LPS-induced Exosomes Derived From Alveolar Epithelial Cells Regulate The Inflammatory Response Of Alveolar Macrophages Via MiR-92a-3p

Objective:To explore the molecular mechanism of lipopolysaccharide-induced alveolar epithelial derived exosomes to regulate the inflammation of alveolar macrophages.Method:The microarray technology was used to compare the expression profiles of miRNAs between normal alveolar epithelial cell-derived exosomes(AEC-Exo)and LPS-induced exosomes(LPS-AEC-Exo).Screened miR-92a-3p which was closely related to inflammation.After co-culturing LPS-AEC-Exo with AM,qRT-PCR technology was used to detect the expression of miR-92a-3p in AEC and AM;To overexpressed or silenced the expression of miR-92a-3p in AEC-Exo,miR-92a-3p mimic or inhibitor was transfected into AEC and co-cultured with AM to detect the expression of TNF-α,IL-1β,IL-6 and NF-κB signaling pathway in each group of AM.Bioinformatics predicts the downstream target genes of miR-92a-3p,After regulating the expression of miR-92a-3p in AEC-Exo and LPS-AEC-Exo,the downstream target genes PTEN was detecting in each group of AM;dual luciferase reporter gene experiment was used to verify the targetd binding of miR-92a-3p to the target gene 3’UTR.Result:1.The microarray results show that significant differential expression of miRNAs between AEC-Exo and LPS-AEC-Exo.Compared with AEC-Exo,there are 26 miRNAs with FC>2 and P<0.05,including 13 up-regulation and 13 down-regulation.In the up-regulated group,the top 10 miRNAs with different multiples were selected to verify the chip results.Using qRT-PCR technology,it was found that the 10 miRNAs screened were highly expressed in LPS-AEC-Exo compared with the AEC-Exo group and the level of miR-92a-3p expression was the highest.2.AEC-Exo,LPS-AEC-Exo and PBS were co-cultured with AM for 12 hours,and qRT-PCR was used to detect the expression level of miR-92a-3p in each group of AM.The results showed that AM treated with LPS-AEC-Exo had higher level of miR-92a-3p expression than normal AM,suggesting that LPS-AEC-Exo could transfer miR-92a-3p to AM.3.qRT-PCR technology detected that overexpression of miR-92a-3p in AEC-Exo can increase the levels of IL-1β,IL-6,TNF-α in AM,activate the NF-κB signaling pathway and induce AM inflammation;Transfecting inhibitor to silence miR-92a-3p in LPS-AEC-Exo can inhibit the NF-κB signaling pathway and weaken the AM inflammation induced by LPS-AEC-Exo.4.Bioinformatics analysis predicts that there is a complementary pairing sequence between the 3’UTR of the PTEN gene and the seed region of miR-92a-3p;double luciferase reporter gene verification found that transfection of miR-92a-3p can make wild type PTEN-3’UTR The luciferase activity of the vector was reduced by about 40%,but it had no significant effect on the luciferase activity of the mutant PTEN-3’UTR vector.5.Western Blot technology found that overexpression of miR-92a-3p in AEC-Exo can inhibit the level of PTEN protein in AM and increase the expression of P-p65 protein,while silencing miR-92a-3p in LPS-AEC-Exo caused PTEN in AM Protein expression is up-regulated and NF-κB nuclear translocation is suppressed.Conclusion:LPS-AEC-Exo inhibits PTEN through miR-92a-3p,activates the NF-κB inflammation signaling pathway in AM and induces AM inflammation.