基于急性髓系白血病患者骨髓单个核细胞中诊断候选基因筛选鉴定及其对预后影响的生物信息学分析和实验验证OA

Objective:To discuss the diagnostic value of differentially expressed genes(DEGs)in bone marrow mononuclear cells of the patients with acute myeloid leukemia(AML),and to clarify their clinical significance and potential functions.Methods:The GSE9476 dataset(bone marrow samples from 26 AML patients and 10 healthy donors)was obtained from the Gene Expression Omnibus(GEO)database;GEO2R was used to screen the DEGs the fold change(FC)was calculated,and|log2 FC|≥2,P≤0.05,and adj.P≤0.05 were the criteria;the DAVID database was used to perform Gene Ontology(GO)functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis;STRING was used to construct a protein-protein interaction(PPI)network;Cytoscape was used to screen core,key,and bottleneck genes;the Venn diagram was used to obtain diagnostic candidate genes by intersection;the GEPIA and Kaplan-Meier Plotter online databases were used to test the expression differences of the candidate genes and their significance with patient survival;real-time fluorescence quantitative PCR(RT-qPCR)was used to validate the related mRNA expression levels in bone marrow mononuclear cells from clinical specimens of newly diagnosed non-acute promyelocytic leukemia(APL)type AML patients and healthy donors;through recombinant plasmid construction,lentiviral packaging and infection,AML cell models overexpressing platelet factor 4(PF4)and pro-platelet basic protein(PPBP)were established;CCK-8 method was used to detect the proliferation activities of the cells in various groups;Western blotting method was used to detect the expression levels of apoptosis-related proteins in the cells in various groups.Results:A total of 496 DEGs were screened,including 69 up-regulated genes and 427 down-regulated genes.The enrichment analysis results showed that the DEGs were mainly involved in immune response,cell cycle,and chemokine signaling pathways.The PPI network analysis identified 10 candidate genes,which were cyclin-dependent kinase 1(CDK1),CD36 molecule(CD36),Runt-related transcription factor 1(RUNX1),CD3 delta subunit of T-cell receptor complex(CD3D),vascular endothelial growth factor A(VEGFA),cyclin B1(CCNB1),amyloid beta precursor protein(APP),Src family tyrosine kinase(FYN),PPBP,and PF4.The results from GEPIA and Kaplan-Meier Plotter online databases showed that compared with control group,the expression levels of APP,CD36,FYN,RUNX1,PF4,and PPBP mRNA in bone marrow aspirate fluid of the patients in AML group were increased(P<0.05),while the expression levels of CCNB1,CD3D,CDK1,and VEGFA mRNA were decreased(P<0.05);high expression of APP,CD36,PF4,PPBP,and RUNX1,as well as low expression of CD3D and VEGFA,were significantly associated with poor prognosis(P<0.05).The real-time fluorescence quantitative PCR results showed that compared with control group,the expression levels of PF4 and PPBP mRNA in bone marrow aspirate fluid of the patients in AML group were significantly increased(P<0.01).The CCK-8 assay results showed that compared with control group,after overexpression of PF4 or PPBP,the proliferation activities of THP-1 cells in OE-PF4 group and OE-PPBP group significantly increased(P<0.05).The Western blotting method results showed that compared with control group,after overexpression of PF4 or PPBP,the expression levels of B-cell lymphoma 2(Bcl-2)protein in THP-1 cells in OE-PF4 group and OE-PPBP group were significantly increased(P<0.05),while the expression levels of Bcl-2-associated X protein(Bax)protein were significantly decreased(P<0.05).Conclusion:The screened PPBP and PF4 are abnormally expressed in AML and are correlated with the patient prognosis;they may participate in AML progression by promoting cell proliferation and inhibiting apoptosis,and have the potential to serve as the diagnostic and prognostic biomarkers.