Sequencing Analysis Of Ricin-induced Macrophage Inflammation Related Genes And Functional Verification Of MiR-155-3p

In recent years about Micro RNA(mi RNA)to participate in the immune response and regulation of the inflammatory response in the process of research,especially of micro RNAs in the development and testing of the inflammatory response in the treatment of also gradually thorough.Generally,the regulation effect of mi RNA on inflammatory response is realized through post-transcriptional level regulation,and then influence the expression of inflammatory factors and the signaling pathway of inflammatory factors.Ricinus communis is a castor plant in the Euphorbia family,as an important oil plant,it is widely planted in China,Brazil,India and other countries.There have been reports of poisoning or death from ingesting castor beans,even a small amount of ingestion will cause strong inflammatory reaction of the body,resulting in tissue damage and threatening life and health.At present,the research and understanding of the regulation mechanism of the inflammatory response caused by castor seed poisoning are not deep enough.Especially,the role of mi RNA in the regulation of gene expression in this process is still unclear and needs to be further explored.This study is based on RNA high-throughput sequencing(RNA-seq)to study the gene expression of macrophages after Ricin toxin treatment.By screening significantly differentially expressed mi RNAs and m RNAs and analyzing their functions,the regulatory mechanism of mi RNA-m RNA regulatory network involved in the inflammatory injury of macrophages was explored,with a view to providing new ideas for exploring the toxic action mechanism of castor seed and clinical prevention and treatment.Research methods:1.The expression profiles of mi RNA and m RNA in RAW264.7 cells treated with RT and control group were sequenced by RNA-Seq,and the sequencing results were compared and annotated and analyzed by bioinformatics analysis.Real-time fluorescence quantitative PCR(q RT-PCR)was used to verify the accuracy of the sequencing results,and to detect the expression of differential mi RNA and m RNA.2.The accuracy of the sequencing results was verified by real-time fluorescence quantitative PCR(q RT-PCR),and the expression of differential mi RNA and m RNA was detected.3.The target genes of mi RNA were predicted by Miranda algorithm,and the differentially expressed m RNA,regulated by mi RNA was obtained by data matching.The mi RNA-m RNA regulatory relationship network was constructed by Cytoscape3.0 software,and the controlled target genes were analyzed by GO functional enrichment and KEGG pathway analysis..4.miR-155-3p overexpression and gene knockout stable cell lines were constructed.The expression of inflammatory cytokine TNF-α protein in mi R-155-3p mimics group,inhibitor group and NC group was detected by ELISA method.,Western blot method was used to detect the changes of inflammatory related protein IκB and p-IκB in mi R-155-3p mimics group,inhibitor group and NC group.5.Double luciferase reporter gene assay verified the binding of mi R-155-3p to the 3’untranslated region of its target gene GAB2;q RT-PCR method was used to study the effect of mi R-155-3p on GAB2 gene expression;Western blot method was used to detect the expression of GAB2 protein in mi R-155-3p mimics group,inhibitor group and NC group.The experimental results are as follows:1.RNA-Seq analysis showed that 14910 m RNA and 1599 mi RNA,were obtained,of which323 m RNA and 19 mi RNA were differentially expressed.The differentially expressed m RNA and mi RNA were verified by q RT-PCR,and the results were basically consistent with the sequencing results.2.2177 differentially expressed mi RNA target genes were predicted by Miranda algorithm,of which 213 could match the differentially expressed m RNA data.The results of GO analysis showed that the regulated differential m RNA was mainly involved in immune response,inflammatory response,cell adhesion and other biological processes.KEGG analysis showed that the regulated differential m RNA was mainly concentrated in JAK-STAT signal pathway,T cell receptor signal pathway and MAPK signal pathway.3.ELISA results showed that mi R-155-3p mimics could significantly inhibit the expression of TNF-α.The results of WB showed that mi R-155-3p mimics significantly down-regulated the level of p-IκB protein,while the result of mi R-155-3p inhibitor group was the opposite.These results suggest that mi R-155-3p plays an inhibitory role in the process of cellular inflammation induced by RT.4.In the target genes of mi R-155-3p predicted by bioinformatics,the gene levels of GAB2 were significantly differentially expressed.Dual-luciferase reporter gene assay confirmed that mi R-155-3p could specifically bind to the 3 ’-terminal non-translated region of GAB2 and regulate its expression.q RT-PCR and WB results showed that overexpression of mi R-155-3p could significantly inhibit the gene and protein expression levels of GAB2,indicating that mi R-155-3p had negative regulation on its target gene GAB2.Conclusion:Multiple inflammation-related signaling pathways were activated in RAW264.7 cells after RT treatment,and mi RNA played an important regulatory role in this process.By targeting GAB2,mi R-155-3p down-regulates the expression levels of its genes and proteins,thus playing a negative regulatory role in RT-induced inflammatory injury.Based on transcriptome sequencing,this study revealed the mechanism of RT-induced cellular inflammation,providing certain experimental basis and theoretical basis for further study on the regulation mechanism of mi RNA in RT-induced cellular inflammation and in-depth understanding of the toxic action mechanism of RT.