The Role Of MiR-27a-3p Regulate Litaf In The Treatment Of Cerebral Ischemia-reperfusion Injury With Biliverdin

PartⅠObjective:To screen microRNA expression profiles and the differentially expressed miRNAs in the treatment of cerebral ischemia-reperfusion injury with biliverdinMethods:After administration with biliverdin to cerebral ischemia/reperfusion injury in rat,the cerebral infarct volume was measured by TTC staining,and the serum inflammatory factors IL-1β,IL-6,TNF-α expression levels were detected by enzyme-linked immunosorbent assay.In addition,the ischemic cerebral cortex in Sham group,IR group,and the BV treatment group were harvested for miRNA chip sequence.Moreover,the heat map was drawn based on miRNA chip sequence result,and the differentially expressed miRNAs was quantificated by qPCRResults:First of all,The cerebral infarction volume in rats treated with BV were reduced(P<0.05),secondly,the inflammatory factors IL-1β,IL-6,TNF-α expression levels in BV treated group were downregulated by Compared with the IR group.As for the miRNA chip sequence result,there are 10 miRNAs were differentially expressed in the BV group compared with the other groups.Finally,the miR-27a-3p expression level was verified by qPCR exhibited similar alterations as detected by miRNA chip sequence.Conclusion:The expression of miR-27a-3p was upregulated in the treatment of cerebral ischemia/reperfusion injury in rat with biliverdin,which indicated that miR-27a-3p expression may be related to the treatment of cerebral ischemia/reperfusion injury in rats with biliverdin.Part ⅡObjective:To predict,analyze and identificate target genes of rno-miR-27a-3p which selected from miRNA chip and provide the basis of further investigation of rno-miR-27a-3p related biological function in the treatment of cerebral ischemia-reperfusion injury.Methods:The target genes of rno-miR-27a-3p were screened through Target Scan,miRDB and miRwalk databases respectively.Then,bioinformatic analysis of these intersection target genes were performed by Gene Ontology analysis and KEGG Pathway analysis by DAVID database.Moreover,these intersection target genes’function and description were Queried through the NCBI database respectively.Results:42 target genes were intersected by three databases,and the biological Process of these genes mainly located in positive regulation of cellular process,cellular protein modification process,response to stress,cell death(P<0.05),etc.Molecular function of these genes were significantly involved in protein binding,protein kinase activity,transferase activity and transcription factor activity(P<0.05),etc.The KEGG Pathway analysis demonstrated that these genes were mainly enriched in p53 signal pathway,Wnt signal pathway,mTOR signal pathway,cGMP-PKG signal pathway,and autophagy pathway(P<0.05).Among them,the lipopolysaccharide-induced tumor necrosis factor alpha involved in the p53 signaling and autophagy pathway.Conclusion:rno-miR-27a-3p may target Litaf gene and then play a bioactive role in the treatment of cerebral ischemia-reperfusion injury in rats with biliverdin.Part ⅢObjective:To Confirm the direct interaction between miR-27a-3p and Litaf.Methods:First of all,predict the binding site between miR-27a-3p and Litaf by Target Scan database,Secondly,basing on the predicted interaction between miR-27a-3p and Litaf,the Litaf-3’UTR-WT,Litaf-3’UTR-MUT and miR-27a-3p mimic were constructed into the pmirGLO plasmid vector.Luciferase activity was measured after 48 h of co-transfection of 293T cells with the miR-27a-3p mimic and the WT or MT reporter vector.Results:There are a direct interaction between miR-27a-3p and Litaf though Target Scan database,The sequencing results showed that Litaf-3’UTR-WT,Litaf-3’UTR-MUT and miR-27a-3p mimic were successfully constructed into the pmirGLO plasmid vector,the dual-luciferase reporter gene assay results showed that the luciferase activity in miR-27a-3p mimic group significantly decreased which Compared to the control(P<0.001).Conclusion:The dual-luciferase reporter gene assay results suggested that Litaf was a direct target of miR-27a-3p.Part ⅣBackground:Ischemia-reperfusion(IR)-induced neuroinflammation,miR-27a-3p was one of miRNA among multiple microRNAs are dys-regulated during the treatment of biliverdin in cerebral ischemia-reperfusion injury,and the Litaf was confirmed as a target gene of miR-27a-3p.However,the roles of miR-27a-3p target Litaf in cerebral ischemia-reperfusion injury are unclear.Objective:Thus,we investigated whether miR-27a-3p is involved in the treatment of biliverdin in IR by regulating the LitafMethods:First of all,in order to establish overexpression and interference models,miR-27a-3p mimic/inhibitor and Litaf were injected into intracerebroventricular under the guidance of a stereotaxic instrument.After intracerebroventricular injection 7d,The IR model was established by Middle Cerebral Artery Occlusion for 2h and reperfusion 24h.The relationship between miR-27a-3p and Litaf was elucidated via RT-PCR,the cellular distribution of Litaf was determined via double immunofluorescence.The effect of miR-27a-3p on the protein expression of Litaf,Bax,Bc12,Caspase3,TLR4,NF-κB,and IL-6 were evaluated using western blotting.Double immunofluorescence staining was performed to delineate the cellular distribution of Litaf and TLR4/NF-κB/IL-6.The effect of miR-27a-3p regulate Litaf on cerebral ischemia-reperfusion injury was examined by TTC staining,TUNEL staining,NISSL staining,mNSS score,and measuring the water content.Results:① miR-27a-3p expression was obviously downregulated with time and reached its lowest levels at24h after IR compared with the levels in the sham surgery group(P<0.05).Likewise,the Litaf expression levels were increased after IR(P<0.05),suggesting a potential negative correlation between miR-27a-3p and Litaf expression(R squared=0.8516,P<0.001).② After intracerebroventricular injection 7d,the expression of miR-27a-3p/Litaf was detected by qPCR,the qPCR results showed that the expression level of miR-27a-3p was significantly increased(P<0.01)in miR-27a-3p mimic group.Likewise,and the expression level was decreased in inhibitor group(P<0.05),which revealed that we successfully established the overexpressed and interference model.③ Double immunofluorescence analysis of Litaf and markers of neuron,microglia in cerebral Cerebral cortex was performed at 24 h after IR,the majority of the e fluorescence signal for Litaf in the IR group was localized in the cells positive for NeuN and Ibal(cells with yellow signals)at 24 h after IR,which indicated the Litaf was upregulated in neurons and microglia.Furthermore,Litaf expression in the injured cortex increased over time,as shown the number of double-labeled cells in the quantitative analysis(P<0.05).④ Compared with the Sham group,intracerebroventricular injection of miR-27a-3p mimic significantly inhibited the Litaf,Bax,Caspase3,TLR4,NF-κB,and IL-6 protein levels at 24 h in the IR group(P<0.05),and these effects were abrogated in the presence of Litaf mimic(P<0.05).⑤Double immunofluorescence analysis shown that compared with the Sham group,intracerebroventricular injection of miR-27a-3p mimic significantly inhibited the fluorescence intensity of Litaf/TLR4,Litaf/NF-κB,Litaf/IL-6(P<0.05).⑥After miR-27a-3p regulates Litaf,the apoptosis cells in rat cerebral cortex were reduced(P<0.05),the Nissl bodies positive cells were increased(P<0.01),Moreover,cerebral infarct percentage also decreased(P<0.05),finally can improve the symptoms of neurological deficits in rats(P<0.05).Conclusion:①The Litaf was upregulated in neurons and microglia after IR-24h.②MiR-27a-3p regulates Litaf has anti-apoptotic and anti-injury role in cerebral ischemia-reperfusion injury.③miR-27a-3p regulate Litaf though TLR4/NF-κB/IL-6 signaling pathway.