Therapeutic Effect Of BFGF Against Focal Cerebral Ischemia/Reperfusion Injury And Its Influence On Penumbral MicroRNAs Expression In Rat

Background and Objective:Ischemic stroke(IS)is a serious acute neurological disorder caused by the abrupt occlusion of intracranial vessels resulting in reduced blood flow to the brain region supplied and recanalization in early stage is the only effective therapy.Cerebral eschemia/reperfusion injury(CIRI)is the most common complication of IS and no effective therapy is available yet.Basic fibroblast growth factor(bFGF)is a multifunctional growth factor.It has been proved to have neuroprotective effect on CIRI,but the exact mechanism is obscure.MicroRNAs(miRNAs)are a class of endogenous non-coding small RNA molecules and widely expressed in brain.MiRNAs have been reported to be involved in CIRI and the potential targets in developing new strategies for IS therapy as their expression profiles are changing abnormally with the occurrence and development of CIRI.However,the influence of bFGF treatment on the expression patterns of miRNAs in brain with IS is unknown.In this study,the miRNA expression profile changes in the ischemic penumbra under the influence bFGF treatment were identified by high-throughput sequencing-based miRNA profiling method.The targets and likely roles of the differentially expressed miRNAs in ischemic penumbra of brain were predicted and analyzed by the combination of different bioinformatics methods.Methods:1.The animal model of focal CIRI was established by MCAO.The rats in the treatment group were treated with 5 μL bFGF by intracerebroventricular injection immediately after reperfusion.The rats in the sham and control groups were treated with equal volume of saline in same way.Neuroprotective effect of bFGF against CIRI was studied by neurobehavior evaluation,magnetic resonance imaging(MRI)and 2,3,5-triphenyltetrazolium chloride(TTC)staining.2.Total RNAs were extracted from the brain tissue of the ischemic penumbral region of rats from both the saline-treated control group and bFGF-treated group at 24 hours after reperfusion.The differentially expressed miRNAs between the control and treatment groups were identified by high-throughput sequencing-based miRNA profiling method and validated by RT-qPCR.3.The softwares of MiRanda,targetscan,RNAhybrid and databases of miRecords,miRTarBase,starBase were used for target gene prediction and screening.The likely roles of the predicted target genes were analyzed by GO(Gene Ontology)and KEGG Pathway(Kyoto encyclopedia of genes and genomes).The biological roles and network communities of the key miRNAs and their target genes were also analyzed with miRNA--gene-networks.Results:1.SD rats with focal CIRI were successfully obtained and treated with exogenous bFGF.2.The scores of neurologic deficit and the volumes of infarction area in the bFGF-treated rats significantly decreased at 24 hours,3 days and 7 days after reperfusion in comparison with the rats of the control group(P<0.05).These results indicated that exogenous bFGF can effectively protect the brain against CIRI.3.28 miRNAs were identified differentially expressed(fold changes>1.5,P<0.05)in the ischemic penumbra of brains between the control and treatment groups by high-throughput sequencing-based miRNA profiling method.Among them,16 miRNAs were up-regulated and 12 miRNAs were down-regulated by bFGF treatment.The difference in the expression levels of miR-10b-5p between the control and treatment groups was the most significant.To validate thehigh-throughput miRNA sequencing results,the expression levels of 4 arbitrarily selected miRNAs(miR-10b-5p,miR-423-3p,miR-329-3p,and miR-124-3p)were examined by RT-qPCR.The results of deep sequencing method and RT-qPCR method indicated that the change tendencies of the above miRNAs expression levels were consistent.3.A total of 2173 genes were predicted to be targeted by the 28 differentially expressed microRNAs.GO and KEGG Pathway analyses results indicated that majority of the targeted genes are associated with membrane-bounded organelle and were related to the regulation regulation of molecule modification,protein binding,metabolism,development,cell differentiation,and intracellular signal transduction..The main pathways involved are protein processing in endoplasmic reticulum and axon guidance.And miRNA-gene-network analysis indicated that miR-138 and miR-10b-5p may be the key miRNAs regulated by bFGF treatment and the key genes targeted by miR-138 in CIRI are MAPKs,AKt3,Plxnb2,Sema4d,Sema4c and the key gene targeted by miR-10b-5p is LDHA.Conclusions:Exogenous bFGF can effectively protect the brain against CIRI by improving neurologic outcome and decreasing brain injury.The neuroprotection of bFGF against CIRI is achieved by changing the expression profile of miRNAs in the ischemic penumbra area of the brains.The key miRNAs that were sensitive to bFGF treatment are miR-138 and miR-10b-5p which might be involved in regulating protein processing in endoplasmic reticulum,axon guidance,RIG-I-like receptor signaling pathway,FoxO signaling pathway,neurotrophin signaling pathway and HIF-1 signaling pathway via targeting the ischemic associated genes such as MAPKs,AKt3,Plxnb2,Sema4d,Sema4c and LDHA.