Screening And Preliminary Analysis Of MiRNAs Expression Profile In Rat During Spinal Cord Ischemia-reperfusion Injury

Spinal cord ischemia-reperfusion injury (SCIRI) is an important pathogenicmechanism of spinal cord secondary injury. Studies show that the secondary injury,such as SCIRI, is an important factor involved in nerve injury. However, there are noeffective treatments and specific drugs towards SCIRI, and the pathogenic molecularmechanism of SCIRI is still unclear now, which are big hindrances to thedevelopments of novel effective treatments and specific drugs. Hence, it is extremelyurgent to underline the molecular mechanism of SCIRI.MicroRNA (or miRNA) is an endogenous non-coding18~24nt short chain RNAmolecule, which has specific mRNA translation inhibitory function, that wouldregulate the gene expression at post-transcriptional level. Recent studies show that,numbers of miRNAs exist in mammalian central nervous system (CNS), includingbrain and spinal cord, where miRNAs were involved in the regulation of CNS injury,repair following the injury and neurotraumatic pathologies. But, the regulatoryfunctions of miRNAs in spinal cord are still unclear. So, study the miRNA regulatorypattern in lesion region of SCIRI would clarify the molecular mechanism of SCIRI,which has great significance on the development of efficient treatments and novelspecific drugs.In our study, we successfully constructed a SCIRI Rat model, and utilizedmiRCURYTM LNA Array (V.16.0) to analyze the miRNA expression profiles oflesion region of spinal cord ischemia injury and SCIRI. QRT-PCR was used to verifythe data of miRNA microarray. Target gene prediction of the differentially expressedmiRNAs (Fold charge over5) was performed based on the data collected from severaldatabases, including microRNA.org、Microcosm、miRBase. Following the prediction,GO analysis and pathway enrichment analysis were performed to identify thebiological function of these differentially expressed miRNAs. Additionally,miRNAs-TFs-Genes regulatory networks were built to visualize the regulatory patternof miRNAs. PurposesBased on SCIRI rat model, high-throughout screen the differentially expressedmiRNAs in lesion tissues. And through the construction of miRNAs-TFs-Genesregulatory networks to gain preliminary analysis of the miRNA regulatory pattern inSCIRI rat model, from a global point of view.Methods1. Construction of SCIRI rat modelSCIRI rat model was induced in mature SD rats by occluding the descendingthoracic aorta. The24mature SD rats were assigned randomly into four groups: shamoperation group; spinal cord ischemia injury group; spinal cord ischemia-reperfusioninjury group (24-hour group and48-hour group). Neurological functions of SCIRI ratmodel were assessed at24and48hour after reperfusion. Then spinal cords wereharvested for hematoxylin-eosin (HE) staining and verified that the SCIRI rat modelwas constructed successfully.2. Screening of rat SCIRI miRNA expression profileWe extracted the total RNA from spinal cord of SCIRI rat model, and utilizedmiRCURYMLNA Array (V.16.0) to detect the miRNA expression profile of spinalcord ischemia injury group and spinal cord ischemia-reperfusion injury groups(24-hour group and48-hour group). QRT-PCR was then used to verify the miRNAexpression profile. Unsupervised hierarchical clustering was performed to analyze themiRNA expression profile and finally we performed the bioinformatics analysis toidentify the biological functions of differentially expressed miRNAs.3. Preliminary analysis of miRNA regulatory pattern in SCIRI rat modelWe combined the miRNA expression profile data of two spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) as a totalmiRNA expression profile of SCIRI rat model. MiRNAs of which fold charge was noless than5were collected for significant miRNA regulatory pattern analysis. Targetprediction of these selected differentially expressed miRNA was performed based onthe data provided in public databases including MicroRNA.org, Microcosm andMiRanda. Then, databases, including DAVID and webgestalt, were utilized toperform GO analysis and pathway enrichment analysis. Finally, intersection of abovethree databases was used for miRNAs-TFs-Genes regulatory networks construction,where the transcriptional factors (TFs) were identified based on the data provided by TRED database. Cytoscape software version3.2.0was used to visualize themiRNAs-TFs-Genes regulatory networks information.Results1. Construction of SCIRI rat model1.1Compared to control group, rats in operation group had significantly lowerlimb function, and would gradually improved in48hours, of which content is obviousin the first24hours.1.2HE staining of spinal cords showed that neurons were rare in spinal cords ofSCIRI rat model, which got worst at the48hour. And interstitial edema increasedgradually until the24hour, which remitted after reperfusion for48hours.1.3Results in this study proved that SCIRI rat model was successfullyconstructed for further studies.2. Screening of rat SCIRI miRNA expression profile2.1115(39up regulated,76down regulated),13(12up regulated,1downregulated) and105（44up regulated,61down regulated) differentially expressedmiRNAs were respectively detected in spinal cord tissues of spinal cord ischemiainjury group and spinal cord ischemia-reperfusion injury groups (24-hour group and48-hour group).2.2Among spinal cord ischemia injury group and spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group),rno-miR-22-3p was steadily high-expressed, and reached the peak in spinal cordischemia injury group.2.3Unsupervised hierarchical clustering analysis showed that spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) can bedistinguished accurately.3. Preliminary analysis of miRNA regulatory pattern in SCIRI rat model3.1We constructed miRNAs-TFs-Genes regulatory networks of spinal cordischemia-reperfusion injury groups (24-hour group and48-hour group) bybioinformatics tools.3.2Spinal cord ischemia injury and SCIRI may share part of miRNA regulatorynetworks, which may regulate cell proliferation and death by transcriptional factorSp4and Cebpb.3.3The specific miRNA regulatory pattern of SCIRI may be involved in cell proliferation, differentiation, survival and death, through the regulation of MAPKsignaling pathway, which would play an important role in the progress of SCIRI.3.4Rno-mir-22-3p would have key roles in cell apoptosis regulatory pathway, bythe regulation of transcriptional factor Tp53.Conclusions1. These differentially expressed miRNAs can accurately identify the SCIRI ratmodel.2. Spinal cord ischemia injury and SCIRI may share part of miRNA regulatorynetworks, which may regulate cell proliferation and death by transcriptional factorSp4and Cebpb.3. The specific miRNA regulatory pattern of SCIRI may be involved in cellproliferation, differentiation, survival and death, through the regulation of MAPKsignaling pathway, which would play an important role in the progress of SCIRI.4. Bioinformatics analysis shows that rno-miR-22expression in SCIRI rat modelspinal cord tissues would play an injury repair role through the regulation of cellapoptosis regulatory pathway.Innovation points1. Based on the systemic biology theory, using bioinformatics method，wepreliminarily analyzed the miRNA regulatory pattern in SCIRI rat model, from aglobal point of view.2. We reported the function of miR-22in SCIRI rat model for the first time.