Molecular Mechanism Of Synthetic Microrna Underlying Chronic Myeloid Leukaemia Cells Proliferation By Targeting The 3’UTR Of ABL1 Component Of The BCR-ABL1 Fusion Gene

Chronic myeloid leukaemia (CML) is a hematologic malignancy driven by the BCR-ABL1 fusion gene, leading to uncontrolled proliferation of myeloid cells. Despite advancement in treatment, resistance to tyrosine kinase inhibitors (TKIs) remains a critical challenge. This study investigates the molecular mechanisms of synthetic microRNAs (miRNAs) in regulating cell proliferation by targeting the 3’ untranslated region (UTR) of the ABL1 as the components of the BCR-ABL1 fusion gene. In-silico analyses using DIANATOOLS, psRNATarget, RNA22, and TargetRank, identified three candidate miRNAs namely, hsa-miR-3131, hsa-miR-891a-3p, and osa-miR1858a/b. These miRNAs were further explored via network and pathway analyses using STRING and Cytoscape to predict their broader cellular impact. Through in-vitro, miRNA transfection efficiency and miRNA-mRNA interactions were determined in K562-s (sensitive) and K562-r (resistant) CML cell lines using TaqMan assays and biotin miRNA pull-downs. Gene expression of ABL1 and BCR-ABL1 post-transfection was assessed by qPCR, while cell viability and cell cycle were measured via MTS assays and flow cytometry, respectively. Additionally, ABL1 protein expression was evaluated through a colorimetric enzyme-linked immunosorbent assay (ELISA), and microarray analysis was performed to identify differentially expressed genes (DEGs) affected by miRNA transfection. Results indicated effective transfection, with osa-miR1858a/b exhibiting high expression in k562-r cells and hsa-miR-3131 in k562-s cells, suggesting cell-type-specific transfection efficiency. The miRNA pull-down assay confirmed binding interactions of hsa-miR-891a-3p and hsa-miR-3131 with target mRNA in k562-r but not in k562-s cells. Notably, Mann-Whitney test shows that hsa-miR-3131 significantly downregulated target BCR-ABL1 in k562-s cells (p<0.05), while no substantial downregulation occurred in k562-r cells. Despite this, no significant changes in ABL1 protein levels were observed post-transfection. Functionally, cell viability of k562-r cells was lowered by over 50.5% ± 1.53, 63.4% ± 0.25 and 61.3% ± 0.23 when transfected with osa-miR1858a/b, hsa-miR-3131 and hsa-miR-891a-3p respectively compared to the control group (p<0.05). Nevertheless, the viability of k562-s cells was decreased to 84.45% ± 0.673 (hsa-miR-3131), 81.45% ± 0.816 (hsa-miR-891a-3p), and 83.86% ± 0.733 (osa-miR-1858a) with p<0.05 compared to the control groups. Additionally, osa-miR1858a/b and hsa-miR-891a-3p induced a cell cycle arrest at the G2/M phase in k562-r cells, with 6.5% ± 0.9 and 8.6% ± 1.1 respectively, compared to the control group (p< 0.05). Furthermore, osa-miR1858a/b induced a cell cycle arrest at the S and G2/M phases in k562-s cells, with 59.74% ± 1.34 and 7.80 ± 0.58, respectively, compared to controls (p<0.05). Microarray analysis revealed no (DEGs) between miRNA-treated and control groups; however, selected genes relevant to CML were further validated by qPCR. Notably, osa-miR1858a/b significantly downregulated IL6 and MAP4K1 in K562-s and K562-r cells, respectively. Both genes are involved in proliferation-related pathways such as MAPK and JAK/STAT. These findings highlight the exceptional ability of the plant-based miRNA osa-miR1858a/b in decreasing cell viability and inducing cell cycle arrest, as well as downregulating key genes such as IL6 and MAP4K1, demonstrating inter-kingdom regulatory potential in-vitro. This research provides novel insights into the therapeutic potential of synthetic miRNAs in CML treatment, emphasizing the unique role of hsa-miR-3131, hsa-miR-891a-3p and especially osa-miR1858a/b in disrupting essential proliferative signalling cascades of CML.