| Objective: B-cell acute lymphoblastic leukemia(B-ALL)is a common type of blood cancer.Although the remission rate of existing treatments has increased,the current treatments for B-ALL often cause adverse reactions and recurrences.Therefore,many scholars have conducted in-depth research on this.In recent years,people have gradually realized that genomics has played a key role in the rapid detection of genes involved in tumorigenesis and development,and it has been proven to be a reliable method for identifying core genes.In this study,our purpose is to find out the molecular biological markers related to the occurrence and development of B-cell acute lymphoblastic leukemia through bioinformatics analysis,and to provide personalized treatment for Bcell acute lymphoblastic leukemia in the future.Studies have found that G protein signal regulator 1(GPSM1)is involved in a variety of cellular events,including membrane protein transport,macroautophagy,Golgi structure and function,mitotic spindle orientation in cerebral cortex progenitor cells,and addiction-related Neuroplasticity,cardiac function and metabolism,polycystic kidney disease,leukocyte migration and cycle progression.Therefore,GPSM1 may serve as a potential therapeutic target,but the role of GPSM1 in B-cell acute lymphoblastic leukemia remains unclear.This topic aims to clarify the expression of GPSM1 in B-cell acute lymphoblastic leukemia and its molecular mechanism,explore the possibility of GPSM1 as a potential therapeutic target,and provide a new theoretical basis for the treatment of B-cell acute lymphoblastic leukemia.Methods:?.Screening of differential genes between acute B lymphocytic leukemia cells and normal human peripheral blood monocyte1.Screen the differentially expressed genes in acute B lymphocytic leukemia cells and normal human peripheral blood mononuclear cells through the Oncomine database.2.DAVID website was used to perform gene ontology(GO)and pathway(KEGG)enrichment analysis of differential genes.3.STRING protein interaction network online database was used to predict the PPI network and MCODE in Cytoscape was used for core module analysis.4.GEPIA website was used to analyze the correlation between candidate gene expression and survival of leukemia patients.5.Oncomine was used to analyze the expression of candidate genes in different cancer types.6.The Blood Spot online public database was used to investigate the expression of GPSM1 in blood disease samples.The Oncomine database was used to extract GPSM1 expression data in acute B lymphocytic leukemia?.GPSM1 regulates the JNK pathway through ADCY6-RAPGEF3 and affects the proliferation,cycle and apoptosis of BALL-1 and Reh cells1.RT-qPCR and Western blot were used to detect the expression of GPSM1 in acute lymphocytic leukemia cell lines.2.Adenovirus was used to construct BALL-1 and Reh cell lines with low GPSM1 expression.In cell function experiments,CCK8 was used to detect cell proliferation in each group.Propidium iodide(PI)staining was used to detect the effect of GPSM1 knockdown on the cell cycle of BALL-1 and Reh.Detect cell apoptosis before and after GPSM1 knockdown by Annexin V-PE / 7AAD double staining and flow cytometry.Western blot was used to detect the expression of apoptosis markers Bcl2 and BAD before and after GPSM1 knockdown.At the same time,differentiationrelated molecules were also tested.RT-qPCR was used to detect the expression of CD19,CD22,CD79 A,CD79B,CD81,and CD200 mRNA before and after GPSM1 knockdown.CD22 expression was detected by flow cytometry.CD200 protein expression was detected by Western blot.3.In the signaling pathway experiment,RT-qPCR and Western blot were used to detect the expression of JNK before and after GPSM1 knockdown.The gene expression data GSE87070 was used to perform gene set enrichment analysis on GPSM1 with GSEA to investigate the way that GPSM1 affects the occurrence and development of acute B lymphocytic leukemia.The correlation between GPSM1 and ADCY6 and RAPGEF3 in LAML was analyzed using UALCAN,and the pathway proteins such as ADCY6 and RAPGEF3 were detected by RT-qPCR and Western blot.Finally,different concentrations of inhibitor ESI-09 was used to inhibit RAPGEF3,then,JNK expression was detected at multiple time points.Results:?.Screening of differential genes between acute B lymphocytic leukemia cells and normal human peripheral blood monocyte1.Oncomine database shows that compared with the normal group,there are 395 highexpressing mRNAs and 421 low-expressing mRNAs.2.GO analysis results show that the biological process(BP)of up-regulating differential genes mainly focuses on hematopoiesis,cell activation,cell adhesion,etc.The biological process(BP)of down-regulation of differential genes mainly focuses on immune response,cell surface receptor signaling pathway,leukocyte migration,etc.The up-regulated differential genes are mainly concentrated in the outer part of the plasma membrane,the plasma membrane,etc.in the cell component(CC).The downregulated differential genes are mainly enriched in cell exosomes,the outer side of the plasma membrane,and the components of the plasma membrane in the cell component(CC).The up-regulated differential genes in molecular function(MF)are mainly enriched in transcription regulation activity,protein kinase activity,protein tyrosine kinase activity,etc.The down-regulated differential genes in molecular function(MF)are mainly enriched in protein binding,receptor activity,and protein homodimerization activity.KEGG PATHWAY analysis showed that the differential genes were mainly enriched in transcriptional dysregulation in cancer,Th17 cell differentiation,Th1 and Th2 cell differentiation,cancer pathway,B cell receptor signaling pathway,cell cycle,cell aging,NF-?B signaling pathway.3.Through the STRING protein interaction network online database,a PPI network of DEGs is constructed.The network includes 739 nodes and 1385 edges.The core genes in the PPI network were obtained by using MCODE in Cytoscape software,including55 nodes and 724 edges.These nodes with the highest scores were selected as the core genes: LCN2,CRISP3,CD34,FUT4,CD9,CD7,ARG1,CD2,CD58,FCGR1 A,CD40,CHI3L1,NCAM1,ORM1,ORM2,CHIT1,CD5,RAG1,CD68,PTPRC,QPCT,MMP8,CAMP,DNTT,JUP,CD24,CD33,OLFM4,MPO,CD1 E,ANPEP,CD79 A,CD19,CD86,TFRC,CD69,MME,BPI,FLT3,CD1 A,KIT,ITGAM,CD48,SLPI,ELANE,SPN,CD1 D,LTF,CD80,LYZ,CD28,SLAMF1,GYPA,DEFA4,IL3 RA.4.The GEPIA database results show that the genes closely related to the survival of LAML patients are: GPSM1,SYT1,SEMA4 D and SEMA4 F.5.The Oncomine database was used to analyze the pan-oncogene expression profile of GPSM1,SYT1,SEMA4 D and SEMA4 F expression in normal tissues and cancer tissues,and the results showed that GPSM1 specifically increased in leukemia.6.The results of Blood Spot showed that GPSM1 expression was relatively high in ALL,GPSM1 expression levels were also high in several BCP-ALL samples of B-ALL.Oncomine data set analysis showed that GPSM1 significantly increased in B-cell children with acute lymphoblastic leukemia,B-cell acute lymphoblastic leukemia,and pro-B acute lymphoblastic leukemia compared with peripheral blood mononuclear cells.?.GPSM1 regulates the JNK pathway through ADCY6-RAPGEF3 and affects the proliferation,cycle and apoptosis of BALL-1 and Reh cells1.Compared with normal lymphoblasts HMy2.CIR,GPSM1 is highly expressed in acute B lymphoblastic leukemia cell lines BALL-1 and Reh.2..In the acute B lymphocytic leukemia cell lines BALL-1 and Reh,after GPSM1 knockdown,cell proliferation was inhibited,the cell underwent S phase arrest of the cell cycle,and cell apoptosis increased.The expression of anti-apoptotic protein Bcl2 was down-regulated,and the expression of pro-apoptotic protein BAD was upregulated.In BALL-1,leukocyte differentiation antigens CD19 mRNA,CD79 B mRNA and CD200 were down-regulated,and CD22 was up-regulated.3.After knocking down GPSM1,JNK was down-regulated in BALL-1 and Reh cells.GSEA analysis shows that GPSM1 is related to calcium signaling pathway,and ADCY is one of the frontier genes in this subset,that is,ADCY is one of the core genes that represent biological importance in this pathway gene set.The results in UALCAN showed that GPSM1 was positively correlated with ADCY6 and RAPGEF3 in LAML.After GPSM1 was knocked down,ADCY6 and RAPGEF3 was downregulated in BALL-1 and Reh cells.The RAPGEF3 inhibitor ESI-09 inhibited JNK expression in a concentration and time dependent manner.Conclusion:1.Our research found that in the acute B lymphocytic leukemia cell lines BALL-1 and Reh,the abnormally high expression of GPSM1 may play a role in the pathogenesis of acute B lymphocytic leukemia through functions such as cell proliferation,cell cycle and apoptosis.2.GPSM1 may regulate JNK pathway through ADCY6-RAPGEF3 and participates in the pathogenesis of acute B lymphocytic leukemia... |
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