LncRNA Gm26917 Regulates Macrophage Differentiation By Enhancing Annexin A1 Ubiquitination In LPS-induced Acute Liver Injury

Background:The liver is the largest gland in the body and plays a central role in metabolism and immune balance.This organ is responsible for more than 200 functions such as detoxification,storage,blood clotting,energy production,nutrient conversion,and hormone balance.These important physiological functions make the liver a critical organ for host survival after severe trauma,such as sepsis.In recent years,more and more studies have found that the polarization of macrophages plays an important role in LPS-induced immune dysfunction,but there is no specific therapeutic intervention for LPS-induced acute liver injury.Therefore,it is of great significance to further understand the molecular mechanism of liver injury to provide molecular biomarkers for its early diagnosis and new treatment strategies.Lnc RNA is a class of RNA molecules with a transcript length of more than 200 nucleotides.Its expression is regulated by development and plays a key role in gene transcription and epigenetic regulation.However,its molecular mechanism in LPSinduced acute liver injury is not entirely clear.Aims: To explore the biological role and mechanism of lnc RNA Gm26917 in LPSinduced acute liver injuryContent:1.High-throughput sequencing was used to screen and verify differentially expressed lnc RNAs in LPS-induced acute liver injury: First,the differentially expressed lnc RNAs in acute liver injury were screened according to the results of RNA-seq,and q PCR was used in LPS-treated liver tissue macrophages.Validation in phagocytes to determine the final research object,the target long non-coding RNA;2.Identification and location of Gm26917 in macrophages:According to the screening results of sequencing and verification in LPS-induced liver injury samples,Gm26917 was determined to be studied.Fluorescence in situ hybridization（FISH）was used to determine the location of Gm26917 in macrophages;3.To explore the role of Gm26917 in LPS-induced acute liver injury in vivo: Gm26917 was knocked down by tail vein injection of lentivirus to observe the liver damage in mice;4.To explore the role of Gm26917 in macropages in vitro: RNA sequenceing and q PCR were used to explore and verify the effects do knockdown of Gm26917 on the inflammatory immune response of macrophages.The effect of knockdown of Gm26917 on macrophage polarization was analyzed by flow cytometry;5.To screen and validate the interaction between Gm26917 and ANXA1: The possible binding proteins of Gm26917 were screened by cat RAPID database and verified by RIP-q PCR,fluorescence in situ hybridization and immunofluorescence co-staining in LPS-treated macrophages;6.To explore the mechanism of Gm26917 promoting the inflammatory response of macrophages: The total protein expression of ANXA1 was detected by knockdown and overexpression of Gm26917,respectively,and the effect of Gm26917 on ANXA1 ubiquitination was detected by western blotting.Luciferase reporter gene assay was used to detect the effect of Gm26917 on NF-κB,and the effect of Gm26917 on the phosphorylation of p-65 and IKK-α were measured by western blotting;7.To investigate FOXM1 can regulate the expression of Gm26917 in macrophages:Western blotting and q PCR were used to detect the knockdown efficiency of FOXM1,the expression of Gm26917 and inflammatory factors（IL-1β,IL-6,TNF-α）after knockdown of FOXM1 was detected by q PCR;CHIP-q PCR confirmed that Gm26197 was regulated by the transcription factor FOXM1.Results:1.Lnc RNA Gm26917 was highly expressed in both LPS-treated hepatic macrophages and peritoneal macrophages.Fluorescence in situ hybridization showed that Gm26917 was localized in the cytoplasm of macrophages;2.Lentivirus-mediated gene silencing of Gm26917 attenuated liverinflammation and protected mice from LPS-induced acute liver injury;3.Gm26917 is an important regulator of macrophage inflammatory response.Si RNAmediated silencing of Gm26917 attenuates the inflammation of macrophages and reduces the phagocytic function and chemotactic ability of macrophages.Gm26917 facilitated macrophage polarization from the proinflammatory M1 phenotype to the alternatively activated M2 phenotype;4.The 3’-truncation of Gm26917 interacted with the N-terminus of Annexin A1;5.Gm26917 knockdown suppressed NF-κB activity by decreasing the ubiquitination of Annexin A1 and its interaction with NEMO.6.Gm26917 affected K6,K11,K29,K33,and K63 branched ubiquitin chain formation,while K27 and K48 branched ubiquitin chains were not affected.7.The expression of Gm26917 in inflammatory macrophages was regulated by the transcription factor forkhead box M1（FOXM1）.LPS treatment dramatically increased the binding of FOXM1 to the promoter region of Gm26917 in macrophages.Conclusion:Gm26917 regulates macrophage differentiation by promoting the ubiquitination of Annexin A1 in LPS-induced acute liver injury.Gm26917 could be a potential therapeutic target for the treatment of inflammatory liver diseases.