This study was approved from the Institutional Review Boards of the Institute of Hematology and Blood Disease Hospital and informed consent was from each patient according to the revised Declaration of Helsinki. Cell lines and thymocytes The human T-ALL cell lines CCRF-CEM, KOPT-K1, MOLT4, JURKAT, LOUCY, SUPT1 and the 293T cell collection were purchased from American Type Tradition Collection (Manassas, VA, USA) and recently identified by DNA fingerprint. represent a DNA-binding website (DBD) whereas the last two zinc-fingers are components of a dimerization website; the latter allows competitive binding between isoforms.8 These domains are encoded by seven different exons, and differential splicing produces different isoforms. Ikaros isoforms that display at least three zinc-fingers in the DBD are considered dominating positive (DP, IK1-3), whereas Ikaros isoforms with less than three zinc-fingers in the DBD are considered dominant bad (DN, IK4-9). DN isoforms are not only defective typically due to decreased/no DNA binding capacity but also may interfere with the activity of practical isoforms. Mice with the heterozygous loss of Ikaros rapidly develop T-cell leukemia.9, 10 microRNAs (miRs) are short noncoding RNAs of 20C22 nucleotides that function to regulate gene expression in the posttranscriptional level. miRs play fundamental tasks in the rules of cellular proliferation, differentiation, and apoptosis. miRs are dysregulated in many types of malignancy, including T-ALL. miRs can function as oncogenes, favoring the initiation and progression of cancers, or as tumor suppressors, avoiding tumorigenesis.11C29 The biological functions of miRs in T-ALL are largely unknown. To better understand T-ALL pathogenesis and determine new therapeutic targets in T-ALL, we previously developed a knockout T-ALL mouse model. 30 In IC-87114 this study, we profiled the miRs in the Pten deficient mouse T-ALL. miR-26b was shown to be aberrantly indicated. Recent studies possess implicated aberrant manifestation of miR-26b in several forms of non-hematopoietic malignancy.31C33 However, the expression level of miR-26b and its part in T-ALL is unfamiliar. In this study, we investigated the expression level of miR-26b in T-ALL, showed its aberrant manifestation, and IC-87114 studied the effects of its modified expression on human T-ALL cells. Materials and Methods Patient samples We obtained 27 bone marrow samples from newly diagnosed T-ALL patients, from 2009 to 2013, accessioned at the Institute of Hematology and Blood Disease Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, PR China. The median individual age was 26 years old (range 18C66). The median percentage of blasts in bone marrow was 92% (range, 80%C98%). The diagnosis of T-ALL in all cases was established on the basis of morphologic findings, and immunophenotypic, cytogenetic, and molecular data according to the World Health Business (WHO) classification and the National Comprehensive Malignancy Network (NCCN) guidelines. Mononuclear bone marrow cells were separated using Ficoll-Hypaque IC-87114 density gradient centrifugation and stored in liquid nitrogen. This study was approved by the Institutional Review Boards of the Institute of Hematology and Blood Disease Hospital and informed consent was obtained from each patient according to the revised Declaration of Helsinki. Cell lines and thymocytes The human T-ALL cell lines CCRF-CEM, KOPT-K1, MOLT4, JURKAT, LOUCY, SUPT1 and the 293T cell collection were purchased from IC-87114 American Type Culture Collection (Manassas, VA, USA) and recently recognized by DNA fingerprint. Two human postnatal normal thymocyte samples were provided by Dr. Andrew Weng (Terry Fox Laboratory, Canada). The mouse T-ALL cell lines (LPN248, LPN236, LPN228) were generated from mouse knock-out T-ALL models and LPN211 was generated from knock-out mice.30 The CCRF-CEM-FFluc cell line was obtained from Dr. Malcolm K. Brenner and was explained previously.34 The cell lines were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 10 mM L-glutamine. 293T cells were cultured in Dulbeccos Modified Eagle Itgb7 Media (DMEM) with 10% FBS. Cells were kept at 37C in 5% CO2 and tested without cytoplasm contamination. miRNA expression profiling RNA labeling and hybridization on miRNA microarray chips were performed as explained.35, 36 Briefly, 5 g of total RNA from each sample was biotin-labeled by reverse transcription using 5 biotin end-labeled random octomer oligo primers. Hybridization of biotin-labeled cDNA was carried out on a miRNA microarray chip (MD Anderson miRNA expression Bioarray Version 5), which contains 2300 miRNA probes, including 1400 human and 900 mouse miRNA genes, in duplicate. Hybridization signals were detected by biotin binding of a streptavidinCAlexa647 conjugate b using Axon Scanner 4000B (Axon Devices, Union City, CA). The images were quantified by GENEPIX 6.0 software.