PADI4Cooperates With Sox4on Inducing Differentiation Of HL-60Cells Into Granulocytic

BackgroundPeptidylarginine deiminase4(PADI4), which converts a protein arginine residue to acitrulline residue are widely distributed in animal tissues. Little is known about PADI4ofhemopoietic cells.We found that PADI4activity in human myeloid leukemia HL-60cells wasinduced with the granulocyte-inducing agents retinoicacid. We cloned and characterized aPADI4cDNA from retinoic acid-induced cells. The cDNA was2,238base pairs long andencoded a663-amino acid polypeptide. Immunoblotting with an antiserum against theenzyme showed that a protein of approximately67kDa increased concomitantly with increaseof mRNA of approximately2.6kilobases during granulocyte differentiation. Duringmonocyte differentiation the same mRNA and protein increased as in granulocytedifferentiation. These results suggested that expression of the PADI4gene is tightly linked tomyeloid differentiate. However, little is known about the precise function and target substratesof PADI4under physiological conditions.Sox gene is a family of SRY-related genes,which constitute large groups of transcriptionfactors related to a DNA-binding domain known as the high-mobility group(HMG)-box．Soxproteins are required for avariety of developmental processes,including central nervoussystem,sex determination,neural crest cell development, chondrogenesis,lens development,cardiac development,and hematopoiesis. Sox4,a47×103protein，encoded by a single exon and has been identified as playing critical role in development.The Sox4transcription factorwas identified as a gene that cooperates with PU.1,CREB,PML/RARα in myeloidleukemogenesis. SOX4was indeed consistently expressed in primary ATL cells.FRA-2/JUND efficiently activated the SOX4promoter via an AP-1site. Knockdown ofSOX4expression by small interfering RNA(siRNA) strongly suppressed cell growth of ATLcell lines. Loss and gain-of-function experiments identified Sox4as a critical activator ofPI3K/AKT and MAPK signaling in ALL cells.Leukemia is the malignant clone disease which occurs in a haematopoietic stem cell andstill can’t completely cured. In recent years, studies found that PADI4expression in variousmalignancies， however,PADI4expression was not observed in benign leiomyomas ofstomach, uterine myomas,endometrial hyperplasias.That suggested PADI4play importantroles in the initiation and progression of malignancies. PADI4was detected in granulocytesand macrophages induced by HL60cells, the enzyme is believed to be involved in thedifferentiation and apoptosis of these cells,and Sox4inhibiting leukemia cell differentiationthrough various channels. Guess there are certain correlation between PADI4and sox4, forthis study, hoping that become a promising target for the treatment of leukemia.On the basis of predecessors’ research, preliminary reasearch PADI4and sox4relation tothe differentiation of leukemia cells, providing new theoretical basis for the clinical treatmentof leukaemia and therapeutic targets.ObjectiveBased on the previous research, we investigate the regulation role and mechanism ofPADI4targeting sox4in directional differentiation of leukemia cells by vitro experiments,focusing on changes of PADI4and sox4expression in HL60cells to granulocytedifferentiation induced by all trans retinoic acid, and the mechanism of PADI4targeting sox4on cell differentiation, verifyingin the regulation function and expression regulationmechianism of PADI4targeting sox4in order to provide a theoretical basis and newtherapeutic targets for the treatment of leukemia. MethodsEstablish all-trans retinoic acid induced HL-60cell model,then the differentiation ofHL-60cells induced by all-trans-retinoic acid (ATRA) was detected by Giemsa staining assayand flow cytometry assay for CD11b. The upstream sox4gene that were related to PADI4expression was detected by RT-PCR and western blot assay. Specific siRNAs targetingPADI4were transfected into HEC293T cells and the PADI4\sox4expression was measuredwith reverse transcription-polymerase chain reaction (RT-PCR) and Q-PCR assay.The in vitroexperiments, the construction of PADI4eukaryotic expression vector and the reporter plasmidpGL3-sox4were transfected into HEC293T cells, luciferase assays were performed inverifying PADI4binding sox4promotor and the chromatin immunoprecipitation (ChIP) assaywas used to reveal the role of PADI4by binding the target gene sox4.In addition, theconstruction of PADI4eukaryotic expression vector and the construction of sox4fourdifferent domians eukaryotic expression vector were transfected into HEC293T cells, CoIPassays were performed in verifying the relationship of PADI4and sox4proteins. Eachexperiment was repeated for3times, the experimental results based on mean±standarddeviation, mean analysis using t test, P<0.05had statistical significance.ResultsInduced by all-trans retinoic acid in HL60cells, after96h, PADI4at the levels oftranscription and translation is showing a consistent elevate, compared with the control groupphase ratio and four time points were compared with two statistics by the gray value (P<0.05);in the contrast, sox4at the levels of transcription and translation is showing a consistentdecrease,compared with the control group and the four time point were compared with twostatistically significant (P<0.05); SiRNA knockdown of PADI4promote the expressionofsox4as compared with that of control cells. Luciferase verified that PADI4as transcriptionfactor can activate sox4promoter. The chromatin immunoprecipitation (ChIP) assay was usedto reveal the role of PADI4by binding the target gene sox4and CoIP demonstated PADI4and sox4had relationship in protein level. ConclusionsPADI4plays an important role in the progression of differentiation in HL-60cells.With the differentiation of HL-60cells increased, PADI4elevated at the levels oftranscription and translation and sox4decreased.so PADI4may control sox4expression.PADI4as transcription factor can bind with sox4promoter.PADI4protein and sox4proteinhas a certain correlation.