The Discovery And Clinical Significance Of Activating Transcription Factor ATF5 In Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a kind of hematopoietic system malignant hyperplasia disease,which derives from hematopoietic stem cell.The disease develops rapidly and the natural course of disease is only a few months.In addition to the special type of acute promyelocytic leukemia,most AML patients are prone to relapse after complete remission,low long-term survival rate,poor prognosis,and AML targeted drugs are also very limited.Therefore,it is important to discover new molecules in the pathogenesis and development of acute leukemia and to excavate new therapeutic targets.Bioinformatics is an important tool to search for molecular markers related to leukemia.In this study,we used bioinformatics to mine the data of acute myeloid leukemia(AML)in gene expression database(GEO),to explore candidate molecules for AML development and prognosis evaluation,to screen out the expression of ATF5 in AML patients at different stages,the correlation with other prognostic indicators,the biological effects in AML cells and the possible mechanisms.So as to evaluate its value as a candidate molecule for AML targeted intervention.Methods1.Differently expressed genes analysis in GEO databaseWe choose GEO dataset which contains both the control group and AML group,and verify the data quality by R studio,then analyze gene expression by using R studio and finally choose the genes that expressed differently between the two groups.Then,we analyze the expression level of objective genes in AML clinical samples and cell lines expression data.2.Verify expression level of ATF5 in AML samplesBone marrow mononuclear cells(MNCs)were extracted from newly diagnosed,complete remission and relapsed AML patients,as well as IDA patients as control group.The expression of ATF5 in different stages of the disease was detected by qRT-PCR,Western Blot and immunofluorescence assay.3.Detect ATF5 expression in AML cell lines induced differentiation in vitro.The expression level of ATF5 in AML cell line differentiation related microarray was analyzed in GEO database.The expression level of ATF5 was detected by Western Blot and qRT-PCR after the induction of differentiation to mature blood cells in AML cell lines by Heminine,DMSO and PMA in vitro.4.Measure cell proliferation and apoptosis in AML cell lines transfection with ATF5 siRNA.After transfected ATF5 siRNA into AML cell lines.CCK8 and Edu were used to detect cell proliferation,and use flow cytometry to detect cell apoptosis.5.Analyze relation between expression level of ATF5 and clinical outcomes of AML patients.The data of TCGA database were collected to analyze the correlation between ATF5 expression level and age,sex,leukocyte count and FAB classification,cytogenetic risk stratification and gene mutation(FLT3,NPM1,DNMT3A,1DH2,RUNX1).Furthermore,the expression level of ATF5 was analyzed in patients with different cytogenetic risk grade,then,the relationship between ATF5 expression level and cytogenetic risk grade was explored.At the same time,we chose gene chip datas of AML patients which have completed gene expression information and clinical survival information,and survival analysis of AML patients was did.After matching the gene expression information with the clinical information of patients,the specimens were divided into high expression group and low expression group according to the expression level of ATF5.The total survival of patients was analyzed by Kaplan-Meier.6.Cluster analysis of differential expression genes related to ATF5In order to explore the potentially related molecular of ATF5.we select the datasets concerning with AML in TCGA database,and rank the datasets expression data according to ATF5 expression level,then the top 25%and the bottom 25%were selected to analyze the differentially expressed genes by R studio.Then.enrichment analysis based on R Studio was processed,including Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)Pathway examination,to discover potential pathways and molecular mechanism caused by ATF5 expression level.Results1.The expression of ATF5 was significantly different in AML compared to the control group.We choose the gene expression data including AML patients and healthy control group in the GEO database to screen the statistically significant DEGs with log2FC?2,p values<0.01,then we obtained 95 DEGs and found that ATF5 in transcription factor family was significantly highly expressed in AML patients.Furthermore.larger-sample gene expression profile data were analyzed,and we found almost all of the ATF family molecules were obviously dysregulated in AML patient.Among them the variation of ATF3,ATF5 and ATF6 was especially remarkable.Data analysis of datasets including both AML samples or cell lines and healthy controls with larger samples,results shows that ATF5 presents higher expression in the AML patient specimens and cell lines.Further analysis showed that ATF5 expression was significantly disitinct in AML patients with different cytogenetic backgrounds,and was especially high-expressed in APL.2.ATF5 is overexpressed in AML clinical specimens and related to disease progressionqRT-PCR,Western Blot,and immunofluorescence(IF)were used to verify ATF5 expression level in bone marrow specimens of AML patients comparing to the healthy control.The results showed that ATF5 was significantly overexpressed inAML samples compared to control group,but it underexpressed in CR patients,while.it was overexpressed again in relapsed AML patients.3.The expression of ATF5 decreased in differentiated AML cell lines.In the GEO database,we selected the AML cell line differentiation related gene chip,analyzed the expression level of ATF5,found that the expression of ATF5 decreased significantly after induced differentiation.and after inducing differentiation of AML cell lines to mature blood cells in vitro,the expression of ATF5 was significantly decreased by qRT-PCR and Western Blot.It is suggested that ATF5 mediates the differentiation of AML cells.4.ATF5 knockdown induces cell apoptosis and inhibits cell proliferation.After the silence of ATF5 expression by transfect ATF5 siRNA into AML cell lines.CCK-8 and Edu experiments found that cell proliferation was inihibited,and flow cytometry revealed an increase in cell apoptosis.5.The high expression of ATF5 was related to poor prognosis of AML.We analysis the relationship between ATF5 expression and age.sex,leukocyte number and FAB typing,gene mutation and so on in TCGA database.It was found that the expression of ATF5 was related to the age of the patients.The expression level of ATF5 was analyzed according to the cytogenetic risk groups in TCGA database.It was found that the expression of ATF5 in the low risk group was significantly lower than that in the middle risk group and high risk group.According to the expression level of ATF5,the high expression of ATF5 was found to be related to the poor prognosis of patients with GEO datasets involving complete survival and gene expression information.6.Pathway enrichment analysis explore potential ATF5 downstream genes.In TCGA database.according to the expression level of ATF5,we perform a differential expression gene analysis,setting log2FC ? 1,p<0.05 for the filter,and finally get 268 DEGs,then enrichment analysis based on R studio was progressed,including Gene Ontology(GO)analysis,Kyoto Encyclopedia of Genes and Genomes(KEGG)and GSEA(Gene Set Enrichment Analysis)pathway enrichment.There are 18 items by Gene Ontology,including 4 GO-BP items,7 GO-CC items,2 GO-MF items and 5 KEGG pathway,as well as 3 items are enriched by GSEA.We speculatethe mechanisms that ATF5 promote AML occurrence may be by influence oxidative phosphorylation.ubiquitin mediated proteolysis,together with the expression imbalance of some important genes.such as RUNXITI.ConclusionsBy analyzing the AML gene chip in GEO and TCGA database,it was found that ATF5 is overexpressed in AML.The high expression of ATF5 was associated with the poor prognosis of AML patients,and the expression level of ATF5 was related to the progression of the disease.Cell line studies showed that ATF5 could be an oncogene by inhibiting the differentiation and apoptosis of AML cells and promoting the proliferation of AML cells.These results suggest that the abnormal high expression of ATF5 in AML may be a potential candidate target for the diagnosis and intervention of AML.