Figure 2

miR-2909 targets 3′UTR of KLF4 in B-ALL but not T-ALL. (A-B) The sequence alignment of the miR-2909 binding site, located at 491-508 nucleotides within KLF4 3′UTR region in pediatric T-ALL (A) and B-ALL (B). The NCBI sequence is shown for comparison. (C) Endogenous expression of hsa-miR-2909 in indicated cancer cell lines representing high expression in HEK 293 cells and low expression in HeLa cells using qRT-PCR. (D) Schematics representing miRNASelect™ pMIR-GFP reporter vector construct containing KLF4 3′UTR and the miR-2909 target site from both B-ALL (pGFP-KLF4-3′UTR-B) and T-ALL (pGFP-KLF4-3′UTR-T) patients into the cloning site of the vector downstream of the green fluorescent protein (GFP) reporter gene under the control of CMV promoter. (E) Representative fluorescent microscopic images and FACS results of HEK 293 cells transfected with either control pGFP vector without 3′UTR insert or pGFP-KLF4-3′UTR-B reporter vector or pGFP-KLF4-3′UTR-T reporter vector following 48 h of transfection. (F) Percentage of cells expressing GFP in transfected cells with indicated constructs was calculated using flow cytometry. Each bar represents mean percentage ± S.D of cells expressing GFP of the experiment performed in triplicate; * P < 0.05 relative to control.