Roles of hsa-miR-12462 and SLC9A1 in acute myeloid leukemia

To the Editor,

MicroRNAs (miRNAs) play important roles in cell proliferation, differentiation, and survival and may be useful for acute myeloid leukemia (AML) diagnosis and prognosis [1‐4]. In this study, we defined a novel miRNA, hsa-miR-12462, through small RNA sequencing of the bone marrow (BM) cells from 128 newly diagnosed subjects with AML (Supplementary Table1-2). Based on 2016 World Health Organization (WHO) criteria, all subjects were grouped into 2 cohorts: (1) those achieving a complete remission (CR) with conventional induction chemotherapy and remaining in CR ≥ 6 months and (2) those not achieving CR after 2 courses of standard induction chemotherapy (refractory) or relapsed in < 6 months after CR (relapsed) [5]. Small RNA sequencing of these samples revealed different miRNA expression profiles between CR and refractory/relapsed (RR) AML patients [6]. One miRNA showed the highest differential expression pattern in this analysis. This miRNA has never been reported in the literature. We named this miRNA as hsa-miR-12462 (Figure S1A). Next, we explored the biological activity of hsa-miR-12462 by overexpressing it in AML cells using a lentiviral vector (Figure S1B). The growth rate of the hsa-miR-12462 overexpressing cells was significantly decreased when compared with those of the wild-type and MOCK controls in both U937 and HL-60 cells (Fig. 1a, Figure S1C). U937 cells were confirmed using an EdU incorporation assay (Figure S1D-E).

Treatment of AML cells with cytarabine for up to 48 h resulted in a lower proliferation of the hsa-miR-12462 overexpressing cells when compared with that of the controls (Fig. 1b, c, Figure S1F-G). The apoptosis rate of the hsa-miR-12462 overexpressing cells was significantly higher than that of the control (Fig. 1d, e, Figure S1I,L) as well. These differences were not seen in U937 cells (Figure S1H,J) or HL-60 cells (Figure S1K) treated with doxorubicin. We hypothesized that these different responses might reflect the effects of hsa-miR-12462 on the cell cycle. Cell cycle analysis revealed that a greater proportion of hsa-miR-12462-expressing cells were adjusted in G₀/G₁ and S-phase when compared with MOCK-transfected cells (Fig. 1f, Figure S1M).

We further studied the biological behavior of the hsa-miR-12462 overexpressing U937 cells in a subcutaneous xenograft mouse model (Fig. 1g). Tumor weight and size in the overexpressing cohort were decreased when compared with those of the MOCK-transfected cohort (Fig. 1h, i). (MOCK vs. OE: P = 0.0057). These differences were further confirmed by both magnetic resonance imaging (MRI; Fig. 1j) and histopathology (Figure S1N-P).

Through RNA-sequencing analysis of hsa-miR-12462 overexpressing and MOCK-transfected U937 cells, 306 genes were identified with differential expressions (Fig. 2a, S2A). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were done as well (Figure S2B-C). Enrichment analysis of the KEGG pathway indicates the involvement of the cAMP signaling pathway (Figure S2C) [7]. In addition, CCNE1, E2F4, and TP53 involved in cell cycle regulation were found significantly downregulated in the RNA-sequencing analysis (Figure S2A). Q-RT-PCR analysis of these genes confirmed the results from the RNA-sequencing analysis (Fig. 2b–d). Combining the data from the MiRDB, an online microRNA database (http://​mirdb.​org) [8], with the RNA-sequencing results, 15 genes were found to share predicted targets (Fig. 2e, f) including SLC9A1, ARRB1, and CHRNA6 (Fig. 2g). SLC9A1 (NHE1), the most common isomer in the Na⁺/H⁺ exchanger family [9], is important in cell transformation [10]. β-arrestins (ARRBs) participate in mediating tumor proliferation and inflammation-induced cancer development [11], whereas nicotinic acetylcholine receptors (CHRNs) are important regulators of tobacco-induced carcinogenesis [12]. The mRNA and protein levels of SLC9A1, ARRB1, and CHRNA6 were inhibited by overexpression of hsa-miR-12462 (Fig. 2h, i, S2D-E). Using a luciferase 3′UTR reporter assay, we found that hsa-miR-12462 bound exclusively to the 3′UTR of SLC9A1 in U937 cells (Fig. 2j; S2F-G). In summary, a higher level of hsa-miR-12462 in AML cells is associated with increased sensitivity to cytarabine chemotherapy via downregulation of SLC9A1.