HOTAIRM1 knockdown enhances cytarabine-induced cytotoxicity by suppression of glycolysis through the Wnt/β-catenin/PFKP pathway in acute myeloid leukemia cells

https://doi.org/10.1016/j.abb.2019.108244Get rights and content

Highlights

  • Knockdown of HOTAIRM1 enhances Ara-C cytotoxicity.

  • Knockdown of HOTAIRM1 suppresses glycolysis and PFKP expression.

  • Knockdown of HOTAIRM1 inactivates the Wnt/β-catenin pathway.

  • β-catenin overexpression counteracts the effect of HOTAIRM1 knockdown on glycolysis and PFKP expression.

  • β-catenin overexpression attenuates the effect of HOTAIRM1 knockdown on Ara-C cytotoxicity.

Abstract

HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a long non-coding RNA (lncRNA) that is highly specific for maturing myeloid cells. Dysregulation of HOTAIRM1 has been found to be implicated in the development of acute myeloid leukemia (AML). However, the role of HOTAIRM1 in the drug resistance in AML remains unknown. The present study aimed to investigate the effect of HOTAIRM1 on the cytarabine (Ara-C) resistance in leukemia cell lines and to explore the underlying mechanism. The leukemia cell lines, HL60 and THP-1, were transfected with HOTAIRM1 specific siRNA (si-HOTAIRM1) or control siRNA (si-ctrl), and then treated with Ara-C for 48 h. The mRNA levels of HOTAIRM1 and platelet-type phosphofructokinase (PFKP) were measured using RT-PCR. Cell viability was evaluated by MTT assay. Apoptosis was determined using flow cytometry and caspase-3/7 activity assay. Glycolysis was evaluated by determining the glucose consumption and lactate production. To activate the Wnt/β-catenin signaling pathway, HL60 and THP-1 cells were transfected with β-catenin overexpressing plasmid (pcDNA-β-catenin). Protein levels of PFKP, β-catenin, and c-Myc were examined using western blot analysis. The results showed that knockdown of HOTAIRM1 enhanced Ara-C-induced reduction of cell viability and increase of cell apoptosis. HOTAIRM1 knockdown suppressed the glucose consumption and lactate production, as well as the expression of PFKP in AML cells. Besides, HOTAIRM1 knockdown resulted in a significant inhibitory effect on the Wnt/β-catenin pathway. Furthermore, activating Wnt/β-catenin pathway mitigated the effects of HOTAIRM1 knockdown on glycolysis and Ara-C cytotoxicity in AML cells. In conclusion, knockdown of HOTAIRM1 enhanced Ara-C cytotoxicity through regulating the Wnt/β-catenin/PFKP signaling pathway. These findings suggested that HOTAIRM1 might be a therapeutic target for overcoming the Ara-C resistance in AML.

Introduction

Acute myeloid leukemia (AML) is the most common acute forms of leukemia with a poor long-term survival [1]. AML is characterized by the rapid growth of immature myeloid cells and suppression of normal hematopoietic cells in the bone marrow, peripheral blood and other tissues [2]. Prognosis of AML is poor with 5-year survival rates ranging from 5 to 65% depending on demographic and clinical features [3]. In recent years, the first-line treatment of AML consists primarily of chemotherapy. Treatment for AML has remained cytarabine (Ara-C) and an anthracycline (most often daunorubicin) given in the standard 3 + 7 regimen [4]. The Ara-C is given as a continuous IV infusion for seven days, while the anthracycline is given as an IV push for three consecutive days. However, acquired resistance to Ara-C is a major challenge in the clinical management of AML [5]. Therefore, a novel therapeutic approach enhancing the Ara-C cytotoxicity is required for complete cure of AML.

It has been demonstrated that aberrant expression of various non-coding RNAs, such as micro-RNAs (miRNAs) and long non-coding RNAs (lncRNAs) is proved to be of seminal importance in the pathogenesis of AML and the development of resistance to chemotherapy [[6], [7], [8], [9]]. The non-coding RNAs act as key players in the regulation of both physiologic and pathologic gene expression profiles. HOX antisense intergenic RNA myeloid 1 (HOTAIRM1) is a lncRNA that is highly specific for maturing myeloid cells. HOTAIRM1 has been found to regulate cell cycle progression during myeloid maturation in NB4 human promyelocytic leukemia cells [10]. Besides, HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway, indicating that HOTAIRM1 may be the potential therapeutic target for leukemia [11]. However, the role of HOTAIRM1 in the Ara-C cytotoxicity remains unknown. In the present study, we investigated the effect of HOTAIRM1 on the Ara-C cytotoxicity in leukemia cell lines and explored the underlying mechanism.

Section snippets

Cell culture and treatments

The AML cell lines HL60 and THP-1 were purchased from American Type Culture Collection (Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 100 μg/ml streptomycin (Solarbio, Beijing, China) and 100 U/ml penicillin (Solarbio). Cells were grown in humidified air with 5% CO2 at 37 °C. For some experiments, cells were incubated with different concentrations of Ara-C (Sigma-Aldrich, St. Louis, MO, USA).

Knockdown of HOTAIRM1 enhances Ara-C cytotoxicity

We have previously showed that HOTAIRM1 was overexpressed in AML patients than healthy controls [12]. To investigate the role of HOTAIRM1 in AML cells, HL60 and THP-1 cells were transfected with si-HOTAIRM1 to knock down HOTAIRM1. As shown in Fig. 1A, the expression of HOTAIRM1 was decreased after transfection with si-HOTAIRM1 as compared to cells transfected with si-ctrl. HL60 and THP-1 cells were incubated with different concentrations of Ara-C (0, 0.25, 0.5, 1, or 2 μM) for 24, 48, and 72 h.

Discussion

HOTAIRM1 plays a key role during myeloid maturation and highly expresses in AML patients. The higher expression level of HOTAIRM1 is associated with worse prognosis of HOTAIRM1 patients. HOTAIRM1 has been identified as an independent prognostic factor in a large series of intensively treated IR-AML patients [15]. These findings suggest that HOTAIRM1 might play a crucial role in AML leukemogenesis and outcome. Ara-C is a chemotherapy medication that is clinically used to treat AML, chronic

CRediT authorship contribution statement

Li Chen: Conceptualization, Investigation, Data curation, Writing - original draft. Ning Hu: Investigation, Methodology, Formal analysis, Writing - review & editing. Chao Wang: Methodology, Formal analysis, Software. Hongmian Zhao: Conceptualization, Project administration, Supervision, Writing - review & editing.

Declaration of competing interest

All authors declare no conflict of interest.

References (29)

  • N.M. Ghahhari et al.

    Interplay between microRNAs and WNT/β-catenin signalling pathway regulates epithelial-mesenchymal transition in cancer

    Eur. J. Cancer

    (2015)
  • M.R. O'Donnell et al.

    Acute myeloid leukemia, version 3.2017, NCCN clinical practice guidelines in oncology

    J. Natl. Compr. Cancer Netw.

    (2017)
  • H. Dohner et al.

    Acute myeloid leukemia

    N. Engl. J. Med.

    (2015)
  • J.A. Barker et al.

    Successful use of high-dose cytarabine in a patient with acute myeloid leukemia and severe hepatic dysfunction

    J. Oncol. Pharm. Pract.

    (2016)
  • J.F. Bishop

    The treatment of adult acute myeloid leukemia

    Semin. Oncol.

    (1997)
  • F. Ferrara et al.

    Current therapeutic results and treatment options for older patients with relapsed acute myeloid leukemia

    Cancers (Basel)

    (2019)
  • A. Zebisch et al.

    Therapeutic resistance in acute myeloid leukemia: the role of non-coding RNAs

    Int. J. Mol. Sci.

    (2016)
  • M.M. Gabra et al.

    microRNAs and acute myeloid leukemia chemoresistance: a mechanistic overview

    Front. Oncol.

    (2017)
  • M. Bill et al.

    Expression and functional relevance of long non-coding RNAs in acute myeloid leukemia stem cells

    Leukemia

    (2019)
  • B.C. Medeiros

    Long non-coding RNAs: another brick in the wall of normal karyotype acute myeloid leukemia?

    Haematologica

    (2017)
  • X. Zhang et al.

    Long intergenic non-coding RNA HOTAIRM1 regulates cell cycle progression during myeloid maturation in NB4 human promyelocytic leukemia cells

    RNA Biol.

    (2014)
  • Z.H. Chen et al.

    The lncRNA HOTAIRM1 regulates the degradation of PML-RARA oncoprotein and myeloid cell differentiation by enhancing the autophagy pathway

    Cell Death Differ.

    (2017)
  • N. Hu et al.

    LncRNA HOTAIRM1 is involved in the progression of acute myeloid leukemia through targeting miR-148b

    RSC Adv.

    (2019)
  • A. Vallee

    Aerobic glycolysis activation through canonical WNT/β-catenin pathway in ALS

    Med. Sci. (Paris)

    (2018)
  • Cited by (0)

    View full text