title: miR-2909-mediated regulation of KLF4: a novel molecular mechanism for differentiating between B-cell and T-cell pediatric acute lymphoblastic leukemias.
abstract: BACKGROUND
  microRNAs (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.
  
  
  METHODS
  Elevated 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), P21CIP, 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.
  
  
  RESULTS
  We 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 P21CIP 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).
  
  
  CONCLUSIONS
  This 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.
authors.forename: Deepti
authors.initials: D
authors.lastname: Malik
authors.affiliation: Department of Experimental Medicine & Biotechnology, Postgraduate Institute of Medical Education & Research, Chandigarh, India. dkaul_24@hotmail.com.
authors.forename: Deepak
authors.initials: D
authors.lastname: Kaul
authors.forename: Nalini
authors.initials: N
authors.lastname: Chauhan
authors.forename: Ram Kumar
authors.initials: RK
authors.lastname: Marwaha
mesh_terms: D000293:Adolescent; D049109:Cell Proliferation; D002648:Child; D002675:Child, Preschool; D003937:Diagnosis, Differential; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D057089:Pathology, Molecular; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology; D054218:Precursor T-Cell Lymphoblastic Leukemia-Lymphoma / Q000235:genetics* / Q000473:pathology; D011401:Promoter Regions, Genetic
chemical_list: C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C000589020:MIRN2909 microRNA, human; D035683:MicroRNAs