Role of long non-coding RNA HOTAIRM1 in the pathogenesis of paediatric acute myeloid leukaemia

Abstract

Background: The role of long non-coding RNA HOTAIRM1 in acute myeloid leukemia (AML) is unknown. We aim to study its expression and mechanism as we hypothesize that HOTAIRM1 is involved in the pathogenesis of paediatric AML. Methods: Six AML cell lines (Kasumi-1, THP1, HL-60, KG1, MOLM-13, MOLM-14) were cultured in 10% fetal bovine serum supplemented RPMI-1640 media. Bone-marrow samples from 26 paediatric AML patients and 10 normal marrows were included in the study. The expression of HOTAIRM1 was quantified using qPCR. Downstream microRNA targets of HOTAIRM1 were predicted using DIANA-LncBase and RNAHybrid. The microRNA binding region on HOTAIRM1 was cloned into the pmirGLO vector, and interaction was studied using dual-luciferase assay. Cell cycle analysis was performed by quantifying 7-AAD and apoptosis assay by the annexin-PI method using flow cytometry. Results: Expression of HOTAIRM1 transcript variant HM1V1 was downregulated in HL-60, MOLM-13, and MOLM-14 cell lines (p<0.05), whereas HM1V2 was downregulated in all cell lines (KG1, Kasumi-1, THP1, MOLM-13 p<0.01 and HL-60, MOLM14 p<0.001). In AML marrow samples, both variants were downregulated more than two-fold (HM1V1 p=0.0007, HM1V2 p=0.0023). Contrarily, the expression of predicted HOTAIRM1 microRNA targets were upregulated in AML cell lines (miR221-3p p<0.001, miR222-3p p<0.001), marrow samples (miR222 p<0.0001; miR221 p=0.0345) and peripheral blood samples (miR222, p=0.0005; miR221 p=0.431). Co-transfection of pmirHM1 and miR222 mimic in HEK293T cells decreased luciferase activity (p<0.01) demonstrating in vitro interaction. Additionally, inhibition of mir222 in a paediatric AML cell line led to cell cycle arrest (p<0.01) and increased apoptosis (p<0.01). Conclusions: The inverse expression of HOTAIRM1 and its predicted target, miR222 indicated a potential regulatory interaction between the two that was validated in-vitro. MiR222 acts downstream of HOTAIRM1 to promote cell cycle turnover of myeloblasts while inhibiting apoptosis thereby supporting our hypothesis for the role of HOTAIRM1-miR222 in the pathogenesis of paediatric AML.