The Role Of LncRNANR₀03508 On Autophagy Of Macrophage Induced By Pseudomonas Aeruginosa Infection

Background and objective:Pseudomonas aeruginosa(PA)is a Gram-negative bacteria frequently involved in healthcare-associated pneumonia,septicemia and other diseases,and the main focus of PA research focus on the innte immune mechanism.Macrophages are primary phagocytes of the innate immune system that destroy invading pathogens through autophagy process.Long non-coding RNAs(LncRNAs)has been shown to be involved in the regulation of immune responses and disease progression.As a member of LncRNAs family,LncRNA NR＿003508 has not been reported in any previous research about its mechanism of regulating PA-induced autophagy.The present study aims to illustrate the potential mechanisms of LncRNA NR＿003508 in PA-induced autophagy,which enriches the basic research content of the mechanism of PA infectionMethods:The expression levels of LncRNA NR＿003508 was detected by qRT-PCR and fluorescence in situ hybridisation(FISH)probe.To silence the LncRNA NR＿003508,we used small-interfering RNA(siRNA).The autophagy-related proteins Beclinl,LC3,Atg5,Atg7,Atg12 and p62 expression in RAW264.7 cells and C57BL/6J mice lung tissues were detected by qRT-PCR,Western blot and immunofluorescence.Simultaneously,autophagic flux was detected by using the mRFP-GFP-LC3 adenovirus,autophagy rate was measured using the flow cytometry,and the expression of inflammatory factors in C57BL/6J mice was detected by ELISA.To further investigate the mechanism of PA on autophagy,we employed bioinformatics software to predict the specific miRNA and mRNA regulated by LncRNA NR＿003508.Furthermore,we conducted luciferase reporter assay and FISH probe to validate this prediction.And immunofluorescence and molecular means were used to study the regulation of LncRNA NR＿003508 on target genes through miRNA.Results:PA infection can induce autophagy in RAW264.7 cells and mice lung tissue,as well as promote the expression of LncRNA NR＿003508.Meanwhile,the bacterial load showed that knockdown of LncRNA NR＿003508 upregulated the bacterial load in RAW264.7 cells and mice lung tissue.In addition,knockdown of LncRNA NR＿003508 significantly inhibited the autophagy rate of RAW264.7 cells with PA infection On the basis of these results,we found that knockdown of LncRNA NR＿003508 significantly upregulated the expression of autophagy related factors LC3,Beclinl,Atg5,Atg7 and Atg12,while LncRNA NR＿003508 knockdown inhibited this phenomenon in vivo and in vitro experiments.At the same time,LncRNA NR＿003508 knockdown inhibited the expression of pro-inflammatory cytokines IFN-y and IL-1β,and up-regulated the expression of anti-inflammatory factors IL-4 and IL-10 in C57BL/6J mice.Mechanistically,we further predicted that LncRNA NR＿003508 interacted with miR-344i and miR-344g-5p by bioinformatics software,and LC3 and p62 could be the target genes of miR-344i and miR-344g-5p,respectively.Luciferase reporter gene assay and FISH probe verified the binding interaction.Further molecular methods showed that LncRNA NR＿003508 could promote the cleavage of its target gene from LC3I to LC3II by sponge adsorption of miR-344i and combine with miR-344g-5p to promote the degradation of p62.Conclusion:LncRNA NR＿003508 can promote autophagy with PA infection through the signaling axis of miR-344i/LC3 and miR-344g-5p/p62.In addition,LncRNA NR＿003508 rapidly improved the inflammatory level to eliminating pathogenic bacteria with PA infection,which may provide theoretical basis and data support for the infection mechanism of PA.