Preliminary Study On The Expression And Mechanism Of Mir-181a During Hepatic Ischemia/reperfusion Injury

Background: Liver transplantation is accepted treatment option for end-stage liver diseases;nevertheless,a shortage of donor graft remains the major obstacles to the development of liver transplantation.Therefore,the method of expanding the application standard of donor graft and applying marginal donor graft in liver transplantation is gradually accepted by people.Compared with ideal donor liver,marginal liver is more vulnerable to injury from ischemia/reperfusion,Reducing ischemia-reperfusion injury in various ways is of great significance for the functional recovery of marginal donor liver transplantation.As a non coding single stranded RNA with a length of about 22 nucleotides,micro RNA plays an important regulatory role in many life activities,including ischemia-reperfusion injury.Mi R-181 a has been confirmed to play an important regulatory role in brain and eye ischemia-reperfusion injury,and its role in liver ischemia-reperfusion injury needs further exploration.Objective: To investigate the expression difference of mi R-181 a in liver ischemia-reperfusion injury and its possible mechanism.Research methods: C57BL/6J mice were used to construct a mouse ischemia-reperfusion injury model,and AML12 and MIHA cell lines were used to construct a cellular hypoglycemia-hypoxia-reoxygenation model.PCR was used to detect changes in the expression of mir-181 a at each time point in the model Happening.Use Targetscan,mi RDB,mi RWalk databases to predict the target genes of mir-181 a.The GO/KEGG enrichment analysis of target genes is carried out through the online website Hiplot and the Clue Go module of the software Cytoscape,the possible protein interaction network of the target gene is predicted by the software Cytoscape,and the key sub-networks are analyzed through the MCODE module.After mir-181a-mimics and mir-181a-inhibitor interfere with the expression level of mir-181 a in cells,a hypoglycemia-hypoxia-reoxygenation model is constructed,and the proteins of Caspase-3,Cleaved Caspase-3,Ki67,and PCNA are detected.Horizontal analysis of the effect of mir-181 a on cell apoptosis and proliferation in the process of cell hypoglycemia,hypoxia and reoxygenation.Results: By constructing a mouse liver ischemia-reperfusion injury model and detecting changes in the expression of mir-181 a with reperfusion time by PCR,it was found that the expression of mir-181 a was significantly increased when the perfusion was restored for 6hours;In the oxygen-reoxygenation-reoxygenation model,AML12 and MIHA cell lines both showed that the expression of mir-181 a was the most significant after 6 hours of hypoglycemia and hypoxia,and 2 hours of reoxygenation and hypoglycemia.238 target genes can be obtained by intersection of the results of Targetscan,mi RDB and mi RWalk databases.The GO enrichment analysis was is performed on them.A total of 41pathways’ p value was less than 0.05,of which 16 pathways were more significant than those with other functions;KEGG enrichment analysis of target genes showed that the life activities of mir-181 a mainly involved MAPK signaling pathway,Longevity regulating pathway-multiple species,Renal cell carcinoma,Rap1 signaling pathway,Insulin resistance,Colorectal cancer,Longevity regulating pathway,etc.The protein interaction network analysis of mir-181 a on target genes shows that 99 target genes have an interaction relationship with other target genes,among which the most critical sub-networks are involved with LONRF1,WSB1,KLHL2,KLHL5,RNF6,CBLB,RNF182,UBE3 C,and CUL3,which have 36 kinds interaction relations.By regulating the level of mir-181 a in cell lines cultured in vitro,after 6 hours of hypoglycemia and hypoxia,it was given glycosylation and reoxygenation for 2 hours.The apoptosis and proliferation-related proteins were detected by Western blotting.It can be seen that mir-181 a promoted cell apoptosis and inhibit their proliferation during the process of hypoglycemia,hypoxia and reoxygenation in vitro culture cells.Conclusion: mi R-181 a expression changes in the process of liver ischemia-reperfusion injury in vivo and in vitro.Through the targeted regulation of many genes and pathways,mi R-181 a can promote hepatocyte apoptosis and inhibit its proliferation.