MiR-27a-3p Aggravates Hepatic Ischemia-reperfusion Injury In Mice By Inhibiting OTULIN

Objectives:1.To investigate the changes in the expression levels of miR-27a-3p and OTULIN in the hepatic ischemia/reperfusion in vivo/vitro model,and to clarify the targeting effect of miR-27a-3p on OTULIN.2.To study the effects of miR-27a-3p and OTULIN on the release of inflammatory factors,liver function damage and liver histological changes in the hepatic ischemia/reperfusion in vivo model.3.To analyze the specific molecular mechanism of miR-27a-3p in the hepatic ischemia/reperfusion in vitro model by targeting OTULIN.Methods:1.Establish an in vivo/vitro model of hepatic ischemia reperfusion,and use qRT-PCR to analyze the changes of miR-27a-3p and OTULIN mRNA expression levels at each time point of the model;The double luciferase reporting assay examined the targeting effect of miR-27a-3p on OTULIN.2.miR-27a-3p antagomir,miR-27a-3p agomir,ADV-OTULIN were used to regulate the expression levels of miR-27a-3p and OTULIN in the hepatic ischemia/reperfusion in vivo model.Serum AST and ALT levels were detected by microplate method,serum IL-6 and TNF-? levels were detected by ELISA,and liver tissue damage was observed by HE staining.3.miR-27a-3p antagomir,miR-27a-3p agomir and ADV-OTULIN were used to regulate the expression levels of miR-27a-3p and OTULIN in the hepatic ischemia/reperfusion in vitro model,and the expression levels of OTULIN,NF-?B signaling pathway related proteins,met1-linked ubiquitination and the changes of p65 nucleation were detected by western blotting.The interaction between OTULIN and HOIP was detected by immunoprecipitation.The levels of supernatant IL-6 and TNF-? were determined by ELISA.Results:1.qRT-PCR results showed that the expression levels of miR-27a-3p in the hepatic ischemia/reperfusion in vivo /vitro model were decreased gradually with time,while the expression level of OTULIN were increased gradually with time.The results of the dual luciferase reporting experiment showed that miR-27a-3p agomir could significantly inhibit luciferase activity in the OTULIN wild-type(WT)co-transfection group.2.The microplate method showed that the down-regulation of miR-27a-3p in the hepatic ischemia/reperfusion in vivo model could significantly reduce the expression levels of serum AST and ALT,and overexpression of OTULIN could reverse the increased expression levels of AST and ALT serum caused by upregulation of miR-27a-3p.ELISA showed that down-regulated miR-27a-3p could significantly reduce the expression levels of serum IL-6 and TNF-?,and Overexpression of OTULIN could reverse the increased IL-6 and TNF-? expression levels caused by upregulation of miR-27a-3p.HE staining results showed that down-regulated miR-27a-3p could significantly alleviate liver tissue damage in the mice model,while overexpression of OTULIN could reverse the aggravation of liver tissue damage caused by up-regulation ofmiR-27a-3p.3.Immunoprecipitation showed that OTULIN could directly bind to HOIP in RAW264.7 cells.Western blotting results showed that down-regulated miR-27a-3p inhibited the expression of NF-?B signaling pathway related proteins,met1-linked ubiquitination levels,p65 nucleation,and increased the expression of OTULIN.While overexpression of OTULIN could reverse the increased expression of NF-?B signaling pathway related proteins,met1-linked ubiquitination levels,p65 nucleation levels,and inhibited expression of OTULIN caused by the overexpression of miR-27a-3p.ELISA showed that down-regulated miR-27a-3p could inhibit the release of IL-6 and TNF-? in the supernatant,while overexpressed OTULIN could reverse the increased secretion of IL-6 and TNF-? caused by up-regulation of miR-27a-3p.Conclusions:1.miR-27a-3p can target on OTULIN directly,and the expression levels of miR-27a-3p in the hepatic ischemia/reperfusion in vivo/vitro model were decreased significantly with time,while the expression levels of OTULIN increased significantly with time.2.MiR-27a-3p can aggravate mice hepatic ischemia/reperfusion injury by targeting OTULIN.3.MiR-27a-3p can aggravate the activation of NF-?B signaling pathway through promoting the ubiquitination level of macrophage RAW264.7 by targeting OTULIN.