Roles Of MicroRNA-210 In Hepatic Ischemia Reperfusion Injury

Hepatic ischemia-reperfusion(IR)injury is a major complication of liver transplantation,resection and hemorrhagic shock.And hepatic IR injury is a complicated process involving multiple kinds of cells and mechanisms.However,the detailed molecular mechanism has not been elucidated,so there is a lack of corresponding effective therapeutic methods in hepatic IR injury.Although protective measures such as ischemic preconditioning,shortened ischemic time and reduced organ transport temperature were used to alleviate liver ischemiareperfusion injury,none of them have achieved the expected effect.Therefore,the mechanism of hepatic IR injury needs to be further studied to provide new methods and ideas for the prevention and treatment.MiRNAs are small noncodingRNAs of 18-22 nucleotides that mediate various physiological and pathological processes.MiR-210 is one of the classical hypoxia-induced microRNAs,and its role in tumor,immune and other organ IR injury have been reported.However,the role and mechanism of miR-210 in hepatic IR injury are still unclear.Objective:1.Explore the role of miR-210 in hepatic IR injury.2.Elucidate the molecular mechanism of miR-210 in hepatic IR injury.Methods:In order to explore the role of miR-210 in hepatic IR injury,we detected the levels of miR-210 in hepatocytes from mice following liver IR by quantitative PCR.And then we established hepatic IR injury model for wild type and miR-210 knockout mice.Pathological analysis,serum biochemical index and inflammatory factor level were used to evaluate liver injury.Subsequently,in vitro,experiments were carried out to verify the role of miR-210 in hepatic IR injury through regulating the expression of miR-210 in cells by transfection with miR-210 mimic or inhibitor.In mechanism research,candidate target genes of miR-210 were predicted by bioinformatics,and then verified by luciferase reporting experiment and biotinlabeled miRNA pull-down experiment.Then the co-transfection experiments were performed to confirme that miR-210 affected hepatic IR injury through repressing the target genes.For the upstream,the candidate transcription factors of miR-210 were predicted by bioinformatics,and then verified by Ch IP and luciferase reporting experiments.The above research methods were designed to explore the role and mechanism of miR-210 in hepatic IR injury.Results:In this study,we found miR-210 was induced in liver tissues from mice subjected to IR related surgeries.miR-210 deficiency remarkably alleviated liver injury,cell inflammatory responses and cell death in a mouse hepatic IR model.In vitro,inhibition of miR-210 decreased HR-induced cell apoptosis of primary hepatocytes and LO2 cells,whereas overexpression of miR-210 increased cells apoptosis during HR.Mechanistically,miR-210 directly suppressed SMAD4 expression under normoxia and hypoxia condition by directly binding to the 3' UTR of SMAD4.The pro-apoptotic effect of miR-210 was alleviated by SMAD4,while sh-SMAD4 abrogated the anti-apoptotic role of miR-210 inhibition in primary hepatocytes,indicating miR-210 regulated cell apoptosis by targeting SMAD4.Further studies demonstrated that hypoxia-induced SMAD4 transported into nucleus,in which SMAD4 directly bound to the promoter of miR-210 and transcriptionally induced miR-210 expression.Conclusions:This study revealed that miR-210 was induced during hepatic IR and aggravated liver injury through promoting hepatocytes apoptosis by targeting repressing SMAD4.Except HIF1?,another transcription factor,SMAD4,was found to regulate the expression of miR-210,forming a negative feedback loop with miR-210.And this study will provide new intervention targets for prevention and treatment of hepatic IR injury.