6290 MOLECULAR MEDICINE REPORTS 16: 6290-6298, 2017 GATA4 is highly expressed in childhood acute lymphoblastic leukemia, promotes cell proliferation and inhibits apoptosis by activating BCL2 and MDM2 QIUGUO HAN1, XIN XU1, JING LI1, JINGGANG WANG1, LI BAI1, AIHONG WANG1, WEI WANG1 and BO ZHANG2 1Department of Pediatrics, Daqing Oilfield General Hospital, Daqing, Heilongjiang 163000; 2Department of Pediatric Neurology, The First Hospital of Jilin University, Changchun, Jilin 130000, P.R. China Received October 30, 2016; Accepted June 16, 2017 DOI: 10.3892/mmr.2017.7369 Abstract. Members of the GATA-binding factor protein lysine methyltransferase 2A, IKARS family zinc-finger 1, family, including GATA1, GATA2 and GATA3, serve an inhib- AT-rich interaction domain 5B, CCAAT/enhancer-binding iting role in leukemia. The present study demonstrated that protein and cyclin dependent kinase inhibitor 2A, may lead to GATA4 was upregulated in children with acute lymphoblastic the development of ALL (7,8). leukemia (ALL). Results from a number of functional experi- The GATA-binding protein family of zinc-finger tran- ments, including cell proliferation analysis, cell cycle analysis, scription factors comprises six members, including GATA1, cell apoptosis assay and Transwell migration and invasion GATA2, GATA3, GATA4, GATA5 and GATA6 (9), which analyses, have suggested that high expression of GATA4 may bind to GATA sequences in the DNA with the consensus facilitate proliferation and metastasis, and suppress apoptosis 5'-WGATAR-3', where W is either T or A nucleotides and R is in ALL cells. Chromatin immunoprecipitation assay and lucif- either G or A (10,11). GATA4 was originally revealed to serve erase reporter assay revealed that GATA4 was a transcription an important role in cardiac development (12,13); subsequent factor that activated mouse double minute 2 homolog (MDM2) studies have reported that GATA4 mediated apoptosis (14,15) and B cell lymphoma 2 (BCL2) expression in ALL cells. and regulated the mRNA expression of B cell lymphoma 2 BCL2 is a key anti-apoptosis protein that was demonstrated (BCL2) (16) and BCL‑XL (17), and serves as a survival factor to suppress cell apoptosis. In addition, GATA4 was revealed to in carcinoma. A number of GATA family proteins have been regulate p53 through the transcriptional activation of MDM2, revealed to serve inhibitory roles in leukemia, including subsequently influencing cell cycle and apoptosis. Results GATA1, GATA2 and GATA3 (18-21). However, the expression from the present study suggested that GATA4 may be a key and function of GATA4 in ALL remains unknown. marker in ALL diagnosis and a potential target of molecular The present study demonstrated that GATA4 expression therapy. was upregulated in ALL and binds to the promoter region of BCL2 and mouse double minute 2 homolog (Mdm2), activating Introduction their transcription. MDM2 protein expression was revealed to negatively regulate the expression of p53, thereby inhibiting Acute lymphoblastic leukemia (ALL) predominantly occurs in apoptosis and promoting cell cycle. In conclusion, the present children between the ages of 2 and 5 years, but may also occur results suggested that GATA4 may be a marker for ALL diag- in adults (1-4). Over the past decade, under different treatments, nosis and a target of molecular therapy. the survival rate of childhood ALL has approached ~90% (1,5); however, the treatment of adult ALL still needs improve- Materials and methods ment (2,6). The activation or inhibition of various genes, including Cell culture and transfection. MOLT-4 adult ALL, Jurkat childhood ALL cell lines and H9 normal human T lympho- cyte cell lines were purchased from American Type Culture Collection (Manassas, VA, USA) and cultured in RPMI-1640 Correspondence to: Dr Bo Zhang, Department of Pediatric Neurology, The First Hospital of Jilin University, 71 Xinmin Street, medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, Changchun, Jilin 130000, P.R. China USA) that contained 1% penicillin-streptomycin and 10% E-mail: [email protected] fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, 5 Inc.) at 37˚C with 5% CO2. Then ~4x10 cells were trans- Key words: GATA-binding factor 4, acute lymphoblastic leukemia, fected with pCMV-Tag 2B empty vector (Youbio, Hunan, apoptosis, B cell lymphoma 2, mouse double minute 2 homolog China) FLAG-GATA4 (pCMV-Tag 2B-GATA4) or small interfering RNA (siRNA) against scrambled (SCR), GATA4 siRNA (siGATA4) or siGATA4 and sip21 simultaneously HAN et al: GATA4 PROMOTES CELL PROLIFERATION AND INHIBITS APOPTOSIS IN CHILDHOOD ALL 6291 using Lipofectamine 2000 reagent (Invitrogen; Thermo 5'-TCA AAC TCC TGA CCT CAA GTG-3' and 5'-TTA CCA TCA Fisher Scientific, Inc.) according the manufacturer's protocol. TAA GCC TAC AGA CC-3'; p53, 5'-GAC CTA TGG AAA CTA Following 48 h transfection, cells were collected by 800 x g CTT CCT G-3' and 5'-CAT TCT GGG AGC TTC ATC TG-3'; p21, centrifugation at 4˚C for 5 min and used for the further experi- GCA TGA CAG ATT TCT ACC AC-3' and 5'-GAC TAA GGC ments. siRNA sequences were as follows: siGATA4#1, AAC AGA AGA TGT AGA G; GAD D45, 5'-CGA GAA CGA CAT CAA CGG CCG CTC ATC AAG CCT; siGATA4#2, AAT CTC GTA CAT CC-3' and 5'-ATG AAT GTG GAT TCG TCA CC-3'; 14-3-3σ, GAT ATG TTT GAC; sip21, AAC TTC GAC TTT GTC ACC 5'-AGA CAA CCT AAC ACT TTG GAC-3' and 5'-AGG AAA GAG. GAA GGA TGA CAC CC-3'; GAP DH, 5'-CAT TTC CTG GTA TGA CAA CGA-3' and 5'-TAC ATG GCA ACT GTG AGG AG-3'. Blood samples. A total of 42 blood samples from patients with Relative mRNA expression levels of indicated genes were ALL and 42 samples from healthy controls were collected from calculated by the 2-ΔΔCq method (22). GAPDH was used as patients at the First Hospital of Jilin University (Changchun, internal control and for normalization. Each experiment was China) between 2010 and 2016. All patients had been informed performed at least 3 times. about the nature of the experiments and provided written informed consent. The study was approved by the Ethics Cell Counting Kit‑8 (CCK‑8) proliferation assay. Following Committee of the First Hospital of Jilin University. 48 h transfection, cells (4x103 cells/well in 100 µl RPMI-1640 medium) were cultured in 96‑well plates at 37˚C. CCK‑8 solu- Western blot analysis. Cells (~4x106) were lysed with radio- tion (10 µl; Beyotime Institute of Biotechnology) was added to immunoprecipitation assay buffer (Beyotime Institute of each well and incubated at 37˚C for 3 h. A microplate reader Biotechnology, Haimen, China) containing a protease inhibitor was used to measure the absorbance at 450 nm; the absorbance cocktail (Beijing Transgen Biotech Co., Ltd., Beijing, China) indicated cells proliferation. Each experiment was performed at 4˚C for 30 min. Protein concentrations were measured using at least 3 times. the Bradford Reagent (Beyotime Institute of Biotechnology). A total of 40 µg protein was loaded in each well and resolved by Apoptosis assay. Adriamycin was purchased from Selleck 8% SDS-PAGE. Proteins were transferred to polyvinylidene Chemicals (Houston, TX, USA) and dissolved in phos- fluoride membranes, which were subsequently blocked with phate-buffered saline (PBS). Following 48 h transfection, 5% non‑fat milk at 37˚C for 1 h. The membranes were incu- Adriamycin (5 µg/ml) was added to the culture medium and bated with the following primary antibodies at 4˚C overnight: incubated for 24 h at 37˚C to induce apoptosis. Subsequently, anti-GATA4 (ab124265; 1:600) anti GADD45 (ab105060; apoptotic rates were examined with the Annexin V/fluorescein 1:3,000), anti-p21 (ab109520; 1:2,000), anti-14-3-3σ (ab14123; isothiocyanate (FITC) Apoptosis Detection kit (Abcam), 1:2,000) and anti-β-actin (1:1,000) from Abcam (Cambridge, according to the manufacturer's protocol. Briefly, approxi- MA, USA); and anti-FLAG (F1804 1:5,000), anti-MDM2 mately 4x105 cells were collected by 800 x g centrifugation at (SAB4300601; 1:2,000), anti-BCL2 (SAB4300340; 1:500) and 4˚C for 5 min, washed with PBS and stained with Annexin V anti-p53 (P9249; 1:1,000) from Sigma-Aldrich (Merck KGaA, and propidium iodide. Attune™ NxT Software (version 1.0; Darmstadt, Germany). Membranes were washed three times Thermo Fisher Scientific, Inc.) was used to analyze apoptotic with TBS + 1% Tween-20 and incubated with horseradish cells using a flow cytometer. Each experiment was performed peroxidase‑conjugated secondary goat anti‑mouse immuno- at least 3 times. globulin (Ig)G (ab6728; 1:5,000; Abcam) or goat anti-rabbit IgG (ab6721; 1:5,000; Abcam) at room temperature for 1 h. Transwell migration and invasion assay. Costar Transwell Immunoreactive protein bands were visualized using the chambers (Corning Inc., Corning, NY, USA) were used to Enhanced Chemiluminescence kit (Beyotime Institute of detect the cell migration and invasion ability. Uncoated Biotechnology). Each experiment was performed at least Transwell inserts were used to assess cell migration ability, 3 times. whereas Transwell inserts precoated with Matrigel (BD Biosciences, San Jose, CA, USA) were used to determine cell Reverse transcription‑quantitative polymerase chain reaction invasion ability. Jurkat cells were transfected for 48 h, resus- (RT‑qPCR). Total RNA was extracted from 3x106 cells using pended in RPMI-1640 without FBS, and plated (2x104 cells) the RNAprep Pure kit (Tiangen Biotech Co., Ltd., Beijing, into the upper chamber; the lower chamber was filled with China), following the manufacturer's protocol. Total RNA 500 µl RPMI-1640 supplemented with 10% FBS. Cells were (2 µg) was reverse transcribed into cDNA using the FastKing incubated at 37˚C with 5% CO2 in a humidified air atmosphere RT kit (Tiangen Biotech Co., Ltd.), according to the manufac- for 24 h.