Systematic Identification And Analysis Of Expressed Profiles Of MRNAs And LncRNAs And The Regulatory Mechanism Of The MRNAs Targeted By HnRNP K In Macrophage Inflammatory Response

Lipopolysaccharide(LPS)is the main component of the cell wall of gram-negative bacteria and plays an important role in the pathogenesis of bacterial infections.As one of the body’s important immune cells,macrophages play a pivotal role in the inflammatory response.There is growing evidence shows that a comprehensive and systematic understanding of transcriptome changes in LPS-induced macrophage inflammation helps us to dissect a new mechanism of inflammatory response.Long non-coding RNAs(lncRNAs),once thought to be transcriptional noise,have been recently shown to regulate a variety of biological processes.However,their roles in the inflammatory response are largely unexplored.In this study,we used RNA-seq technology to systematically investigate LPS-mediated transcriptomics changes of macrophage inflammatory responses,including messenger RNA(mRNA)and lncRNAs.Under the condition that the expression level of RNA accorded with the false discovery rate(FDR)less than 0.05 and Fold Change(FC)more than 4 times,we found that a total of 1187 mRNAs and 325 lncRNAs were significantly changed in LPS induced macrophage inflammation.By performing the Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichments analysis of the above-mentioned differentially expressed genes,the present study found that the functional enrichment of significantly altered mRNA and lncRNA all involved in the inflammatory response,immune response and cytokine regulation and other processes.The co-expression network profile for mRNAs and lncRNAs from the immune category among 70 differentially expressed mRNAs and 23 dysregulated lncRNAs suggests that lncRNAs play an important role in the regulation of functional mRNA expression in LPS-induced inflammation,which provides us a new direction for further research on inflammatory responses.In order to further explore the mechanism of transcriptional changes in inflammatory response,we used RNA co-immunoprecipitation combined with high-throughput sequencing(RIP-seq)to study the transcriptional regulation of hnRNP K.hnRNP K,which is an important RNA binding protein,plays an important role in the biological processes of DNA damage repair,transcription,mRNA transport and degradation.RIP-seq was used to enrich its bound mRNA.Screening the significantly mRNAs using the standard of P_value<0.01 and FC>5 screening,we found that a total of 188 kinds of mRNA were upregulated and 131 kinds of mRNA were downregulated.We also took GO analysis and found that up-regulated mRNAs are mainly enriched in the immune response,regulation of cytokines and inflammatory response and other processes,but the biological process of down-regulated mRNAs enrichment is complex,suggesting that its function is extensive.This result demonstrates that under normal physiological conditions,hnRNP K plays an important role in maintaining cell survival.While cells undergo inflammation,hnRNP K functions mainly act as a regulator of inflammation-related mRNAs and thus protects macrophages.Compared with the WT group,the expressed level of Chstl and Cxcl3 after stimulation with LPS significantly increased in the KD group,but the level of Cd40,Birc3,Trafl decreased.Cytosol-nuclear isolation and Western Blot demonstrated that the phosphorylation of hnRNP K was increased and the phosphorylated protein was mainly localized in the cytoplasm in response to LPS stimulation.When phosphorylation of hnRNP K was inhibited by ERK,the targeted mRNA level by hnRNP K were significantly changed.PI staining showed:LPS stimulation significantly increased the number of death cells in KD group compared with WT group.In conclusion,the present study used the RNA-seq technology to explore the expression profiles of mRNAs and lncRNAs and the regulatory mechanisms of gene expression by lncRNAs in LPS-induced macrophage inflammatory response.Through identification and analysis of hnRNP K-associated mRNAs by RIP-seq in macrophages treated with LPS,we explore its regulation mechanism for target genes:HnRNP K undergoes phosphorylation through the ERK signaling pathway,contributing to modulate the expression of targeted genes,which inhibit death and protect the body.