Background

Acute lymphoblastic leukemia (ALL) is widely recognized as the most prevalent pediatric leukemia [1]; however, the genomic mechanisms responsible for the uncontrolled cell proliferation coupled with cell immortalization remain unknown [2]. In this context, the genes for apoptosis-antagonizing 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 also encodes the novel microRNA (miRNA) miR-2909, which regulates genes involved in inflammation, cell cycle, and immune response [4–6]. KLF4, a member of the SP1/KLF transcription factor family, is characterized by three highly conserved C2H2-type zinc-finger motifs at its carboxyl terminus, which are crucial for its interaction with its target DNA [7]. The KLF4 gene acts as both an oncogene and a tumor suppressor, depending on its genetic and cellular contexts [8]. The tumor-suppressive role of KLF4 and its involvement in regulating apoptosis, proliferation, and differentiation in B-cell malignancies suggest that KLF4 may play a critical role in leukemogenesis [34]. B- and T-cells were purified using MiniMACS™ Separator Kit.

RNA Extraction, cDNA synthesis and qRT-PCR

Total RNA including the small RNA was extracted from patient samples using miRNeasy mini kit in accordance with the manufacturer’s instructions. The quality and quantity of extracted RNAs were analyzed using electrophoresis and optical density measurement at 260 nm; cDNA synthesis was performed via miScript Reverse transcription kit as per suppliers’s instructions. For assaying gene expression, miScript SYBR Green Mix and the Real-time PCR (Stratagene, San Diego, CA, USA) were used. The qRT-PCR reaction was performed with a starting temperature of 95°C for 10 min, followed by 35 cycles of 45 s at 94°C, 30 s at 56°C, and 45 s at 72°C. The small non-coding nuclear RNA U6 and β-actin were used as an invariant controls for normalizing the expression of miR-2909 and other genes respectively. The 2-ΔΔCT method was used to calculate the relative expression of target genes.

Immunoblotting

Total cellular protein was extracted using Laemmli’s buffer [35] and the protein levels of KLF4, p21CIP, SP1, MYC and CCND1 was determined through western blotting using appropriate antibodies as described previously [36]. β-actin antibody was used as an internal control. Scion Image Analysis software was used for densitometry analysis and the results were expressed as intensity ratio of target protein to β-actin protein taken as arbitrary unit.

DNA sequencing

Primer sets were designed to amplify the full coding region and 3′untranslated region of KLF4 in ALL samples using Pfu polymerase. The resultant PCR products were purified using Qiaquick PCR purification kit and sequenced to detect the presence of any genetic aberration(s) in KLF4 in samples from pediatric patients with ALL. The sequence data was analysed using Cluster X 2.0.12 Software (http://www.clustal.org/clustal2) [37].

Plasmid constructs and reporter assays

Full length 3′UTR of KLF4 in B-ALL was cloned into miRNASelect™ pMIR GFP reporter vector; designated as pGFP-KLF4-3′UTR-B which carried no substitution of nucleotides within miR-2909 target site in KLF4 3′UTR. Mutant 3′UTR of KLF4 present in T-ALL was named as pGFP-KLF4-3′UTR-T with substitution of nucleotides within core binding site in KLF4 3′UTR. The plasmid constructs were transfected in HEK-293 cells. After 48 h, fluoresence microscopy and FACS analysis was performed to quantitate the number of cells expressing GFP. For p21CIP promoter analysis, promoter sequence of p21CIP with putative KLF4 binding site was cloned into pBlue TOPO reporter vector with subsequent transfection of β-gal construct into control and T- lymphoblasts. For analysis of SP1 transcriptional activity, B- and T-lymphoblasts with reporter plasmids containing SP1 response elements were transfected. β galactosidase activity was measured 72 h after transfection. To knockdown miR-2909 expression, leukemia cells were transfected with miRCURY LNA™ miR-2909 power inhibitor. To increase miR-2909 expression, HeLa cells were transfected with the PMIRH-2909 expression vector. All the transfections were performed with Lipofectamine 2000 transfection reagent according to the manufacturer’s instructions.

Cell cycle analysis and apoptosis assays by Flow cytometry

Cell cycle analysis and apoptotic assays was done on leukemia cells transfected with antagomiR-2909 (50 nM) and scrambled RNA (50 nM) for 48 h in RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin under 5% CO2 at 37°C. For cell cycle experiments, cells were fixed in 70% ethanol and stained with PI. Cells percentage at different phases were analysed with FACSCalibur cytometer and Cell Quest Pro software (Becton Dickinson, NJ, USA). For apoptosis assays, cells were stained with FITC Annexin V coupled with propidium iodide and apoptosis was measured using BD FACS Diva Software (Becton Dickinson, FACS Canto II).

KLF4 structural model & docking with target DNA

The structural models of zinc finger motifs of wild-type and mutant KLF4 were modeled with template PDB ID: 2WBUA. The Homology models were built using MODELLER (9.9) [38]. Model validation was performed using Verify-3D (http://nihserver.mbi.ucla.edu/Verify_3D/) [39] and PROCHECK [40]. The quality of the final models was evaluated from Ramachandran plot (Additional file 3: Figure S3B). Molecular visualization and structural alignment was done using CHIMERA http://bioinformatics.org/wiki/Chimera[41] and PYMOL http://www.pymol.org/[42]. Target DNA sequence (5′-cgggcggggc-3′) in p21CIP promoter was modeled into B-FORM using DNA analysis servers [43]. Docking studies were performed using High Ambiguity-Driven bimolecular Docking (HADDOCK) under solvated conditions [44, 45]. Cation-π interactions were analysed with CAPTURE http://capture.caltech.edu/[46].

Statistical analysis

Statistical analyses were performed by SPSS Windows version 19. Data was expressed as mean ± S.D of the experiments performed in triplicate. Student’s t test or Mann-Whitney-Wilcoxon test was performed to determine the significance of difference between two groups. Differences were considered significant at p < 0.01 and p < 0.05.