| Background: Leukemia is a malignant clonal disease of hematopoietic stem cells,and cell differentiation is blocked at an early stage so that it does not possess mature cell functions.Induced differentiation therapy achieves therapeutic goals by inducing the differentiation and maturation of tumor cells.Chinese scholars first successfully treated all-trans-retinoic acid(ATRA)to differentiate and treat acute promyelocytic leukemia(APL).However,ATRA induction therapy still has the problems of unstable therapeutic effect and easy relapse in some patients.Therefore,further elucidation of its mechanism of inducing differentiation of leukemia cells will provide new targets and approaches for clinical treatment.Signal transducer and activator of transcription 1(STAT1)is one of the most important members of the STATs family.It is mainly activated by interferon(IFN),participates in anti-infection,and regulates cell proliferation,differentiation,growth,apoptosis,and plays an important role in inhibiting the development of tumors.Studies found that retinoic acid induced differentiation of human histolytic lymphoma U-937 cells is closely related to the serine 727 phosphorylation of STAT1,but its role in the differentiation of human acute myeloid leukemia HL-60,NB4 cells to granulocytes is still unclear.MiRNA is a type of small-molecule,non-coding,single-stranded RNA that binds specifically to the 3’UTR region of the target gene and negatively regulate its expression.MiRNA participate in various physiological processes and the occurrence and development of various diseases such as cell growth and differentiation,proliferation,apoptosis and signal transduction.Recent studies have found that miRNA expression is elevated in various solidtumors and hematologic malignancies,which regulates tumor cell differentiation.However,the role of miRNA in ATRA-induced leukemia cells to granulocytes differentiation is still unclear.Further exploration in this area is expected to elucidate the mechanism of leukemia cell differentiation disorders from the perspective of epigenetic regulation and provide new targets and ideas for clinical treatment.Objective: To investigate the role of STAT1 signaling pathway in ATRA-induced differentiation of human leukemia cells into granulocytes and the miRNA target regulation mechanism,to explain the causes of leukemia cell differentiation disorders from the perspective of epigenetic,and to provide new targets and approaches for clinical diagnosis and treatment.Methods: Human acute myeloid leukemia cell(HL-60)and acute promyelocytic leukemia cell(NB4)were induced by ATRA to differentiate into granulocytes.Cells were randomly divided into two groups,the observation group cells were induced to differentiate into granulocytics with ATRA(2μmol/L),while the control group was given the same concentration of DMSO.Morphologic changes of cells were observed by Wright-Giemsa staining.Cell surface marker CD11 b was detected by flow cytometry.The mRNA expression of STAT1 and related transcription factor mRNA levels were detected by q-PCR,the total protein and phosphorylation level of STAT1 were detected by western blot assay.After Fludarabine was used to inhibit STAT1 activity,flow cytometry was used to detect its effect on cell differentiation,and the mRNA expression of transcription factor were detected by q-PCR.Differential miRNAs were detected in a high-throughput microarray screening system,and q-PCR was used to verify the expression of differential miRNAs;the expression of candidate miRNA was regulated to observe their effects on leukemia-derived granulocyte differentiation and STAT1 signaling pathways.The luciferase reporter system determines the differential miRNA binding site to STAT1.Results: HL-60 and NB4 were sensitive leukemia cell lines induced by ATRA-induced granulocytes differentiation.Compared with the control group,the number of HL-60 cells were decreased after ATRA induction,the shape became irregular,cytoplasm increased,nuclear concentrated,nucleolus became smaller or disappeared,and the number of rod or leaf nuclei increased significantly.The NB4 cells became smaller,the clusters were scattered andirregularly shaped,the ratio of nucleoplasm to cytoplasm decreased,and rod-shaped nucleus and lobar nucleus increased significantly.The expression of CD11 b on the cell surface was significantly increased after ATRA induction compared with the control group(P<0.05),suggesting that HL-60 and NB4 cells can differentiate into granulocytes after the induce of ATRA.The expression of STAT1 mRNA,total protein,and p-STAT1 protein were significantly increased(P<0.05),suggesting that activation of STAT1 signaling pathway plays an important role in ATRA-induced differentiation of HL-60 and NB4 cells into granulocytes.The expression of C/EBPε,C/EBPβ,and PU.1 were significantly increased(P<0.05).After Fludarabine inhibited STAT1 protein activity,leukemia-derived granulocyte differentiation was significantly decreased(P<0.05),while C/EBPε mRNA expression was significantly decreased.Pearson correlation analysis showed that there was a significant positive correlation between STAT1 and C/EBPε mRNA expression(P<0.05),suggesting that activating STAT1 and promoting C/EBPε transcription may be one of the key mechanisms for ATRA-induced leukemic cell differentiation into granulocytes.High-throughput microarray analysis and q-PCR validation showed that miR-155-5p expression was decreased after ATRA induction(P<0.05).Pearson correlation analysis showed that STAT1 mRNA was significantly negatively correlated with miR-155-5p(P<0.05).Targetscan predicted a potential binding site between miR-155-5p and STAT1.Up-regulation of miR-155-5p expression significantly decreased STAT1 mRNA,C/EBPε mRNA levels and leukemia-derived granulocyte differentiation ratio(P<0.05).The luciferase reporter assay confirmed that miR-155-5p has complementary binding to the 3’ UTR of STAT1 mRNA.The above results suggest that ATRA may promote the differentiation of leukemia cells into granulocytes by decreasing the expression of miR-155-5p and promoting the activation of STAT1 signaling pathway.Conclusions: ATRA promotes the activation of STAT1/C/EBPε signaling pathway through targeted regulation of miR-155-5p expression,which in turn induces differentiation of leukemic cells into granulocytes.Targeted regulation of miR-155-5p/STAT1 signaling pathway may be an effective target and pathway for inducing differentiation of leukemia cells into granulocytes. |
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