Research On The Regulation Of MiR-221-5p In Macrophage Inflammation Induced By Ricin

In recent years,studies on the regulatory role of MicroRNAs in immune response and cellular inflammatory response have gradually increased.Studies on the regulation of the occurrence and development of inflammation in the process of inflammatory response and the treatment of detecting inflammation have also been gradually deepened.Previous studies have found that MicroRNAs affect cellular inflammatory response mainly by regulating the expression of inflammatory cytokines and the protein expression of inflammation-related signaling pathways at the post-transcriptional level.Castor is an important oil crop widely distributed in tropical and subtropical areas.Ricin is a protein toxin derived from the castor bean plant.It is one of the known deadly toxins.Inflammation is one of ricin’s toxic reactions,leading to tissue damage.However,the research on tissue inflammatory reaction after ricin poisoning is still not in-depth,especially the regulation of MicroRNAs gene expression in this inflammatory reaction process has not been clarified,which still needs further exploration.To further explore the regulatory role and mechanism of miR-221-5p in ricin-induced inflammation of macrophages,this study used bioinformatics methods to conduct differential expression analysis of transcriptomic sequencing data of ricin-induced inflammation of macrophages in our previous study group.The expression of differentially expressed MicroRNAs and mRNA was detected by q RT-PCR to verify the reliability of the analysis results.The target genes of MicroRNAs were predicted by software,and the regulatory network diagram of MicroRNAs mRNA was constructed according to the predicted results.Stable cell lines with miR-221-5p overexpression and knockout were constructed,and the transfection effect was verified by q RT-PCR.The protein expression of IL-1β and TNF-α in macrophages treated with ricin was detected by ELISA,and the protein expression of PI3K/AKT signaling pathway was detected by WB.The regulatory effects of miR-221-5p on the transcription and translation levels of target gene Col4a5 were verified by q RT-PCR and ELISA experiments.Double luciferase reporter gene assay verified the binding of miR-221-5p and Col4a5 and determined the specific way that miR-221-5p regulated the expression of Col4a5 gene.Results were as follows:1.Further analysis of the previous sequencing data of our group showed that there were 14 significantly differentially expressed MicroRNAs(9 up-regulated,5down-regulated)and 323 significantly differentially expressed mRNA(312up-regulated,11 down-regulated)in RAW264.7 cells treated with ricin.The significant differentially expressed MicroRNAs and mRNA were verified using q RT-PCR,and the results were consistent with the data analysis results.2.GO analysis and KEGG analysis were used to analyze the functions and involved signal transduction pathways of the significantly differentially expressed mRNA in ricin-treated macrophages.The results showed that the functions of differentially expressed mRNA were mainly the regulation of cytokines,nuclear composition,and protein interconnection,and were mainly enriched in PI3K/AKT signaling pathway.3.Miranda 3.3a software was used to predict the target genes of MicroRNAs and construct the microRNAs-mRNA regulatory network diagram.The results showed that miR-221-5p and Col4a5 had a regulatory relationship in ricin-induced macrophages.4.ELISA results showed that overexpression of miR-221-5p could promote the expression of IL-1β and TNF-α.WB results showed that overexpression of miR-221-5p could up-regulate the expression level of PI3 K protein and the phosphorylation of AKT protein.The results after inhibition of miR-221-5p were opposite to those of overexpression.Our data suggest that miR-221-5p plays a role in promoting ricin-induced inflammatory response in macrophages.5.q RT-PCR results showed that miR-221-5p significantly regulated Col4a5 gene expression at the transcriptional level.ELISA results showed that miR-221-5p had a regulatory effect on Col4a5 at the translational level,but the significance was not high.Dual luciferase reporter gene assay showed that there were complementary sequences between the transcript sequences of miR-221-5p and Col4a5 but no binding interaction between them.These results suggested that miR-221-5p was regulated in the same way as siRNA,by silencing the Col4a5 gene to inhibit its expression in cells.In conclusion,miR-221-5p suppressed the expression of Col4a5 in cells by silencing the gene.PI3K/AKT signaling pathway was activated by up-regulating the expression level of PI3 K protein and the phosphorylation level of AKT protein in the downstream PI3K/AKT signaling pathway of Col4a5.It can up-regulate the expression of pro-inflammatory cytokines IL-1β and TNF-α in macrophages and promote ricin-induced inflammatory response in macrophages.