The Novel H19/miR‐675‐5p/LNC000093‐Mediated Non‐Coding RNA Pathway Regulates Treatment Response in BCR‐ABL1‐Positive CML Cells

Abstract

Introduction In recent years, considerable research has been done on the mechanisms of long non-coding RNAs (lncRNAs) modulating various cellular processes, especially in pathological condition such as tumorigenesis. Currently, however, little is known about the functional characteristics of these non-coding elements in the genome. In the present study, we aim to explore any mechanisms that involve novel lncRNA and microRNA (miRNA) axes participating in drug response of chronic myelogenous leukemia (CML). Methods Novel lncRNAs were identified via mRNA sequencing (RNA-seq) in CML cell lines with or without imatinib (IM) resistance. The sequence of a selected novel lncRNA candidate named LNC000093 was confirmed by cycle sequencing in several cell types. The expression level of LNC000093 in different cell lines and human samples was detected by RT-qPCR. In silico analysis and luciferase reporter assays were performed to identify miRNA binding regions on LNC000093. Knockdown and overexpression of H19/miR-675 were performed to examine their regulation on LNC000093 and cell viability upon IM treatment in CML cells. The potential interaction of H19/miR-675 and LNC000093 with RUNX1 was also investigated to study their interrelationship in IM-resistant of CML. Results A significant downregulation of newly identified LNC000093 was found in IM-resistant CML cells while an inverse expression pattern was observed in blood cells from IM-sensitive CML cases. Conversely, H19 and its derived miR-675 were upregulated in IM-resistant CML cells. Deletion of H19 led to upregulation of LNC000093 while miR-675-5p overexpression but not miR-675-3p reversed the elevated expression of LNC000093. In silico analysis revealed LNC000093 may contain three putative binding regions for miR-675-5p close to the 3’ end of its transcript. Subsequent luciferase reporter assays showed the interaction between LNC000093 and miR-675-5p via direct binding through the seed sequence GGUGCG on miR-675-5p. CRISPR-Cas9-mediated deletion of H19 caused downregulation of miR-675 and facilitated CML cell death upon IM treatment especially in IM-resistant cells, while overexpression of miR-675-5p reversed the enhancement of cell death in H19-deleted cells. Western blot assay revealed a downregulation of RUNX1 expression in IM-resistant cells. Overexpression of miR-675-5p could also result in RUNX1 downregulation and this was rescued by co-expression of LNC000093. Ectopic expression of LNC000093 could further increase RUNX1 expression and enhance cell death of IM-resistant cells. Conclusions Increased H19/miR-675-5p levels could contribute to IM resistance in CML cells through regulation of RUNX1 expression, and the newly identified lncRNA LNC000093 may serve as a ceRNA for miR-675-5p. This finding supports a possible linkage between the LNC000093-H19/miR-675-RUNX1 axis and IM resistance in CML.