Expression Of Autophagy-Associated Protein And MiRNA After Liver Impact Injury In Rats

Part 1:Bioinformatics analyzed the regulation of autophagy associated protein LC3 and Beclin1Objective: Bioinformatics analyzed the regulation of autophagy associated protein LC3 and Beclin1Methods: In this study,through the use of a variety of bioinformatics software and database,it determines the autophagy genes,and to explore the related regulation of miRNAs,clarify the existence of downstream target genes of these transcription factors,miRNAs and its upstream feedback and feedforward pathway,as well as understand the role of miRNAs in mechanism in the process of autophagy in order,and the next step for the experimental verification mechanism the molecular regulatory networks provide clues.Results: LC3 and Beclin1 two autophagy related genes were screened by Pubmed database as the research object.A total of 1191 miRNAs predicted to interact with LC3 were predicted by mi RWalk software.Among them,99 miRNAs were predicted by four prediction softwares and 10 miRNAs were related to autophagy.Beclin1 and 2132 miRNA binding sites exist,of which 114 miRNA prediction software for the four co-predicted from17 miRNA involved in autophagy process.After comparing the number of papers reported,LC3 selected mi R-140-3p,mi R-214-3p and mi R-301a-5p as candidate target miRNA,and the target miRNA of Beclin1 was mi R-124-3p,mi R-21-3p and mi R-30a-5p.Consite software were transcription factor prediction of 6 candidate miRNA,confirming that a total of 46 transcription factors were combined with 6 candidate miRNA,including E47 A,AML-1,n-AMY,Snail,C-REL,c-FOS,IRF-1,CREB,E2 F,NF-Y,p65,NF-KB 12 transcription factors involved in autophagy.All related genes constitute a regulatory network and regulate each other,playing an important role in the occurrence and development of autophagy.Conclusion: 12 transcription factors including E47 A,AML-1,n-AMY,Snail,C-REL,c-FOS,IRF-1,CREB,E2 F,NF-?B,p65 and NF-Y influence respectively mi R-214-3p,mi R-301a-5p,mi R-124-3p,mi R-21-3p and mi R-30a-5p in order to regulate the expression of LC3 and Beclin1 and participate in the occurrence and development of autophagy,which provides a new theoretical basis for clarifying the mechanism of autophagy,but also provides a good guide for the follow-up experimental verification.Part 2:Expression of Autophagy Related Protein and Mi RNA in Liver Tissues of Rats after Hepatic InjuryObjective: To investigate the effect of autophagy on hepatic impact injury in rats and its molecular mechanism inovlved.Methods: Sixty healthy male Sprague-Dawley(SD)rats(clean grade)were randomly divided into two groups,the normal group and the model group(n=30 in each group).An impact injury model of rat liver was established by impacting rat liver with 200 k Pa impact using BIM-V biological impactor.The enzyme activity of ALT and AST in blood serum was measured by rate method;the changes of TNF-?,IL-8 and IL-2 levels were detected by ELISA;morphological changes were observed by microscope after routine HE staining;the formation of autophagosomes was observed by laser scanning confocal microscopy;protein expression levels were detected by Western blotting and immunohistochemical methods,and the relative expression levels of genes were determined by RT-PCR;Bioinformatics software was used to analysis the regulate miRNAs of microtubule-associated protein l light chain 3(LC3)and Beclin1;a double-fluorescent reporter gene vector was constructed to detect the interactions between genes.Results: The serum levels of ALT,AST,TNF,IL-8 and IL-2 in the model group were markedly higher than those in the normal group,and the difference was statistically significant(p<0.01).The morphology of the liver tissue in the normal group was normal,versus that in the model group was damaged to different degrees,for example,the sheet necrosis occurred in the surrounding area of the central veins of hepatic lobules where a large number of necrotic cells were observed.The expression of LC3 and Beclin1 proteins of liver tissues in the model group was significantly increased(p<0.05).Mi R-124-3p negatively regulated the Beclin1 expression and mi R-140-3p negatively regulated the LC3 expression.Conclusion: Autophagy plays an important role in the hepatic impact injury in rats,and mi R-124-3p and mi R-140-3p.2 may act as negative regulators of Beclin1 and LC3 expressions,respectively.