| Objective:To determine the expression profile of miRNA in rat lung post severe burn-blast combined injury and to explore the mechanisms of certain differentially expressed miRNAs regulation of lung injury.Methods of part I:Healthy male SD rats were randomly assigned into sham control(CT)group and burn-blast combined injury(BB)group.Micro-CT,lung wet/dry ratio and HE staining were employed to observe lung injury.The miRNA expression profiles of the lungs from both groups were detected with gene chips and compared with each other.Several miRNAs were selected for Real-time PCR to validate the array results.Bioinformatics analysis was conducted to speculate most possibly affected biological processes and pathways.TUNEL staining was used to detect apoptosis in the lung.Methods of part II:1.Healthy male SD rats(200-220g)were randomly assigned into CT group and BB group(n=45).The rats were sacrificed at pre and 6h,12h,24h,36h,48h post injury,respectively.The lung tissue was collected and the serum was kept for in vitro experiment.SP-C immunohistochemistry was done to affirm AT2 damage.While double-labeled immunofluorescent staining was used to verify AT2 apoptosis.SP-C and miR-486-5p expression were determined and their correlation were analyzed.IGF-1R protein and mRNA expression were determined.2.According to the plasmids and serum used,in vitro model was divided into:negative control plasmid + CT serum(NS)group;negative control plasmid + BB serum(NI)group;miR-486-5p mimics plasmid + CT serum(MS)group;miR-486-5p mimics plasmid + BB serum(MI)group;miR-486-5p inhibitor plasmid + BB serum(II)group.miR-486-5p and IGF-1R mRNA were determined by RT-PCR.IGF-1R and its downstream proteins in FoxO pathway were determined by western blotting.Flow cytometry was used to detect apoptosis in different conditions.Results of part I:Lung injury was obvious in BB group.Nine up-regulated miRNAs and two down-regulated miRNAs were detected by array test in BB group when being compared to CT group.RT-PCR validated the array results.Bioinformatics analysis highlighted transcription related GO biological processes and cell fate related pathways.Compared to CT group,apoptosis was significantly up-regulated in BB group.Results of part II:Positive rate of SP-C IHC staining in BB group was significantly lower than in CT group,indicating AT2 damage.Double-labeling immunofluorescence showed co-dyeing of TUNEL and SP-C,which illustrated that apoptosis had been a form of AT2 damage.Down-regulation of IGF-1R protein in BB group was significant when being compared CT group.While no significant difference was detected in IGF-IR mRNA level between CT and BB group.Transfection of miR-486-5p mimics plasmid brought similar effects on IGF-1R to A549 cell.In vitro model showed:BB serum significantly up-regulated apoptosis in cells transfected with negative control plasmids when being compared to CT serum;miR-486-5p up-regulation could exacerbate A549 cell apoptosis induced by BB serum;miR-486-5p up-regulation alone partially mimicked the effects of BB serum on A549 cell;miR-486-5p inhibition partially alleviated A549 cell apoptosis induced by BB serum.Expressions of IGF-1R and its downstream proteins in FoxO pathway changed in accordance with the situation.Conclusion:Burn-blast combined injury resulted in severe lung injury,which leaded to the change of miRNA expression profile.One of the up-regulated miRNA,miR-486-5p,caused AT2 apoptosis via PI3K/Akt/FoxO pathway by targeting IGF-1R.This at least partially explained how miR-486-5p leaded to lung injury. |
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