A decrease in cellular microRNA-27a content is involved in azacytidine-induced P-glycoprotein expression in SKM-1 cells

Highlight

• MDR SKM-1/AzaC cell variant by long-term passaging with AzaC was established.

• Massive overexpression of P-gp in SKM-1/AzaC cells was observed.

• Downregulation of miRNA-27a in SKM-1/AzaC cells was detected.

• Downregulation of P-gp by transfection of these cells with miRNA-27a was observed.

Abstract

We established an azacytidine (AzaC)-resistant human acute myeloid leukemia (AML) cell line (SKM-1/AzaC) by culturing SKM-1 cells in the presence of increasing amounts of AzaC for six months. Because AzaC is not a substrate of P-glycoprotein (a product of the ABCB1 gene; ABCB1), ABCB1 was not responsible for AzaC resistance; nevertheless, it was notably upregulated in SKM-1/AzaC cells. In addition, the transcription of the Nfkb1 gene, which encodes a member of the canonical NF-kappaB regulatory pathway, was downregulated, and the transcription of the Nfkb2 gene, which encodes a member of the non-canonical NF-kappaB regulatory pathway, was upregulated in SKM-1/AzaC cells. Here, we investigate whether miRNA-27a and miRNA-138 (both of which are known to be regulators of ABCB1 expression) are involved in the regulation of ABCB1 expression in SKM-1/AzaC cells. We observed decreased levels of miRNA-27a but of not miRNA-138 in SKM-1/AzaC cells compared with SKM-1 cells. The transfection of SKM-1/AzaC cells with a miRNA-27a mimic induced the downregulation of the ABCB1 mRNA. This was associated with an increase in Nfkb1 and a decrease in Nfkb2 transcript levels in SKM-1/AzaC cells. Taken together, these data indicate that the downregulation of miRNA-27a is involved in the upregulation of ABCB1 expression in SKM-1/AzaC cells, and this effect is associated with a switch between the canonical and non-canonical NF-kappaB pathways.

Introduction

Myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) are characterized by the presence of CD33-positive immature myeloid cells in the bone marrow and peripheral blood (Jilani et al., 2002). The activation of the canonical NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) regulatory pathway is typically observed in the myeloblasts of high-risk MDS and AML patients and is thought to be associated with the downregulation of apoptosis that is induced by anti-cancer substances (Grosjean-Raillard et al., 2009).

The cytosine analogue azacytidine (AzaC), which was described as a cancerostatic agent in 1964 (Sorm et al., 1964), can be incorporated into RNA and DNA and can block DNA methylation (Griffiths and Gore, 2008). AzaC was proven to have beneficial effects in the treatment of MDS (Buckstein et al., 2011) and AML (Ivanoff et al., 2013). It not only inhibits the replication of DNA methylation traces during cell cycle progression, but it also disrupts nucleic acid and protein metabolism by incorporating into mRNA, tRNA and rRNA, leading to apoptosis (Stresemann and Lyko, 2008). Recently, AzaC was shown to reduce the expression of the M2 subunit of ribonucleotide reductase, the enzyme that provides the deoxyribonucleotides required for DNA synthesis and repair (Aimiuwu et al., 2012). Although AzaC was effective in the treatment of MDS and AML patients, some patients developed resistance to the drug (Fenaux et al., 2009, Fenaux et al., 2010). AzaC resistance worsens the prognosis of patients with high-risk MDS or AML that developed from MDS (Prebet et al., 2011, Prebet et al., 2012). Because the mechanisms underlying this resistance have not been fully elucidated, we performed molecular studies of this phenomenon using leukemia cell lines that had been selected for AzaC resistance by passaging in medium containing a clinically relevant concentration of the drug. We derived AzaC-resistant SKM-1/AzaC cells from the human AML cell line SKM-1 using this method (Messingerova et al., 2015). The resistant SKM-1 variants differ from the parental cells because they display a massive elevation in P-glycoprotein expression and efflux activity that confers cross-resistance to P-glycoprotein substrates. P-glycoprotein, a product of the ABCB1 gene, is a plasma membrane drug transporter (ABCB1, a member of the ABC transporter family) with broad substrate specificity that is responsible for reduced cellular sensitivity to large groups of structurally unrelated compounds (reviewed in Breier et al., 2005, Breier et al., 2013). Although AzaC is not an ABCB1 substrate, it is able to induce ABCB1 expression in SKM-1 cells and in another human AML cell line, MOLM-13 (Messingerova et al., 2015). The mechanism of this induction is not understood, but it may involve the effects of AzaC on microRNA (miRNA) regulatory activity, the methylation of the promoters of ABCB1 and other regulatory proteins active in drug resistance, or the function of different nuclear receptors involved in the regulation of the expression of specific proteins that protect cells against chemical stress. Here, we investigate whether miRNA-27a and miRNA-138, both of which are known to reduce the post-transcriptional processing of ABCB1 mRNA (Feng et al., 2011, Katayama et al., 2014, Zhao et al., 2010), are involved in regulating ABCB1 expression in SKM-1/AzaC cells.