The Role And Mechanism Of MicroRNA-27a In Liver Ischemia Reperfusion Injury

Objective: Liver ischemia-reperfusion(I/R)injury is an important clinical problem as related to liver transplantation,hepatic resection and trauma.A large number of studies have shown that the mechanism of liver I/R injury involve complex and multiple pathways,including oxidative stress,inflammation,and cell apoptosis.Recent years,several studies have suggested that endoplasmic reticulum stress(ERS)plays a critical role in the progression of liver I/R injury,inhibiting ERS may provide a novel insight into the treatment of liver I/R injury.Accumulating evidence has demonstrated that microRNAs(miRNAs)have been implicated in various biological and pathological processes,and play significant role in various biological processes including cell proliferation,differentiation,apoptosis,and stress response.In addition,several reports have suggested that miRNAs could contribute to liver I/R injury by regulation of several key signaling pathways.Previous studies have showed that miR-27a induced cell apoptosis is associated with the ERS in HEK293 T cells.Recent studies have found that miR-27a inhibitors can reduce the expressions of GRP78 and CHOP,which are involved in ERS response,and thus participate in the process of diabetic nephropathy.In addition,multiple sutides have suggested that miR-27a participates in the progression of several diseases through targeting peroxisome proliferator-activated receptor gamma(PPAR?),and PPAR? is proved to be involved in liver I/R injury.However,the potential role of miR-27a in liver I/R injury is still unclear.Thus in the present study,the role and potential mechanism of miR-27a during liver I/R injury was investigated from the perspective of ERS in both vivo and vitro.Methods: 1.In vivo experiment: The models of liver ischemia-reperfusion injury in rats were established,with the ischemia time of 1 hour and reperfusion time of 6 hour.The liver function and the degree of liver damage were evaluated through detection of serum indicators of liver function and haematoxylin-eosin(HE)method.Serum alanine transaminase(ALT)and aspartate transaminase(AST)were determined by the Automatic biochemical analyzer.Real-time PCR was applied for the measurement of serum miR-27a level in liver I/R rats.To study the therapeutical effectof miR-27a on liver I/R injury in vivo,the I/R rats were treated with miR-27a antagomir and miR-27a NC by intraperitoneal injection.The relative expressions of miRNA-27 a mRNA were detected by Real-time PCR.TUNEL staining was performed to detect the apoptosis of the liver cell in each group.The protein level of GRP78,CHOP and PPAR? were detected using Western blot.2.In vitro experiment: The target gene of miR-27a was predicted by Targetscan,and was further verified using luciferase reporter assay.To further confirm the effect of miR-27a on liver I/R injury,H/R-induced cell model in AML12 was established,with the hypoxia time of 4 hours,and the reoxygenation time of 12 hours.MiR-27 a mimics,miR-27a inhibitors and PPAR? siRNA were transfected into AML12 cells using Lipofectamine 2000,and the cell viability was determined by the CCK-8 assay.Additionally,the flow cytometry was applied for the determination of the cell apoptosis.The relative expressions of miRNA-27 a mRNA in each group were detected by Real-time PCR.The protein level of GRP78,CHOP and PPAR? were detected using Western blot.Besides,the content of MDA and the activity of SOD in each group were measured to investigate the effect of miR-27a on oxidative stress.Results: 1.The serum level of ALT and AST was significantly increased after liver I/R surgery compared with Sham group.And HE staining of livers revealed large areas of hepatocyte necrosis and inflammatory infiltration in the I/R group.These results suggest that we successfully constructed a rat model of I/R in this study.2.In vivo experiment,we found that the serum miR-27a level and the protein levels of GRP78 and CHOP were increased significantly after liver I/R surgery.It suggested that liver I/R surgery induced the endoplasmic reticulum stress(ERS)of rats,and miR-27a may play an important role in the I/R-induced liver injury.3.In vitro experiment,we observed that the expression of miR-27a was significantly increased treatment compared with control cells.AML12 cells were transfected with miR-27a mimics and inhibitors,and the cell viability was detected by CCK8.The results showed that miR-27a inhibitors significantly improved cell viability compared with control group.W further detected their effect on cell apoptosis by flow cytometry.Compared with the control group,the apoptotic cells were markedly increased in H/R group,which could be effectively attenuated by miR-27a inhibitor.These results suggest that suppression of miR-27a may exert a protective effect against liver I/R injury.In addition,the low expression of miR-27a significantly inhibited the increase of MDA content and alleviated the decrease of SOD activity induced by H/R.Similarly,the low expression of miR-27a significantly reduced the expression levels of GRP78 and CHOP.Our data suggest that miR-27a regulates oxidative stress and ERS signaling in the H/R-induced hepatocyte injury.4.The target gene of miR-27a was predicted by Targetscan,and it was found that PPAR? was one of the candidate target genes of miR-27a,which was further verified by luciferase reporter technology.Western blot results showed that miR-27a significantly inhibited the H/R-induced elevation of PPAR?.Silencing of PPAR? significantly blocked the inhibitory effect of miR-27a inhibitor on ERS pathway.5.To study the therapeutical effect of miR-27a on liver I/R injury in vivo,the I/R rats were treated with miR-27a antagomir and miR-27a NC by intraperitoneal injection.The results suggested that serum ALT and AST levels were significantly decreased in Rats models after miR-27a antagomir treatment,and miR-27a antagomir markedly decreased I/R-induced apoptosis.In addition,after miR-27a antagomir treatment,the PPAR? level in rat liver was significantly up-regulated,and the I/R induced oxidative stress and ERS signaling pathway were inhibited.Conclusion: Taken together,these findings demonstrate that suppression of miR-27a protects against liver I/R injury by targeting PPAR? and inhibiting ERS pathway.This study provides new ideas and a new target for alleviating the liver I/R injury in clinic.