (F) The DKK3 siRNA markedly decreased apoptosis, even in the presence of the miR-708 inhibitor. (G). Representative plots of apoptosis in the cell lines. The data are presented as the means ± SDs of three different experiments. The miR-708 inhibitor NC was used as a control for the miR-708 inhibitor. The miR-708 inhibitor and the siRNA NC were used as controls for the miR-708 inhibitor and DKK3 siRNA, respectively. # and * means P<0.05; ** and ## means P<0.01.

Figure 6. Effects of the miR-708 inhibitor and/or 5-aza on the methylation state of the DKK3 promoter

(A) Genomic structure of the DKK3 gene and locations of the primers used in this study. (B) Detection of the unmethylated and methylated statuses of the DKK3 gene using the MSP method. (C and D) Methylation status of the DKK3 gene promoter region in CpG islands in Nalm- 6 (C) and BALL-1 (D) cells treated with the miR-708 inhibitor and/or 5-aza, as determined by sequencing after bisulfite modification of the genomic DNA. The solid spots means methylated CpG dinucleotide, the hollow spots means unmethylated CpG dinucleotide.

Figure 7. 5-Aza and the miR-708 inhibitor increased DKK3 expression and inhibited the Wnt signaling pathway in the B-ALL cell lines

(A and B). DKK3 mRNA and miR-708 expression were altered when the cells were treated with 5-aza and the miR-708 inhibitor. (C and D) Expression of β-catenin (cytoplasmic and nuclear), cyclin D1, GSK3β and p-GSK3β in the B-ALL cell lines after treatment with 5-aza and the miR-708 inhibitor, as determined by western blotting. The graphs show the corresponding band intensities of β-catenin, cyclin D1, GSK3β and p-GSK3β normalized to GAPDH and compared with the control. (E and F). The images represent β-catenin (cytoplasmic and nuclear), cyclin D1 GSK3β and p- GSK3β expression in BALL-1 cells after treatment with 5-aza and the miR-708 inhibitor/mimics or negative controls (NC), as determined by western blotting. The data are presented as the means ± SDs of triplicate experiments. *P<0.05; **P<0.01.