Anti-apoptotic Effect Of MiR-27a-5p On Hepatic Ischemia/Reperfusion Injury

Background:Hepatic ischemia-reperfusion injury(IRI)is an important cause of liver injury,which can induce cell apoptosis and lead to cell dysfunction.Bach1 is a mammalian transcription factor that inhibits the production of Hmox1,while Hmox1 can encode heme oxygenase-1(HO-1),HO-1 can play a role in anti-apoptosis,antiinflammatory and other protective effects in IRI.MiR-27a-5p is expressed in many kinds of cells,and it has been proved that it can be targeted to a variety of apoptosis related genes.However,it is not clear whether miR-27a-5p can regulate the expression of Bach1-HO-1 in hepatic ischemia-reperfusion injury.Objective:Using the dual luciferase reporter system to verify whether Bach1 is the target gene of miR-27a-5p,to investigate whether miR-27a-5p-Bach1 pathway participates in mice liver ischemia-reperfusion injury,and establish the in vitro hypoxia / reoxygenation(H/R)models to further explore the anti-apoptosis mechanism.Methods:In vivo experiment: 1)Established the models of liver ischemia-reperfusion injury in mice.The warm ischemia time was 1h,and the time of reperfusion was 0h,12 h respectively.2)HE staining was used to detect the damage of hepatocytes.3)The level of caspase-3 protein was detected by immunohistochemistry.4)Tunel method was used to detect the apoptosis of hepatocytes in each group.5)The expression levels of Bach1 and HO-1 were detected by Western blot.6)RT-qPCR was used to detect miR-27a-5p expressions at different time points.In vitro experiment: 1)Dual luciferase reporter system was used to detect whether miR-27a-5p is combined with Bach1 3'-UTR region to target on it.2)Hypoxia / reoxygenation models were established in mice liver AML12 cells.The hypoxia time was 1h,and the time of reoxygenation was 0h,12 h respectively.3)RT-qPCR was used to detect mi R-27a-5p expressions at different time points in cell models.4)MiR-27a-5p mimic,inhibitor and Bach1 siRNA were transfected into the cell models.5)The expression levels of Bach1,HO-1,anti-apoptotic gene Bcl2 and pro-apoptotic gene caspase-3 in the cells were detected by Western blot.6)Using Annexin V-FITC method to analyze the changes of cell apoptosis in different transfection groups of 12 h reperfusion.Result:1.A search of targetscan7.0 database showed that there were two miR-27a-5p seed match sites in the 3'-UTR of Bach1 mRNA and it was found that miR-27a-5p significantly reduced the luciferase activity of Bach1 in the region of 3'-UTR by duel luciferase reporter system.2.For liver injury in mice,the results of HE staining found that with the increase of reperfusion time,liver sinus congestion were more obvious,the hepatic lobule structures disordered,inflammatory cell infiltration increased and there were cell ballooning even partial eosinophilic necrosis.Cell apoptosis also increased by detecting of Tunel,immunohistochemical detection of caspase-3 has the similar results.3.The expression levels of miR-27a-5p increased with the prolongation of reperfusion,the highest expression was at 12 h and the expressions of HO-1 also peaked at 12 h of reperfusion,at this time,the Bach1 levels were at the least.4.Transfected with mi R-27a-5p mimic in hypoxia / reoxygenation models and found that by overexpressioning the level of miR-27a-5p,compared with the control group,the expression of Bach1 decreased,HO-1 levels increased and expression of anti-apoptotic gene Bcl-2 protein levels increased,the expression of pro-apoptotic gene caspase-3 decreased(P<0.05),transfected with miR-27a-5p inhibitor got the opposite result(P<0.05),at the same time,transfected with Bach1 siRNA and compared with the control groups,the expressions of HO-1,Bcl-2 increased(P<0.05),caspase-3 protein level decreased(P<0.05),the difference was statistically significant.5.Select 12 h reoxygenation time point,compared with the control group,we could find that overexpression of miR-27a-5p and transfected with Bach1 siRNA reduced the expressions of Bach1(P<0.05),increased the levels of HO-1 mRNA(P<0.05),down-regulated of miR-27a-5p can have the opposite results.6.Select 12 h reoxygenation time point,compared with the control group by Annexin V-FITC method we could find that mi R-27a-5p mimic and Bach1 siRNA groups had lower apoptosis rate(P<0.05),transfected with miR-27a-5p inhibitor increased apoptosis rate(P<0.05).Conclusion:1.MiR-27a-5p target at Bach1 by duel luciferase reporter system.2.MiR-27a-5p and its regulatory pathway of Bach1-HO-1 participate in the process of hepetic ischemia-reperfusion injury in mice.3.This study suggests that overexpression of miR-27a-5p may play an important role in anti-apoptosis in hepatic ischemia-reperfusion injury.