CT3Promotes HL-60Cells Differentiation By Suppressing MiR-155Expression Through STAT3

The leukemia inhibitory factor (LIF), as a member of the IL-6family of cytokines, hasbeen reported that it can induce differentiation of the M1murine myeloid leukemia cell line.LIF exerts biological activities through a specific cell-surface receptor (LIFR), which ismultimeric shares a subunit, the low-affinity LIF receptor α-chain (LIFRα), and an additionalsubunit, IL-6related signal transducer (gpl30). Cytoplasmic domain of LIFRα contains threemotifs: BOX1, BOX2and BOX3. The YXXQ sequences in every motif are necessary foractivating signal molecules within cells. Previous studies proved the accumulation of YXXQmotifs of LIFRα-CT3by gene transfection can induce differentiation and inhibit proliferationof HL-60cells. Considering the limitation of the motifs in clinical use due toliposome-derived or lentivirus-mediated genetic modifications, we tried to take advantage ofthe HIV-trans-activating transduction domain (TAT-PTD) which can deliver the proteinsacross the cell plasma membrane to cytoplasm. The TAT-mediated LIFRα-CT3eukaryoticexpression fusion protein (TAT-CT3) was produced for peptide-targeting leukemia therapy. Itcould remarkably reduce the cell viability and induce myeloid differentiation in HL-60. Dueto the low expression level of eukaryotic expression system, we produce fusion protein byprokaryotic expression system and hypothesize that prokaryotic TAT-CT3could also achievea therapeutic effect on differentiation of HL-60cells.Signal transducer and activator of transcription (STAT) proteins are activated bytyrosine phosphorylation through the action of receptor-associated Janus family tyrosinekinases (JAKs). STATs have been confirmed to be related to tumor cell proliferation，invasionand metastasis and it is over-expressed in the cancer tissue and serum of cancer patient.Because constitutive activation of STAT3presents in cancer, blocking JAK/STAT3signalingpathway may be potential therapeutic implications for cancer. Intriguingly, because aberrantactivation of STAT proteins was also reported in leukemia and the data showed thatconstitutive STAT3phosphorylation occurred in some AML cells, we presume that reducedSTAT3phosphorylation is associated with induction of HL-60myeloid differentiation. And itis possible that inhibition of STAT3phosphorylation will affect downstream signaling whichcan trigger the differentiation program.MicroRNAs (miRNAs) are endogenous small noncoding RNAs that regulate gene expression in cellular growth and differentiation. They are also functionally linked totumorigenesis. Some of the miRNAs are oncogenic and associated with the pathogenesis ofacute myeloid leukemia (AML). MiR-155is transcribed as a primary transcript from the thirdexon of the B cell integration cluster gene referred to as bic. It was found to be over-expressedin the bone marrow of patients with certain subtypes of AML.Both STAT3and miR-155are considered as oncogene and oncomiR respectively,however JAK/STAT3signaling pathway directly involved in the regulation of miR-155expression is less clear in leukemia. And C/EBPβ that is important in myeloid differentiationhas been identified for target of miR-155. These may suggest that the JAK/STAT signalingpathway could mediate the induction of miR-155during TAT-CT3-induced differentiation ofHL-60cells.The purpose of this study is to observe the phosphorylation of STAT3and miR-155indifferentiation of HL-60cells treated with TAT-CT3fusion protein expressed in prokaryoticexpression system, and to investigate the mechanism on them.In this study, we labeled the fusion protein with fluorescein isothiocynate (FITC) using5-FITC protein labeling kit (Anaspec). Next, HL-60cells were transduced withTAT-CT3-FITC and FITC at a concentration of50μM for1h. And then we observed the cellsunder fluorescence microscope. The results demonstrated that FITC-TAT-fused proteinpenetrated most of the cells. We also analyzed the levels of surface promyeloid differentiationmarkers and cell proliferation. The results suggested that TAT-CT3was able to inducegranulocytic differentiation and induced growth arrest. These data implied that the prokaryoticexpression TAT-CT3fusion protein had biological activity.We next examined the p-STAT3, microRNA-155/BIC and its downstream gene C/EBPβof HL-60cells by qRT-PCR and Western Blotting after treatment with TAT-CT3for4days.We found that decreases in p-STAT3and miR-155/BIC and increases in C/EBPβ and SOCS-1.To determine whether STAT3directly targets bic gene, we performed ChIP analysis onHL-60cells treated with TAT-CT3. The results revealed that the enrichment of p-STAT3wassignificantly decreased at the promoter of BIC in cells transduced with TAT-CT3.In brief, we report for the first time that prokaryotic expression TAT-CT3fusion proteincan reduce the cell viability and induce myeloid differentiation in human myeloid leukemiaHL-60cells with a decrease in STAT3phosphorylation through SOCS1. We also found that miR-155was down-regulated in response to TAT-CT3through direct binding of STAT3to thepromoter of BIC.