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Enhanced Apoptotic Effect of miR-222 Inhibition Combined with Cytarabine in HL-60 Acute Myeloid Leukemia Cells

Abstract

Background: Acute myeloid leukemia (AML) remains an aggressive hematologic malignancy in which resistance to apoptosis limits the efficacy of standard chemotherapy. MicroRNA-222 (miR-222) has been implicated in leukemic cell survival and proliferation, and its inhibition may sensitize AML cells to cytotoxic treatment. Objective: To evaluate whether inhibition of miR-222 enhances the anti-leukemic effect of cytarabine in HL-60 cells and to determine associated changes in apoptosis-related and AML-relevant gene expression. Methods: HL-60 cells were transfected with locked nucleic acid anti-miR-222 (LNA-anti-miR-222). Following pilot optimization of cytarabine concentration (IC50 1.8 μM) and LNA dose (50 pmol selected from 10-100 pmol titration), cells were assigned to five groups: untreated control, scrambled LNA control, cytarabine alone (1 μM), LNA-anti-miR-222 alone (50 pmol), and LNA-anti-miR-222 plus cytarabine. A 3×3 concentration-response matrix was also tested. Relative miR-222 expression was quantified by SYBR Green real-time PCR using U6 normalization. Cell viability was assessed by MTT assay at 72 hours. Apoptosis was evaluated by Annexin V/7-AAD flow cytometry at 48 hours. Expression of BAX, BCL-2, MCL-1, WT1, C-KIT, and CEBPA was measured by real-time PCR (GAPDH normalization). Statistical comparisons for qPCR were performed on ΔCt values. Results: LNA-anti-miR-222 effectively suppressed miR-222 expression in HL-60 cells (by approx. 74%, p<0.01). Cytarabine alone reduced cell viability to 68.3% and induced 19.4% total apoptosis, whereas miR-222 inhibition alone reduced viability to 51.6% and induced 27.5% apoptosis. The combination of LNA-anti-miR-222 and cytarabine produced the greatest reduction in viability (29.8%) and the highest apoptotic fraction (48.6%, p<0.001 vs either alone). A 3×3 concentration matrix confirmed the robustness of the enhanced effect across multiple doses, and two-way ANOVA revealed a significant interaction between the two agents (p=0.008). Combination treatment was associated with marked upregulation of BAX (4.1-fold), downregulation of BCL-2 (0.31-fold) and MCL-1 (0.52-fold), and substantial suppression of C-KIT expression (0.23-fold). WT1 showed a modest decrease (0.71-fold), whereas CEBPA did not change significantly. Conclusion: Inhibition of miR-222 enhanced the cytotoxic and pro-apoptotic activity of cytarabine in HL-60 cells across a range of concentrations. These findings support miR-222 as a potential therapeutic target in AML and suggest that miR-222 blockade may improve chemosensitivity, pending protein-level validation of the proposed mechanisms.

Keywords: acute myeloid leukemia; miR-222; cytarabine; apoptosis; HL-60; C-KIT; BAX; BCL-2
Copyright © 2025 Claudio Ponticelli. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.