Effect And Mechanism Of MiR-20a-5p Expression On Hepatocytes Injury Induced By Hypoxia/Reoxygenation

Background and objectiveIschemia-reperfusion injury is a more common pathological process in clinical practice.In tissues or organs,blood perfusion was resumed after ischemia for a period of time,Their function not only does not recover,but damages further.Sometimes in liver surgery it still need to take hepatic blood inflow occlusion to reduce the risk of bleeding and reduce the difficulty of operation.However,the residual liver will undergo ischemia-reperfusion injury after restoration of blood supply,and even lead to liver failure.Especially in long time ischemia,such as liver transplantation,ischemia-reperfusion injury is more likely to occur.Therefore,it is of important clinical value to study the mechanism of hepatic ischemia-reperfusion injury.There are variety of mechanisms involved in ischemiareperfusion injury,including mitochondrial dysfunction,lactic acid accumulation and acidosis,calcium overload,reactive oxygen species,free radical production,and neutrophil activation,etc.A large number of studies have found that mi RNA play an important biological role in cells.miRNA can specifically identify 3 ' non encoding region(3'-UTR)of mRNA to combine with the degradation of target mRNA or block the translation of the target mRNA and reducing the protein.The purpose of this study is to investigate the role of mi RNA in hepatic ischemia-reperfusion injury and provide a theoretical basis for the prevention and treatment of hepatic ischemia-reperfusion injury.In the early experiments,we established the hepatocyte hypoxia / reoxygenation model with human liver cell line L02 to simulate the ischemia-reperfusion process in the liver.After modeling,biochip technique was used to analyze the changes of all miRNA and mRNA expression in the cells before and after hypoxia / reoxygenation.The results showed that miR-20a-5p and HSP70 mRNA had significant changes both before and after hypoxia / reoxygenation.Above all,The aim of this study was to investigate whether miR-20a-5p and HSP70 interact in the injury of hepatocytes induced by hypoxia / reoxygenation,And their effects on the injury of hepatocytes after reoxygenation,to provide a theoretical basis for the prevention and treatment of hepatic ischemia-reperfusion injury in clinic.Methods1.Human liver cell lines L02 was cultured in vitro.Hypoxia and reoxygenation time gradients were established.Cell apoptosis was detected by flow-cytometry to establish hypoxia / reoxygenation model;2.The cell apoptosis was detected by CCK8 test,and the cell model was validated;3.The changes of ALT and AST expression in cell culture medium before and after hypoxia / reoxygenation were examined;4.Gene chip technology was used to detect the expression of all miRNA and mRNA in the control group,hypoxia group and reoxygenation group;5.The changes of mi R-20a-5p and HSP70 mRNA in the control group,hypoxia group and reoxygenation group were detected by Real-time PCR technique;6.Western blot was used to detect the change of protein HSP-70 in the control group,hypoxia group and reoxygenation group;7.miR-20a-5p mimic was transfected with Lipofectamine 2000 into L02 cells to upregulate the expression of miR-20a-5p,and miR-20a-5p inhibitor was transfected into cells to down regulate the expression of miR-20a-5p;8.After transfection of miR-20a-5p mimic and miR-20a-5p inhibitor,the changes of cell apoptosis before and after hypoxia / reoxygenation were investigated by CCK-8 and flowcytometry;9.Using Targetscan bioinformatics analysis to predict the target genes of mi R-20a-5p;10.After transfection of miR-20a-5p mimic and miR-20a-5p inhibitor,the changes of HSP70 in cells before and after hypoxia / reoxygenation were detected by Westen blot;11.Dual-luciferase reporter assay was used to detect the binding of mi R-20a-5p to HSP70 mRNA 3 '-UTR;12.The synthetic HSP70 siRNA was transfected with Lipofectamine 2000 into L02 cells to knock down the expression of protein HSP70,and the transfection efficiency was detected by Western blot;13.Simultaneous transfection of miR-20a-5p inhibitor and HSP70 siRNA into L02 cells,then CCK8 test and flow-cytometry were used to detect the damage of cells after hypoxia / reoxygenation,testing whether the protective effects of miR-20a-5p inhibitor on liver cells in hypoxia / reoxygenation injury can be inhibited by HSP70 si RNA,to verify the regulation of miR-20a-5p on HSP70;14.miR-20a-5p mimic and miR-20a-5p inhibitor were transferred into L02 cells respectively,followed by hypoxia / reoxygenation treatment,and the ATP content in each group was detected by ATP detection kit;15.miR-20a-5p mimic and miR-20a-5p inhibitor were transferred into L02 cells respectively,followed by hypoxia / reoxygenation treatment,then the expression of apoptosis associated proteins Bcl-2,Bax,and Cyt-c in cytoplasm were detected by Western blot.Results1.The results of flow-cytometry showed that the damage of L02 cells was most obvious after hypoxia 12 h and reoxygenation 12 h,and hypoxia / reoxygenation model of L02 cells was established by this condition;2.The result of gene chip showed that 2588 miRNA were detected,and 19783 mRNA were detected,and mi R-20a-5p and HSP70 mRNA showed significant changes in hypoxia group and reoxygenation group;3.The results of Real-time PCR showed that mi R-20a-5p was down regulated in hypoxia group and up-regulated in reoxygenation group,and the expression of HSP70 mRNA was up-regulated in hypoxia group and decreased in reoxygenation group;4.The results of Western blot showed that the expression of protein HSP-70 was upregulated in hypoxia group and decreased in reoxygenation group;5.CCK8 assay and flow-cytometry results showed that,compared with the control group,after hypoxia/ reoxygenation treatment in transfected miR-20a-5p mimic group,the hepatocyte damage became more serious,and in transfected miR-20a-5p inhibitor group,cell damage was significantly reduced;6.Target Scan bioinformatic analysis revealed that there was a possible binding site between miR-20a-5p and HSP70 mRNA 3 '-UTR;7.The results of Western blot showed that miR-20a-5p inhibitor inhibited the downregulation of HSP-70 after hypoxia / reoxygenation;8.Dual-luciferase reporter assay showed that the binding site of miR-20a-5p existed in the 3 '-UTR of mRNA HSP70 gene.mi R-20a-5p inhibits the expression of protein HSP70 by acting on the 3 '-UTR of the HSP70 mRNA;9.Compared with transfection of miR-20a-5p inhibitor,in the group,which miR-20a-5p inhibitor and HSP70 siRNA were transferred at the same time,the cell injury was aggravated after hypoxia / reoxygenation.These results indicate that,in the process of hypoxia / reoxygenation,the protective effects of miR-20a-5p inhibitor on hepatocytes can be reversed by HSP70 siRNA;10.Compared with only treated by hypoxia / reoxygenation,in the group,which miR-20a-5p inhibitor were transferred before hypoxia / reoxygenation treatment,the expression of ATP in cells increased.This suggests that up regulation of miR-20a-5p leads to dysfunction of mitochondrial function after hypoxia / reoxygenation;11.Compared with only treated by hypoxia / reoxygenation,in the group,which miR-20a-5p inhibitor were transferred before hypoxia / reoxygenation treatment,The expression of Bax and Cyt-c in cytoplasm were down regulated,but the changes of Bcl-2 in hypoxia / reoxygenation group were not obvious.This suggests that up regulation of miR-20a-5p inhibit the expression of protein HSP70 after hypoxia / reoxygenation,then the expression of mitochondrial related damage proteins increased,which indirectly lead to disorders of mitochondrial function,and hepatocyte injury was aggravated.ConclusionDuring the liver cell injury induced by hypoxia / reoxygenation,the expression of miR-20a-5p was down regulated during hypoxia,then the expression of miR-20a-5p increased rapidly after reoxygenation;After reoxygenation,the up-regulation of miR-20a-5p inhibited the expression of protein HSP70 which can guard protect mitochondria,then hepatocytes injury aggravated.