Integrative Proteome,transcriptome And DNA Methylome Analysis Of Regulatory Networks During U937-derived Macrophage Polarization From An M2 To M1 Phenotype

The first part of macrophage culture and in vitro induction and identification of M1 and M2 cell phenotypesOBJECTIVE:Macrophages are derived from the differentiation of monocytes in the blood and are an important part of the body’s immune system.They have the functions of recognizing and presenting antigens,regulating immune processes,removing foreign bodies,immune surveillance and phagocytosis,which affect the growth and development of human cells.Features.In the specific microenvironment of the body,macrophages can be derived into two different cell phenotypes according to function and morphology,namely M1 and M2 phenotypes.The former belongs to the classical activation pathway of macrophages,which has antigen killing and inhibits tumor progression.The latter;the latter belongs to the alternative activation pathway of macrophages,which can participate in inflammatory response,promote angiogenesis and tissue reconstruction,etc.It is clinically considered that M2 type cell function is to promote the development of cancer cells.This study aims to establish two polarized macrophage models,M1 and M2,in vitro,which will lay the foundation for further research on polarized macrophages.METHODS:U937 cells were stimulated with PMA,followed by IL-4 and IL-13 to induce the M2 phenotype,followed by lipopolysaccharide and IFN-γ to induce the M1 phenotype;q PCR analysis by TGF-β,Arg-1 and TNF-α Expression levels identify the phenotype of macrophages;Western blot analysis is used to quantify protein expression of Arg-1 and i NOS;in addition,enzyme-linked immunosorbent assay(ELISA)is used to detect IFN-γ,TGF-β and IL-10 The level of secretion.RESULTS:Compared with undifferentiated macrophages,M1 macrophages showed irregular expression or long spindle shape and highly expressed TNF-α,i NOS and IL-12;M2 macrophages were mostly round or round,clustered Growth,high expression of Arg-1,TGF-β and IL-10.CONCLUSION:This indicates that similar to the previous studies,the M1 and M2 phenotypes of macrophages have different levels of protein and gene expression,and successfully developed macrophages with different activation states for the following experimental procedures.Part Ⅱ:Binding proteomics of macrophage polarization,differences between transcriptome and methylationOBJECTIVE:The development of proteomics,transcriptomics,and genomics indicates that we are entering the post-gene era,which promotes more in-cell research in the medical field,which will lead to the development and further research of precision medicine in the future.The occurrence and development of diseases is of great significance.METHODS:Microarrays(lncRNA and mRNA),proteomics analysis(TMT),DNA methylation immunoprecipitation sequencing(Me DIP-SEQ),quantitative PCR and Me DIP-q PCR quantification,enzymes for three omics Combined immunosorbent assay(ELISA),Western blotting and other methods for statistical analysis.RESULTS:A total of 5572 proteins,20757 transcripts and 80,550 DNA methylation peaks were identified.The results showed that 1039 proteins,2286 genes and 260 methylated regions in U937-derived macrophages from M2 poles were identified.Differentiated expression in the process of transformation into the M1 phenotype.CONCLUSION:Partial proteomics,transcriptomics,and genomics components can influence the regulation of macrophage polarization into classically activated macrophages(M1)or alternatively activated macrophages(M2),tumor microenvironment and tumor metastasis.Part Ⅲ:Correlation between proteome,transcriptome and DNA methylation groupsOBJECTIVE:With the deepening of research on cells,research on RNA,protein and DNA methylation is constantly deepening,and many studies can directly or indirectly show that they can influence each other’s progress and thus influence Cell metabolism and growth.To this end,exploring the clear relationship between them is of great significance for the future regulation of cell prestige activities,especially for the research level of disease.METHODS:Through three groups of bioinformatics GO,Reactome and other databases,microchip detection,gene cluster analysis,protein expression profiling,and the differential data obtained in the second part,integrated analysis,combined with clinical The HCC samples were validated for the lncRNAs RP11-191G24.1 with significantly differential expression.RESULTS:Combined with GO enrichment analysis,differential proteins and genes were mainly enriched in immune response regulation,extracellular structural organization and receptor domains;5 of 18 receptors that were consistently down-regulated in proteomic data(ITGAM,ITGAL),ITGAE,NRP2 and CD44)also consistently showed down-regulation in transcriptome data;CD163 and CD69 m RNA expression correlated with observed protein expression;165 differentially expressed transcripts at both proteome and transcriptome levels Differential expression,post-transcriptional regulation revealed a large degree of diversity in the overall relationship between transcriptome and proteome in M1/M2 cells;and 17 lncRNAs were found to be significantly associated with DNA methylation changes;Corresponding to polarization,the acquisition or loss of DNA methylation in the promoter region is very significant,suggesting that cis-and trans-acting patterns may be key regulators;for epigenetic regulation and cancer Several representative regions of the signaling pathway were identified as being associated with M1/M2 polarization.To further examine the potential of these 3,703 differentially expressed lncRNAs as biomarkers,clinically detected HCC tissue samples and their corresponding adjacent tissues showed RP11-191G24.1 expression(P=0.036)and lymph node metastasis(p-value=0.06)associated with microvascular invasion(P=0.00).CONCLUSION:Detection of post-transcriptional regulation revealed a large degree of diversity in the overall relationship between transcriptome and proteome in M1/M2 cells;RP11-191G24.1 has potential as a prognostic biomarker for HBV-associated HCC;Methylation is a key regulatory mechanism regulating lncRNA expression.In summary,the proteome,transcriptome,and DNA methylation groups are interrelated and affect the polarization process of macrophages.