AML-₄₉₉ Long Non-Coding RNA HOTAIRM1 Plays a Role in Paediatric AML Pathogenesis Potentially via miR-₂₂₂ Mediated Regulation of Proliferation and Apoptosis

Abstract

Context Long non-coding RNAs (lncRNAs) regulate leukemic cell proliferation as well as apoptosis and play a role in paediatric acute myeloid leukemia (AML) pathogenesis via unknown mechanisms. Investigating this axis of lncRNA-AML pathogenesis would open the possibility of novel and improved therapeutic interventions. Objective To study the role of long non-coding RNA HOTAIRM1 in paediatric AML pathogenesis. Design/Methods We prospectively enrolled paediatric AML patients (n=48) and collected bone marrow aspirate samples. A control group with normal bone marrow (n=10) was included. Additionally, we conducted an experimental study using six AML cell lines (THP-1, HL-60, KG-1, Kasumi-1, MOLM-13, and MOLM-14) cultured in RPMI-1640 media supplemented with 10% fetal bovine serum. HOTAIRM1 expression was quantified using real-time PCR. HOTAIRM1 was cloned into pcDNA3.1+ and transfected using a cationic lipid-based method. Cell cycle and apoptosis were analysed using flow cytometry (7-AAD and Annexin-7-AAD methods, respectively). Predicted downstream microRNA targets of HOTAIRM1 were identified using DIANA-LncBase and RNAHybrid. The microRNA binding region on HOTAIRM1 was cloned into pmirGLO vector (pmirHM1), and a dual-luciferase assay was performed to study the interactions. Results HOTAIRM1 expression was significantly decreased in paediatric AML patients (P=0.0005) and six AML cell lines (KG-1, P=0.002; THP-1, P=0.003; Kasumi-1, P=0.002; HL-60, MOLM-13, MOLM-14, P=0.001). Overexpression of HOTAIRM1 in AML cell lines caused cell cycle arrest in Kasumi-1 cells (P=0.0001), which harbour the t(8;21) translocation, but not in THP-1 and MOLM-13 cells with MLL-rearrangement. miR-222, a predicted microRNA target of HOTAIRM1, was significantly upregulated in AML cell lines (P<0.001) and paediatric AML patients (P<0.0001). Cotransfection of pmirHM1 and miR-222 mimic in HEK293T cells led to decreased luciferase activity (P<0.01), indicating an in vitro interaction. Moreover, inhibition of miR-222 in a paediatric AML cell line resulted in cell cycle arrest (P<0.01) and increased apoptosis (P<0.01). Conclusion Patients with AML had significantly lower HOTAIRM1 expression, and its overexpression induces cell cycle arrest in Kasumi-1 cells, suggesting a potential tumor suppressor role. HOTAIRM1 interacts with miR-222 through complementary base pairing, and miR-222 inhibition reduces AML cell proliferation while promoting apoptosis, indicating the involvement of the HOTAIRM1-miR-222 regulatory network in AML proliferation and apoptosis pathways.