LEF 1 induces DHRS 2 gene expression in human acute leukemia
T cell acute lymphoblastic leukemia (T ALL) is an aggressive disease resulting from the accumulation of genetic changes that affect the development of T cells. The precise role of lymphoid enhancer binding factor 1 (LEF1) in T ALL has been controversial since both overexpression and inactivating LEF1 mutations have been reported to date. Here, we have investigated the potential gene targets of LEF1 in human T cell leukemia cell line, Jurkat. We have successfully knocked down LEF1 in Jurkat cells using small interfering RNA (siRNA) technology and then compared the gene expression levels in the LEF1 knockdown cells with non targeting siRNA transfected and non transfected cells by employing microarray analysis. We identified DHRS2, a tumor suppressor gene, as the most significantly downregulated gene in LEF1 knockdown cells, and further confirmed its downregulation by real time quantitative polymerase chain reaction (qRT PCR) in mRNA and by western blotting in protein level. Our results revealed that DHRS2 is positively regulated by LEF1 in Jurkat cells which indicates the capability of LEF1 as a tumor suppressor, and together with previous reports, suggest that LEF1 exhibits a regulatory role in T ALL via not only its oncogenic targets but also tumor suppressor genes. un co r ct d p roo f Introduction T cell acute lymphoblastic leukemia (T ALL) is a malignancy associated with a significant risk of relapse and poor prognosis [1] T ALL represents approximately 25% of the adult and 15% of pediatric ALL patients [2] Although the prognosis of T ALL has gradually improved over the years due to the modern treatment protocols, resistance and relapse still remain major challenges in the treatment. Thus, our understanding of molecular pathogenesis and classification of the patients can improve treatment outcomes and thereby increase success rate [3, 4] Activating mutations in NOTCH1 or inactivating mutations in its negative regulator (FBXW7) occur in about 60% of T ALL cases [5 8] Activation of NOTCH signalling pathway cooperates with loss of p16/INK4A and p14/ARF. In addition, translocations in oncogenes, such as LIM only domain (LMO) genes, homeobox (HOX) genes, MYC and MYB, frequently place these genes under the control of strong T cell specific enhancers, thus causing aberrant overexpression [2, 5] Lymphoid enhancer binding factor 1 (LEF1) a downstream transcriptional regulator of the Wnt/b catenin pathway, regulates many cell cycle regulatory and cellular proliferation genes [9] LEF1 can also modulate gene transcription independently [10] Previous studies have shown that LEF1 plays a crucial role in normal hematopoiesis [9, 11] Defective pro B cell survival and proliferation have been shown in LEF1 knockout mice. Overexpression of LEF1 in bone marrow progenitors results in B lymphoblastic and acute myeloid lymphoma in recipient animals [11] In leukemia and solid tumors, abnormal changes in LEF1 expression have been reported in several studies [12 15] The findings on prognostic significance of LEF1 expression show inconsistency among previously reported studies. For example, LEF1 expression has been found to be associated with poor prognosis in adult precursor B cell acute lymphoblastic leukemia (preB ALL) and chronic lymphocytic leukemia (CLL) [14, 16, 17] while overexpression of LEF1 has been determined as a favorable prognostic factor in childhood ALL, acute myeloid leukemia (AML) [13, 18 20] Heretofore, many gene targets of LEF1 and their associated pathways have been identified. However, its precise role in T ALL has not been clarified yet. While some studies have shown an increased expression of LEF1 in both premalignant thymocytes and T ALL [16] others have reported the deletion of the LEF1 gene accompanied with NOTCH1, PTEN mutations, biallelic INK4A/ARF (CDKN2A) deletions, or activating PI3K or AKT gene mutations in TALL [16, 21, 22] These contradictory findings necessitate further studies to understand the molecular mechanism of LEF1 in T ALL. In this study, we have investigated LEF1 regulated genes in Jurkat, a well characterized human T acute lymphoblastic leukemia cell line that is widely used in variety of studies to understand T cell biology and T cell signalling. The aim of our study was to identify LEF1 regulated potentially critical genes as well as related molecular signalling pathways using Jurkat as model cells. Materials and Methods Cell Culture Jurkat cells were cultured at 37degC with 5% CO2 in RPMI 1640 medium (Lonza, Basel, Switzerland) containing 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany) 100 U/ml penicillin, 100 mg/ml streptomycin (Gibco, Thermo Fisher Scientific, Waltam, MA, USA) and 2mM L glutamine. LEF1 Small Interfering (siRNA) Transfection Jurkat cells were transfected with 100 nM LEF1 siRNA (Smartpool on target plus siRNA, un rre t d p oo f Dharmacon) which targets both long (transcript variant 1, NCBI ID: NM_016269.5) and short isoforms (transcript variant 2, 3, 4; NCBI IDs: NM_001130713.2, NM_001130714.2, NM_001166119.1, respectively) of LEF1 or 100 nM non targeting siRNA (Smartpool ontarget plus siRNA, Dharmacon) with HiPerFect transfection reagent (Qiagen GmbH, Hilden, Germany) according to the manufacturer's protocol and cultured for 24 and 48 hours. RNA Isolation Total RNA was isolated from Jurkat cells using the RNeasy Mini kit (Qiagen) in accordance with the manufacturer's instructions. RNA concentrations were measured using a spectrophotometer (NanoDrop ND 1000, Thermo Scientific, IL, USA) Real Time Quantitative PCR (qRT PCR) LEF1 siRNA knockdown and microarray results were confirmed by qRT PCR. Reverse transcription was performed using random hexamers, total RNA and the Transcriptor First Strand cDNA Synthesis Kit (Roche Life Science, Germany) following the manufacturer's manual. To quantitate the gene expression, primers specific to LEF1 gene, DHRS2 gene and housekeeping gene TATA binding protein (TBP) were designed. qRT PCR was performed using LightCycler 480 SYBR Green I Mix (Roche, Germany) and LightCycler 480 Instrument II (Roche Life Science, Germany) under following PCR conditions: 95 degC for 5 min, 95 degC for 20 s, 64 degC for 20 s and 72 degC for 15 s (45 cycles) Forward and reverse primers were (5' 3' as follows TBP forward: ACT TGA CCT AAA GAC CAT TGC AC and TBP reverse: CTT GAA GTC CAA GAA CTT AGC TGG; DHRS2 forward: CGA CTT CCT GGT GTG CAG and DHRS2 reverse: GTT CTC CAT GTA GGG CAG C; LEF1 forward TGG TGC AGC CAT CCC ATG and LEF1 reverse CGT GAT GGG ATA TAC AGG CTG ACC. Quantification was performed using the relative standard curve method. Each experiment was performed in triplicate. Gene expressions were normalized using the housekeeping gene TBP. Microarray Microarray experiments were performed using Affymetrix's GeneChip(r) 3' IVT Express Kit (Affymetrix, Santa Clara, CA) The sample preparation was conducted in accordance with the manufacturer's protocol. Fragmented end labelled cDNA was hybridized onto the Affymetrix's GeneChip(r) HG U133 Plus2.0 Array according to Affymetrix's standard procedure. After hybridization, the chip was stained and washed in a GeneChip Fluidics Station 450 (Affymetrix) and scanned by GeneChip Array scanner 3000 G7 (Affymetrix) Expression signals were extracted and normalized using the Expression Console (Affymetrix) applying the Robust Multichip Average (RMA) normalization method. The microarray expression data generated in this study is available at the NCBI's Gene Expression Omnibus (GEO; htt:/www.ncbi.nlm.nih.gov/geo/ database [23] under accession number GSE129917. Microarray data analysis Differential gene expression analyses were performed using limma package in R. A one way ANOVA was applied to the RMA expression values in order to determine whether genes were differentially expressed between three groups. A multiple testing correction was applied to the p values of the F statistics to adjust the false discovery rate [24] Expression level differences with p value (FDR corrected) 0.05 and a fold change 2 were considered significant. Morpheus (https:/software.broadinstitute.org/morpheus) was used for the heatmap visualization of gene expression level differences. The web based tool Database for Annotation, Visualization, and Integrated Discovery (DAVID) [25, 26] was used for the biological interpretation of differentially expressed genes. The identified genes were classified based on the Gene Ontology Resource [27] annotations and associated pathways were determined using the Kyoto Encyclopedia of Genes and Genomes (KEGG) [28] Protein isolation and western blotting u co rr cte pr oo f Western blotting was performed to detect LEF1 and DHRS2 protein expression in the cells. All protein samples were prepared from a pool of siRNA treated culture cells (three wells) which were homogenized and treated with RIPA lysis buffer system (Santa Cruz Biotechnology, Santa Cruz, CA, USA) on ice. b actin was used as an internal control. The protein concentrations were quantified using Pierce BCA Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA) A total of 15mg proteins were separated in 4 12% bis tris gels (Nupage Novex, Life Technologies, Bleiswijk, Netherlands) and then transferred onto a nitrocellulose membrane using i Blot Gel transfer Stacks (Novex, Life Technologies) After incubation with blocking buffer (5% BSA) for one hour at room temperature, western blotting was performed using primary antibodies against p53 (dilution, 1:100, DO 1 sc126 Santa Cruz) LEF1 (dilution, 1:250, sc8592 Santa Cruz) DHRS2 (dilution, 1:200, abcam, ab83254) and b actin (1:1000, I 19R sc1616K, Santa Cruz) by overnight incubation at 4degC. After washing step, the HRP conjugated secondary goat anti mouse antibody for p53 (1:3,000, ab97023, abcam) rabbit anti goat ab for LEF1 (1:2,000 abcam, ab6741) goat anti rabbit for b actin and DHRS2 (1:5,000, Abbkine A21020 1, Redlands, CA, USA) were added and inc