miR106a-363 Cluster Has Oncogenic Potential in Childhood T-Cell Acute Lymphoblastic Leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy originating from T-cell precursors and is characterized by high genetic, immunophenotypic, and clinical heterogeneity. MicroRNAs (miRNAs) belong to the class of small noncoding RNAs and are implicated in the regulation of hematopoiesis and in the development of leukemia. miRNAs control expression of their target genes at the post-transcriptional level by blocking translation of messenger RNAs (mRNAs) or promoting their degradation. Some miRNAs are encoded within clusters, giving rise to policistronic transcripts. Such miRNAs are co-expressed and may co-regulate the expression of genes involved in certain biological processes and pathways. In our recent study we performed miRNA profiling in pediatric T-ALL using Next-Generation Sequencing (Dawidowska M et al. Blood 2017; 130:1443) and identified miRNAs differentially expressed in T-ALL. The set of overexpressed miRNAs included, among others, miR-20b-5p, miR-363-3p and miR-92a-2-5p, belonging to a cluster of six miRNAs: miR-106a-363 (ChrXq26.2). miR-106a-363 cluster is a paralog of miR-17-92 cluster (Chr13q31.3), a prototypic oncogenic cluster of eminent importance in human hematopoietic cancers, with reported role in T-ALL pathogenesis (Mavrakis KJ et al., Nature Cell Biology 2010, 12:4). Despite the similarity of seed sequences between miRNAs from miR-17-92 and miR-106a-363 clusters, the significance of miR-106a-363 cluster in T-ALL remains to be elucidated. In this study we investigated the expression of the miR-20b-5p, miR-363-3p and miR-92a-2-5p in children with T-ALL, healthy donor thymocytes, normal bone marrow samples and 6 T-ALL cell lines. RT-qPCR analysis (TaqMan Advanced miRNA Assays; Thermo Fisher Scientific) confirmed overexpression of 2 miRNAs from cluster miR-106a-363 (miR-20b-5p and miR-363-3p) in children with T-ALL and in T-ALL cell lines, suggesting their oncogenic function. To predict potential target genes of overexpressed miRNAs belonging to miR106a-363 cluster, we applied 8 target prediction algorithms and pathway enrichment analysis. This revealed the enrichment of miR-20b-5p and miR-363-3p target genes in GO term: positive regulation of apoptosis. We further validated predicted miRNA-mRNA interactions (Dual Luciferase Reporter Assays; Promega) confirming the majority of them (e.g. PTEN, FBXW7, BCL2L11). Finally, we assessed the effect of mimicry/inhibition (miRVana, Thermo Fisher Scientific) of overexpressed miRNAs from miR-106a-363 cluster on proliferation, cell cycle distribution and apoptosis in 3 T-ALL cell lines. Overexpression of miR-20b-5p and miR-363-3p in CCRF-CEM, DND-41 and P12-Ichikawa cells resulted in increased proliferation and inhibited apoptosis. To summarize, in this study we showed that miRNAs belonging to miR-106a-363 cluster directly interact with mRNAs implicated in the regulation of apoptosis and that miR-20b-5p and miR-363-3p have pro-proliferative and anti-apoptotic effects in T-ALL cells in vitro. These results indicate that miR-106a-363 cluster may have an oncogenic role in the pathogenesis of T-ALL via suppression of pro-apoptotic genes.

Research funded by National Science Centre, Poland grants: 2014/15/B/NZ2/03394, 2017/25/N/NZ2/01132 and National Centre of Research and Development (NCRD) grant STARTEGMED3/304586/5/NCBR/2017.