The Role Of Vesicle Transport Factor USO1 In ATPR-induced Differentiation Of Myelodysplastic Syndrome Cells And Its Possible Mechanisms

Myelodysplastic syndromes（MDS）is a heterogeneous group of myeloid neoplasms that different from a solid tumor,which are characterized by impaired peripheral blood cell production with abnormal cell morphology in common.And it is also hallmarked by bone marrow dysplasia,ineffective erythropoiesis and a high propensity to progression into acute myeloid leukemia（AML）.In addition,age-induced genetic epigenetic and immune-mediated changes in hematopoietic stem cells（HSC）lead to proliferation and amplification of myelodysplastic stem cells,and defective differentiation is a typical feature of MDS.Therefore,inhibition of myelodysplastic uncontrolled proliferation of stem cells and promote their differentiation is an effective method for clinical treatment of MDS.In recent years,the differentiated treatment for malignant tumor is a hot spot in clinical treatment.All trans retinoic acid（ATRA）,as a kind of mature differentiation inducer,was used in the treatment of early acute promyelocytic leukemia（APL）achieved good therapeutic effect.But clinical therapy found that ATRA have serious drug resistance and retinoic acid syndrome,limits its application.Therefore,this research carries on the structure modification,synthesis of a new type of retinoic acid derivatives 4-amino-2-trifluoromethyl-phenyl retinate（ATPR）,preliminary pharmacodynamics screening and pharmacology studies have demonstrated that ATPR has higher solubility and lower toxicity compared with ATRA,and it can be used to suppress a variety of malignant tumor cells and hematologic disease cells proliferation,promote apoptosis and induce differentiation.Our previous studies have shown that ATPR could inhibit the proliferation,induce the differentiation and promote apoptosis in myelodysplastic syndrome SKM-1 cells Therefore,we used proteomic analysis to observe differentially expressed genes after ATPR treated in order to further explore the potential target of ATPR.In this paper,the vesicle transport factor USO1 in MDS cell lines induced by ATPR cycle block and the differentiation of concrete effect and possible mechanism are discussed.This subject is divided the content into the following three parts.1.Expression of USO1 in SKM-1 cells and other leukemia cell lines after ATPR treatmentTo explore the influence of the expression of USO1 in myelodysplastic syndrome and other leukemia cell lines KG-1,Jurkat,MOLM-13,and HL–60 after ATPR treatment.Western blot assay showed that,compared with ATRA,USO1 expression were significantly inhibition by different degree after ATPR treatment.Then use ATPR at same concentration(10^-6M)at different times（0 h,12 h,24h,48 h,72h,96h）and at different concentrations(10^-5M,10^-6M,10^-7M,10^-8M,10^-9M)with same time（72h）.The results showed that the level of USO1 presented a time and concentration-dependent reduction and the best effect was achieved at the time of 72h and concentration of 10^-6M.2.The role of USO1 in ATPR-induced cell cycle blocked and differentiation of myelodysplastic syndrome cellsIn order to investigate the role of USO1,we silenced USO1 with small interfering RNA,then treated with ATPR at the optimal concentration and time,and examined the level of protein USO1,PCNA,CDK2,CDK4 and PU.1 by western blot.Wright’s-Giemsa staining examined the cell morphological changes.Ki67 staining was to detect cell proliferation and NBT reduction assay to detect cell differentiation.Flow cytometry was used to detect cell apoptosis and differentiation marker CD14.The results indicated that USO1-silencing reduced the protein expression of cell cycle CDK2,CDK4 and proliferating cell nuclear antigen（PCNA）while transcription factor connection with myeloid differentiation PU.1 was lower.Wright’s-Giemsa staining results showed that kidney-shaped shrinkage of nuclear and lessened nuclear:cytoplasmic（N:C）ratio.immunofluorescence assay indicated that the expression of cellular proliferation marker Ki67 was notably declined when USO1 was silenced.NBT reduction assay demonstrated that NBT positive cells were obviously increased after USO1 silencing.Flow cytometry revealed that surface molecular markers CD14 was elevated and cell apoptosis ratio was declined.For the purpose of deeply confirm the role of USO1,p CMV-MCS-3*FLAG-USO1 was applied to overexpress USO1.Then treated with ATPR at the optimal concentration and time,and detected the protein expression level of USO1,PU.1,PCNA and CDK4 by western blot.Wright’s-Giemsa staining examined the cell morphological changes.Ki67staining was to detect cell proliferation and NBT reduction assay to detect cell differentiation.Cell apoptosis and differentiation marker CD14 was detected by flow cytometry.Western blot results presented that p CMV-USO1 could accelerate cell cycle progression and promote cell proliferation by heightening the expression of CDK2 and PCNA.Wright’s-Giemsa stain indicated that p CMV-USO1 could block ATPR-induced cell morphologically mature changes.Cell proliferation revealed by Ki67 staining manifested that expression of USO1 in SKM-1 cells arrested ATPR-induced decrease of cell proliferation.ATPR-induced the count of NBT positive cells markedly decreased after USO1-overexpression.Flow cytometry results showed that CD14 expression were prevented and apoptosis of cells was decreased with overexpression of USO1.3.Raf/ERK signaling was involved in ATPR-induced cell cycle arrest and differentiation in myelodysplastic syndromeThere being research showed that protein kinases play an important regulatory part in various aspect of cell life.It could regulate cell cycle and proliferation,cell migration and invasion,differentiation and transcription.Ras promotes the formation of active homodimers and heterodimers of c-Raf through a complicated process.Then these protein like threonine/serine kinases further enhance the phosphorylation and activation of ERK.Prior study showed that the sustained activation of ERK could promote cell differentiation in leukemia.We observed the activity changes of Raf/ERK signals after the silencing and overexpression of USO1 respectively after treated with ATPR at the optimal concentration and time.The results showed that the activity of Raf-ERK pathway was also enhanced by USO1-silencing as shown by the hyperphosphorylation of Raf and ERK while p CMV-USO1 could bring the inverse impact.The protein expression level of USO1,Raf,p-Raf,ERK,p-ERK,c-myc,cyclin D1 were detected by western blot.Wright’s-Giemsa staining examined the cell morphological changes.Ki67staining was to detect cell proliferation and NBT reduction assay to detect cell differentiation.Cell surface differentiation marker CD14 was examined by flow cytometry.The result illustrated that the inhibition of ERK by PD98059 alleviated the ATPR-induced cell cycle arrest and differentiation.