The Role And Mechanism Of Long Non-coding RNA Mirt2 In Inflammation

Part ?:Mirt2 is involved in the LPS-induced macrophage polarization and inflammationObjective:Toll-like receptor 4 is a member of the pattern recognition receptor family,which play a central role in innate immune response.Macrophage polarization induced by TLR4 binding with LPS on plasma membrane is the initiation of various inflammation related pathological process.Recent studies on long non-coding RNA indicate their novel regulation abilities in many physiological activities.However,the relationship between lncRNA and innate immune remains rarely explored.Here we propose series of experiments aimed at the function of lncRNA in the innate immune response.Methods:Samples from activated primary cultured peritoneal macrophages induced by LPS and PBS were separately collected and analyzed by microarray study.Those differentially expressed lncRNAs were selected as candidate genes for further research.The expression level of Mirt2 according to the stimulation of LPS or IL-4 was confirmed by qRT-PCR respectively.Fluorescence in situ hybridization(FISH)was performed to identify its subcellular location.The endotoxemia induced by LPS administration was used to study Mirt2 function in vivo.The expression levels of Mirt2 in different tissues were evaluated by qRT-PCR and FISH.Results:lncRNA microarray profile shows 98 lncRNAs were up-regulated in LPS stimulated macrophages,and 47 were decreased.Mirt2 was the most highly induced among them,and was highly expressed in macrophage at basal line.LPS promoted Mirt2 expression while IL-4 inhibited it.Mirt2 was expressed mostly in cytoplasm and colocalized with CD68,which is specifically existed on macrophage cell membrane in tissues.LPS administration induced Mirt2 expression in many tissues,including the heart,liver,spleen,lung and serum.Conclusions:Mirt2 is abundantly expressed in macrophage and up-regulated in cytoplasm during macrophage phenotype switch towards M1 macrophage.Mirt2 is down-regulated when stimulated by IL-4.In the endotoxemia mice,the Mirt2 levels in various tissues are increased due to the up-regulated Mirt2 in tissue macrophage cells rather than parenchymal cells.Part ?:Mirt2 regulates TRAF6 by inhibiting its oligomerization and autoubiquitinationObjective:Previous works have revealed the potential connection between lncRNA Mirt2 and LPS related inflammation.This section explained the partner molecule and the role of Mirt2 played in cell signal pathway.Methods:To determine the potential interacting protein partner of Mirt2,a RNA-pull down assay was performed in macrophage.TRAF6 was chosen to be a candidate partner according to the mass spectrometry following the pull-down assay.Additionally,its relationship with LPS induced polarization was taken in to consideration.Their interaction was confirmed by co-immune precipitation,RNA immunoprecipitation,FISH and immunostaining.The truncation of Mirt2 and TRAF6 was induced into cells respectively followed by co-IP assay to determine their domains essential for interaction.Mirt2 overexpression and knockdown were achieved by recombinant adenovirus infection.The oligomerization and ubiquitination of TRAF6 was evaluate by co-IP and western blot followed by processed as indicated.The phosphorylation of p65,jnk,which play as downstream of TRAF6,was evaluated by western blot.Results:TRAF6 can bind to Mirt2 in macrophage cells.Either TRAF6 RING domain or zinc finger motifs defected causes its binding ability dysfunction.Exogenous Mirt2 inhibited K63-ubiquitination of TRAF6 but left the K48-linked ubiquitination uninfluenced.Knockdown Mirt2 resulted in elevation of the phosphorylation of p65 and jnk,which can be rescued by TRAF6 knockdown.Conclusions:TRAF6 is a key point for Mirt2 regulation function.Mirt2 interacts with TRAF6 via binding to RING domain and zinc-finger motifs.Mirt2 diminishes the oligomerization and K63-ubiquitination of TRAF6.Mirt2 promotes the phosphorylation of p65 and jnk in a TRAF6-depended manner.Part ?:Mirt2 regulates macrophage polarization and attenuates inflammationObjective:As for the crucial role Mirt2 played in the signal pathway involved in inflammation was discussed above.This part explored the effects of Mirt2 on macrophage polarization and inflammation in vivo and in vitro.Methods:Exogenous Mirt2 overexpression in vivo was achieved by administration of Mirt2 recombinant adenovirus via tail vein.After pretreatment of adenovirus administration,intraperitoneal injection of LPS was performed to imitate the endotoxemia.Survival rate was recorded,concentration of serum proinflammatory factors was measured by ELISA.Central molecule in inflammation signal pathways was evaluated by western blot.The degree of acute inflammation was assessed by pathology studies of the lung and liver tissue.Mirt2 levels were measured in the myocardium of AMI(acute myocardial infarction)mice,the aortas of atherosclerotic mice,and the kidneys and myocardium of STZ(streptozotocin)induced diabetic mice,the adipose tissues of ob/ob mice,HFD(high fat diet)induced NAFLD(nonalcoholic fatty liver disease),CC14(carbon tetrachloride)induced acute liver injury,and MCD(methionine choline deficient diet)induced NASH(nonalcoholic steatohepatitis),compared with the levels in their normal littermates.Results:Overexpression of Mirt2 in macrophage inhibited its classically activation induced by LPS.M1 related proinflammation factors was attenuated by Mirt2 overexpression.The LPS-activated signal pathway MAPK and NF-?B was alleviated by Mirt2.Mirt2 diminished p65 and jnk phosphorylation in vivo.Exogenous Mirt2 via adenovirus injection resulted in a profound decrease in the plasma concentrations of the proinflammatory cytokines(IL-1?,IL-6,and TNF?)compared to the negative control group.The pathology of diverse tissues shows less organ injury associated with inflammation due to the Mirt2 overexpression.Mirt2 was upregulated in lesion tissues from AMI,obesity,STZ-induced diabetes,atherosclerotic mice.Mirt2 was colocalized mainly with iNOS+M1 macrophages not Argl+M2 macrophages.Conclusions:Mirt2 regulates classically activated macrophage polarization.Mirt2 alleviated MAPK and NF-?B signal pathway in vivo and in vitro.Mirt2 had anti-inflammatory effects on endotoxemia and protected organs from LPS challenge.Mirt2 is involved in several inflammatory diseases.