MicroRNA-1246 by Targeting AXIN2 and GSK-3β Overcomes Drug Resistance and Induces Apoptosis in Chemo-resistant Leukemia Cells

Abstract

Background and objective: Chemotherapy plays an important role in the treatment of leukemia. Multidrug resistance (MDR) induced by chemotherapy always leads to treatment failure and disease recurrence. MicroRNAs (miRNAs) have been verified as crucial components in carcinogenesis, including chemo-resistance of tumor cells, which has not been fully understood. In this study, we aimed to identify the potential candidate miRNA, miR-1246, and reveal its regulatory role in chemo-resistance of leukemia cells. Methods: Candidate miRNAs were selected by microarray analysis, screened by bioinformatics tools and verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Chemo-resistant phenotypes, including cell viability, apoptosis, adriamycin (ADM) efflux and in vivo oncogenicity of leukemia cells following transfected with miR-1246 mimics or inhibitor were checked with or without ADM treatment to make clear the relationship between miR-1246 and chemo-resistance. RT-qPCR, western blot and dual luciferase reporter assay were performed to measure the expression of related genes and address the potential regulatory mechanism of miR-1246 in chemo-resistance. Results: The expression of miR-1246 was significantly higher in chemo-resistant leukemia K562/ADM cells, HL-60/RS cells and recurrent primary leukemia cells. Loss of miR-1246 inhibited proliferation, induced apoptosis, altered cell cycle distribution, inhibited ADM efflux in chemo-resistant leukemia cells, while overexpression of miR-1246 showed the opposite role in chemo-sensitive leukemia cells. Both bioinformatics prediction and luciferase assay indicated that AXIN2 and glycogen synthase kinase 3 beta (GSK-3β) were the direct targets of miR-1246 in leukemia cells. Inhibition of miR-1246 could up-regulate AXIN2 and GSK-3β and inactivate Wnt/β-catenin pathway, accompanied with inhibiting the expression of β-catenin and further influencing the expression of P-glycoprotein (P-gp) in the chemo-resistant leukemia cells. Conclusions: Chemo-resistant ability of MDR leukemia cells is attenuated by loss of miR-1246 via negatively regulating AXIN2 and GSK-3β to inactivate Wnt/β-catenin pathway and suppress P-gp expression, these mean that targeting miR-1246-AXIN2/GSK-3β-Wnt/β-catenin axis may be beneficial to overcome the chemo-resistance in relapse and refractory leukemia patients.

Keywords: AXIN2; GSK-3β, Wnt/β-catenin pathway.; chemo-resistance; leukemia cells; miR-1246.

Figure 1

miR-1246 was highly expressed in chemo-resistant leukemia cells and recurrent primary leukemia cells. A and B, miR-1246 was detected in K562, K562/ADM cells and HL-60, HL-60/RS cells using RT-qPCR. C, RT-qPCR assay was used to testify the expression difference of miR-1246 between the first diagnostic primary leukemia cells and recurrent primary leukemia cells in patients (** P < 0.01, comparing to their parental cells or the first diagnostic samples). D and E, The expression levels of the candidate miRNAs in K562/ADM cells compared to their parental cells.

Figure 2

Loss of miR-1246 in drug-resistant leukemia cells could improve their drug sensitivity and induce cell apoptosis. A, The expression of miR-1246 was inhibited in chemo-resistant leukemia cells (* P < 0.05, comparing to their negative control, respectively). B, The cell survival and proliferation rate was measured using MTT assay after the miR-1246 was repressed in K562/ADM and HL-60/RS cells (* P < 0.05, ** P < 0.01, comparing to the NC inhibitor transfected cells treated with the same concentration of ADM, respectively). C, Morphological alterations of K562/ADM and HL-60/RS cells transfected with NC inhibitor or miR-1246 inhibitor and treated with or without ADM. (a) Morphological changes detected by phase-contrast microscopy (×200). (b) Ultrastructural changes of cells dectected by transmission electron microscopy (×10,000). D, The cell apoptotic rate was examined using flow cytometry staining with Annexin V-FITC and PI (* P < 0.05, comparing to their negative control. # P < 0.05, comparing to their negative control treated with the same concentration of ADM). E, After the cells were fixed and stained, flow cytometry was used to check the cell cycle distribution. F, Relative positive rate and mean fluorescence intensity were analysed to investigate the ADM efflux ability of cells using flow cytometry. (* P < 0.05, ** P < 0.01, comparing to their negative control, respectively).

Figure 3

Overexpression of miR-1246 could improve the chemo-resistant ability of drug-sensitive leukemia cells. A, miR-1246 was checked using RT-qPCR after K562 and HL-60 cells transfected with miR-1246 mimics or NC mimics (* P < 0.05, comparing to their negative control, respectively). B, MTT assay was used to examine the cell survival and proliferation rate of miR-1246 mimics or NC mimics transfected drug-sensitive cells treated with different concentration of ADM (* P < 0.05, ** P < 0.01, comparing to their negative control treated with the same concentration of ADM, respectively). C, After the cells were stained with Annexin V-FITC and PI, flow cytometry was used to detect their cell apoptotic rate (* P < 0.05, comparing to their negative control. # P < 0.05, comparing to their negative control treated with the same concentration of ADM). D, ADM efflux ability was measured using flow cytometry after the cells were incubated with ADM (* P < 0.05, ** P < 0.01, comparing to their negative control, respectively).

Figure 4

Animal studies were performed and the results were analyzed. A, The weight of mice in all groups were recorded and compared. B and C, The tumors dissected from all groups were photographed and the statistics results of their weights were analyzed. D, The difference of radiance recorded from the tumor of different groups' mice.

Figure 5

miR-1246 regulated drug-sensitivity of leukemia cells by targeting AXIN2 and GSK-3β and further influencing P-gp activity. A, The mRNA expression level of AXIN2 and GSK-3β in K562/ADM and HL-60/RS cells was analyzed comparing to their chemo-sensitive parental cells (a, b, c, d). B, The protein immunoblotting was used to test the expression of AXIN2 and GSK-3β in K562, K562/ADM and HL-60, HL-60/RS cells. C, Comparing to NC inhibitor transfected chemo-resistant leukemia cells, mRNA (a and b) and protein (c) level of AXIN2 and GSK-3β in miR-1246 inhibitor transfected chemo-resistant leukemia cells were analysed (* P < 0.05, comparing to their negative control, respectively). D AXIN2 and GSK-3β were detected using RT-qPCR and western blot in miR-1246 mimics and NC mimics transfected chemo-sensitive leukemia cells. E, Position 313-320 of AXIN2 3'UTR and position 510-516 of GSK-3β 3'UTR were identified as potential targets of miR-1246. F, Dual luciferase reporter assay was performed to verify the interaction between miR-1246 and AXIN2 or miR-1246 and GSK-3β. pRL-TK renilla luciferase plasmid was co-transfected for normalization (* P < 0.05, comparing to their negative control, respectively). E, Protein immunoblotting assay was used to check the differences of multidrug resistant protein (P-gp) and key factors in Wnt/β-catenin pathway and its downstream factors with or without miR-1246 inhibitor transfection.