Identification And Functional Analysis Ofnovel MicroRNA Associated With Ischemic Penumbra In MCAO Model Of Rats

Vascular recanalization is an effective therapeutic strategy for acute ischemic stroke,and the ischemic penumbra represents the theoretical background for the application of this clinical therapy.Accurate detection of the penumbra is important for predicting whether patients will have a favorable response to recanalization therapy.At present,determination of the ischemic penumbra mainly depends on the imageological methods,however,the special equipment,exorbitant prices,sophisticated techniques and time cost make it popularize difficultly.A minimally invasive,easily manipulated,convenient and lowly expensed biomarker or a panel remains to be established.MicroRNAs(miRNAs)are endogenous small nucleic acid molecules.miRNAs play various roles in physiological or pathological processes of ischemic stroke,and were potential circulating biomarkers for penumbra.This study was designed to investigated the microRNA profiles associated with the penumbra in the different phases of ischemic in the permanent middle cerebral artery occlusion(p-MCAO)model of rats,to identify and characteristic the novel miRNAs and to explore the potential value of blood markers associated with ischemic penumbra.Part 1 The identification of novel miRNAs associated with ischemic penumbra in MCAO model of rats[Objective]To explore the microRNAs profiles of the penumbra and normal brain tissues in MCAO model of rats.And to predict the novel miRNAs,seek and verify the differential expressed miRNA?[Method]We applied a permanent,middle cerebral artery occlusion(p-MCAO)model in rats.We explored the microRNA(miRNA)profiles correlated with the penumbra in three different phases of ischemic stroke.Healthy male Sprague-Dawley rats were divided into 4 groups randomly:?sham,? 1 hour after ischemic,?2 hours after ischemic;?3 hours after ischemic;A 2-mm coronal section was cut from the optic chiasma in the caudal direction,and the penumbra was located in the area between the longitudinal line approximately 2 mm from the midline and the transverse diagonal line at the "2 o'clock" position.The tissue and peripheral blood total RNAs were extracted using TRIzolTM and TRIzolTM LS reagent,respectively.The screening of differentially expressed novel miRNAs was conducted using edgeR package of R 3.3.2.Their expression levels were detected by real-time quantitative polymerase chain reaction(RT-qPCR).[Results]Using deep sequencing analysis,32,89%sequences could not be mapped with the available non-coding RNA databases,we identified 9 significantly novel miRNA candidates in tissues.rno-miR-686-3p has a stable secondary folding molecule structure and conserved mature sequence across mammals suggest that it is a true miRNA.It was down-regulated at 1 h(p=0.042),3 h(p =0.032),and 4 h(p=0.007)after p-MACO in penumbra.rno-miR-686-3p was significantly expressed in the pancreas and brain tissues.In the peripheral blood specimens of these groups,we found that a sharp and great increase in the expression level in the 1-h group(p=0.015),but there were no significantly differences in the 3-h and 4-h groups compared with sham group.[Conclusion]rno-miR-686-3p is a novel miRNA,is abundant,in the brain tissue,and the expression level is significantly deregulated in the penumbra progressively with time and upregulated in peripheral blood at 1 h after p-MACO.Part 2 The localization of mir-686-3p in the ischemic penumbra and its related functional bioinformatics analysis[Objective]To explore expression and distribution of rno-mir-686-3p in the ischemic penumbra in different groups using In situ hybridization.The target genes of rno-mir-686-3p were predicted by bioinformatics,and the predicted results were analyzed with functional annotation clustering and pathway enrichment analysis.[Method]The SD rats were divided into four groups according to the method in the part I.After sham,A cardiac perfusion with 4%polyoxymethylene after sham,1 hour after p-MCAO,2 hours after p-MCAO,3 hours after p-MCAO,respectively.About 2 mm-thick coronal slices were obtained from optic chiasm to 2 mm posterior.In situ hybridization reaction was performed by using digoxin labeled oligonucleotide probe(LNA).The mean optical density(MOD)of each image was calculated using imageJ 1.48v image analysis software.The target genes predicted using the miRDB web tool.Molecular function annotation clustering and Kyoto Encyclopedia of Genes and Genomes(KEGG)signaling pathway enrichment analysis of target genes were conducted by DAVID online tool.[Results]rno-miR-686-3p,which localized in the cell nuclei of the cortex.Using deep sequencing analysis,the hybridization signal of miRNA 686-3p gradually attenuated in the sections of the ischemic penumbra over time.There were significant differences in each group MOD(p<0.001).Compared with the sham,the MOD was significantly decreased in 1 hour(p=0.001),3 hours and 4 hours were decreased(p<0.001).Compared with the 1 hour,the MOD were both significantly decreased in 3 hours and 4 hours(p=0.003 p<0.001,respectively).Compared with the 3 hours,the MOD was significantly decreased in 4 hours(p=0.022).A total of 297 potential target genes were predicted.Moreover,functional annotation clustering and pathway enrichment analysis suggested that rno-miR-686-3p might participate in transcription regulation,Wnt and cAMP signaling pathway.[Conclusion]rmo-miR-686-3p was associated with the penumbra:It was down-regulated at 1 h,3 h,and 4 h post p-MACO in the ischemic penumbra.miRNA target prediction,functional annotation clustering and pathway enrichment analyses suggested that rno-miR-686-3p may participate in transcription regulation and may modulate the Wnt signaling pathway and the cAMP signaling pathway.Furthermore,it may serve as a possible new biomarker with potential value for detecting the existence of the penumbra and predicting the curative effect of recanalization.