MicroRNA Expression Changes And Functional Analysis In Lung Ischemia Reperfusion Injury In Mice

Objective: To study the changes of expression profile of micro RNA in lung and lung ischemia-reperfusion injury model in mice and its related functional analysis by using high-throughput sequencing platform.Methods: Eight BALB / c mice were randomly divided into two groups,including 5 in experimental group and 3 in control group.In the experimental group,non-invasive microvascular clip was used to block the hilar in the left side of the experimental group for 45 minutes.After a 45-minute ischemic phase,the microvascular clip was removed and the mice's chest were closed,the left lung tissue was taken after 24 hours of reperfusion.While in the control group,we did not block the hilar when the left chest was cut open,then after 45 minutes the chest was closed,and the left lung tissue samples were taken after 24 hours.The lung tissues were stained with HE to observe the degree of lung inflammation and tissue damage under light microscope to evaluate whether the model of lung ischemia-reperfusion injury was established successfully.The expression of microRNA in the experimental group and the control group were detected by Illumina assay,Three microRNAs were detected by RT-qPCR method.The microRNAs were significantly different between the experimental group and the control group.The miranda software was used to predict the target genes of the significant differences of microRNAs.GO enrichment and KEGG pathway were used to predict microRNA target genes and the target genes involved in the pathway.Results: By using illumina high-throughput sequencing platform,20,613,567 and 22,970,296 clean reads were sequenced separately from the small RNA library constructed from the experimental group and the control group.From the identified 2601 known microRNAs and screening out 12 significant differences between the experimental group and the control group of microRNAs,of which five microRNAs were up-regulated more than twice,seven microRNAs were down-regulated and twice the above.The miR-122-5p,miR-129-1-3p and miR-410-3p were significantly different between the experimental group and the control group detected by Illumina sequencing platform by RT-qPCR.The validation results showed that the results of the two methods Match.Using miranda software to predict 2476 target genes,these target genes were mapped to 7261 loci.GO enrichment analysis showed that the predicted target genes can be classified into 6063 categories,among which 4634 GO terms were annotated to biological processes,975 were cellular components,and 854 were classified into molecular functions;KEGG pathway analysis showed that the predicted target genes could be classified into 222 biological pathways.Conclusion: The Illumina high-throughput sequencing(HiSeq)platform has provided us with good technical support for studying the expression changes of microRNAs in mice lung ischemia-reperfusion injury model.HiSeq have the advantages of high throughput,high repeatability,low signal to noise ratio and so on.Through the model of ischemia-reperfusion injury in mice reperfusion 24 h after ischemia for 45 min,we constructed small RNA library of experimental group and control group to obtain highquality microRNA expression profile and identified the use of traditional microarray technology.New microRNA that can not be found.In the experiment,a total of 12 microRNAs showed significant difference in expression between the experimental group and the control group.Functional annotation and pathway analysis in the GO enrichment and KEGG pathway analysis indicated that microRNA may be involved in each pathophysiological process of the lung ischemia-reperfusion injury,Western Blot results further verify this hypothesis.These findings will deepen our understanding of the role of microRNAs in lung ischemia-reperfusion injury and provide a theoretical basis and diagnostic strategy for treating various causes of lung ischemia-reperfusion injury.