Preliminary Screening Of Molecular Targets Promoting M2-type Polarization Of Mouse Macrophages In Acidic Tumor Microenvironment

Objective:Preliminary screening of molecular targets promoting m2-type polarization of macrophages in acidic tumor microenvironment will provide theoretical basis for tumor immunotherapy based on remodeling immune function of macrophages in clinical practice.Methods:1.Identification of macrophages:Mouse peritoneal macrophages were extracted and the expression levels of F4/80~+and CD11b~+cells were detected by flow cytometry.2.Effect of acidic tumor microenvironment on polarization of macrophages:macrophages were divided into 3 groups and treated with conditioned medium with p H7.3(0m M),p H6.5 and 20m M lactic acid for 24h,respectively.Collecting cells,Real-time quantitative PCR detection(RT-PCR)was used to detect the expression levels of hypoxia inducible factor-1α(HIF-1α),vascular endothelial growth factor(VEGF),interleukin-10(IL-10)and arginase-1(Arg-1).The secretion levels of VEGF and IL-10 were detected by enzyme-linked immuno sorbent assay(ELISA).3.Transcriptome analysis:Transcriptome sequencing technology was used to study the effect of 20m M lactic acid concentration on macrophages,screen differentially expressed genes.The cellular components,molecular functions and biological processes of differentially expressed genes were analyzed by gene ontology(GO).The kyoto encyclopedia of genes and genomes(KEGG)analyzed the signaling pathways involved in differentially expressed genes.The corresponding differentially expressed proteins were obtained through the screened differentially expressed genes.Cytoscape software was used to display the interaction network diagram between differentially expressed proteins,and the key differentially expressed proteins were screened according to the number of nodes.4.Preliminary screening of molecular targets:Selecting two potential molecular targets:tumor protein translationally controlled regulator 1(Tpt1)and metastasis-associated transcripts of lung adenocarcinoma(Malat1),Tpt1-si RNA and Malat1-si RNA were constructed by RNA interference technique to inhibit their expression,the expression levels of VEGF,Arg-1 and CD11c,markers of macrophage polarization,were detected by RT-PCR.Results:1.The positive rates of F4/80~+and CD11b~+expressed in mouse peritoneal cells were more than 90%.2.The expression levels of HIF-1α,VEGF,Arg-1 and IL-10 in macrophages increased in acidic microenvironment,and the secretion levels of VEGF and IL-10 increased.3.The expression of 6295 genes in macrophages was changed by 20m M lactic acid treatment for 24h(|log2(fold change)|>1),and the corresponding differentially expressed proteins were mainly involved in bacterial defense response,cytoplasmic gene translation,ribosomal biosynthesis,placental development and other biological processes;The involved signaling pathways include PI3K-Akt,MAPK,NOD-like receptor,and so on.The up-regulated differential expression proteins MALAT1 and TPT1 and down-regulated differential expression proteins HES6 and SLC4A7 were screened.4.Silencing Malat1 or Tpt1 can up-regulate the expression of CD11c,a marker of macrophage polarization,and down-regulate the expression of VEGF and Arg-1 in acidic tumor microenvironment.Conclusion:Acidic microenvironment promoted M2-type polarization of macrophages by up-regulating Malat1 and Tpt1 expression.