Abstract
BackgroundmicroRNAs (miRNAs) play both oncogenic and oncostatic roles in leukemia. However, the molecular details underlying miRNA-mediated regulation of their target genes in pediatric B- and T-cell acute lymphoblastic leukemias (ALLs) remain unclear. The present study investigated the relationship between miR-2909 and Kruppel-like factor 4 (KLF4), and its functional relevance to cell cycle progression and immortalization in patients with pediatric ALL.MethodsElevated levels of miR-2909 targeted the tumor suppressor gene KLF4 in pediatric B-cell, but not pediatric T-cell ALL, as detected by pMIR-GFP reporter assay. Expression levels of genes including apoptosis-antagonizing transcription factor (AATF), MYC, B-cell lymphoma (BCL3), P21 CIP , CCND1 and SP1 in B- and T-cells from patients with pediatric ALL were compared with control levels using real-time quantitative reverse transcription polymerase chain reaction, western blotting, and reporter assays.ResultsWe identified two novel mutations in KLF4 in pediatric T-ALL. A mutation in the 3′ untranslated region of the KLF4 gene resulted in loss of miR-2909-mediated regulation, while mutation in its first or third zinc-finger motif (Zf1/Zf3) rendered KLF4 transcriptionally inactive. This mutation was a frameshift mutation resulting in alteration of the Zf3 motif sequence in the mutant KLF4 protein in all pediatric T-ALL samples. Homology models, docking studies and promoter activity of its target gene P21 CIP confirmed the lack of function of the mutant KLF4 protein in pediatric T-ALL. Moreover, the inability of miR-2909 to regulate KLF4 and its downstream genes controlling cell cycle and apoptosis in T-cell but not in B-ALL was verified by antagomiR-2909 transfection. Comprehensive sequence analysis of KLF4 identified the predominance of isoform 1 (~55 kDa) in most patients with pediatric B-ALL, while those with pediatric T-ALL expressed isoform 2 (~51 kDa).ConclusionsThis study identified a novel miR-2909-KLF4 molecular axis able to differentiate between the pathogeneses of pediatric B- and T-cell ALLs, and which may represent a new diagnostic/prognostic marker.
Highlights
MicroRNAs play both oncogenic and oncostatic roles in leukemia
Increased miR-2909 expression was always accompanied by significant downregulation of Kruppel-like factor 4 (KLF4) mRNA and protein in pediatric B-Acute lymphoblastic leukemia (ALL) compared with controls, indicating that miR-2909 may regulate the expression of KLF4 by targeting its 3′untranslated region (UTR) (Figure 1C and D)
To the best of our knowledge, the results provide the first evidence for two novel mutations in the KLF4 gene in T-cell lineage acute lymphoblastic leukemia (T-ALL): a mutation in the 3′UTR resulted in loss of miR-2909-mediated regulation, and mutation in the First zinc finger motif (Zf1)/Third zinc finger motif (Zf3) motif rendered KLF4 transcriptionally inactive
Summary
MicroRNAs (miRNAs) play both oncogenic and oncostatic roles in leukemia. the molecular details underlying miRNA-mediated regulation of their target genes in pediatric B- and T-cell acute lymphoblastic leukemias (ALLs) remain unclear. Acute lymphoblastic leukemia (ALL) is widely recognized as the most prevalent pediatric leukemia [1]; the genomic mechanisms responsible for the uncontrolled cell proliferation coupled with cell immortalization remain unknown [2]. In this context, the genes for apoptosisantagonizing transcription factor (AATF) and Kruppel like factor 4 (KLF4) have assumed importance. AATF provides a critical link between cell cycle progression, check-point control, and apoptosis [3], and encodes the novel microRNA (miRNA) miR-2909, which regulates genes involved in inflammation, cell cycle, and immune response [4,5,6]. KLF4 mRNA has been shown to be targeted by miR-130a and 135b in M1 acute myeloid leukemic blasts, and silencing of KLF4 arrested the maturation of blood cells at an early progenitor stage [10]
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