Screening Of Key Genes And Expression Of LncRNA Tnxa-ps1 In Neuron After Ischemia/reperfusion Injury

Part ? Gene expression profiles of neuron after neuronal ischemia/reperfusion injuryObjective:Neonatal hypoxic ischemic encephalopathy(HIE)involves hypoxic ischemia and subsequent reperfusion,which can lead to irreversible brain damage.However,its underlying pathomechanism is still under exploration.In order to observe the cellular autonomic mechanism involved in HIE comprehensively and in real time,we used the Next-Generation Sequencing(NGS)technique to illustrate the gene expression changes of the primary cultured hippocampal neurons under the condition of oxygen-glucose deprivation/reperfusion(OGD/R)and seek for the key genes involved.Method:1.Primary culture of rat hippocampal neurons and establishment of an in vitro hypoxia ischemia cell model oxygen-glucose deprivation model(OGD/R).2.RNA-Seq analysis of the neurons in each group:control group(OGD 0 minutes/R 0 hours),oxygen glucose deprivation for 45 minutes(OGD 45 minutes/R 0 hours),oxygen glucose deprivation for 45 minutes and reperfusion for 45 minutes,6,12 hours and 18 hours(OGD 45 minutes/R 6 hours,OGD 45 minutes/R 12 hours,OGD/R OGD hours).3.Bioinformatics analysis of the differentially expressed genes(DEGs),including gene ontology(GO),Kyoto Encyclopedia of Genes and Genomics(KEGG)analysis,gene interaction network and gene co-expression analysis.The novel key genes and pathways involved in the pathology OGD/R were screened.4.Verification of the screened key genes by qRT-PCR.5.Prepare the rat model of Middle cerebral artery occlusion/reperfusion(MCAO/R)and verify the success of the model by TTC.6.QRT-PCR and Western blot were used to detect the expression of the selected Itga5 and ErbB4 genes in the brain of MACO/R rats.7.The expression of Itga5 was knocked down by transfection of siRNA into the cultured neurons.QRT-PCR and Western blot were used to detect the effect of siRNA.8.MTT assay was used to detect the viability of siRNA transfected neurons.TUNEL assay was used to detect the apoptosis of siRNA transfected neurons.Result:1.The gene expression level of each sample was calculated by RPKM method and compared after RNA-Seq analysis.There were significant differences in gene expression profiles between groups(OGD 0 hours/R 0 hours vs OGD 0 minutes/R 0 h,OGD 45 minutes/R 6 hours vs 45 minutes 0 hours,45 minutes 45 minutes 12 hours)(FDR? 0.001 and multiple variation>2).2.The functional enrichment analysis of the differentially expressed genes(DEGS)showed that OGD induced strong stress and apoptosis response in GO analysis.KEGG analysis further showed that both TNF signal transduction pathway and Toll like receptor signal transduction pathway were involved in the inflammatory response after stimulation.The activated MAPK pathway was significantly enriched at the initial stage of OGD,and gradually disappeared after reperfusion.3.Through the construction of co-expression network,the key genes in different OGD/R groups were screened out.The results of qRT-PCR were consistent with those of the bioinformatics analysis.4.The MACO/R model of rats was successfully prepared.The results of qRT-PCR and Western blot showed that the selected Itga5 and ErbB4 genes by OGD/R neuron model had significant changes in the brain tissues of MCAO/R rats at different time points.5.MTT and TUNEL assays showed that blocking of Itga5 in cultured neurons by siRNA could significantly inhibit neuronal apoptosis after OGD.Conclusion:In this study,the RNA-seq technology was used to sequence genes of the neurons in OGD/R model and a large number of differentially expressed genes were screened.By the bioinformatics analysis,these differentially expressed genes were enriched to clarify the biological process and signal pathways involved.The novel key genes were selected by the co-expression network.Among these genes,Itga5 and ErbB4 were verified to have significant changes in the MACO/R model in vivo,indicating that the neuron OGD/R model could simulate the pathological process of cerebral ischemia/reperfusion.Meanwhile,the up regulation of the key Itga5 gene promoted the apoptosis of the neurons.Part ? Change of IncRNA Tnxa-psl expression after neuronal ischemia/reperfusion injuryObjective:After cerebral hypoxic ischemia/reperfusion,the transcriptome of neurons can undergo extensive changes.Many studies have focused on the transcriptome changes of mRNA associated with cerebral hypoxic ischemia/reperfusion,little is known about the changes of long non-coding RNAs(lncRNAs)that play a key role in cell homeostasis and how long the lncRNAs expression changes last.In this study,we analyzed the changes of lncRNAs expression in the primary cultured hippocampal neurons after the oxygen-glucose deprivation/reperfusion(OGD/R)for 0h,6h,12h and 18h and its relationship with mRNAs by high throughput screening.We also aimed to find the effect of lncRNA on neuronal apoptosis.Method:1.Primary culture of rat hippocampal neurons and establish an in vitro hypoxia ischemia cell model oxygen-glucose deprivation/reperfusion model(OGD/R).2.The neurons in each group(control group(OGD 0 minutes/R 0 hours),oxygen glucose deprivation 45 minutes(OGD 45 minutes/R 0 hours),oxygen deprivation 45 minutes after 45 minutes,6,12 hours and 18 hours(OGD 45 minutes/R 6 hours,OGD 45 minutes/R 12 hours,OGD/R OGD hours))were sequenced by RNA-Seq technology.3.The differentially expressed lncRNAs were screened and part of the lncRNAs were verified by qRT-PCR.4,The siRNA were constructed and transfected to the neurons to knock down the expression of lncRNA Tnxa-psl.The activity of the transfected neurons was detected by MTT assay.The apoptosis of the neurons was detected by TUNEL assay.5.The coding and non-coding co-expression networks were analyzed combined with the results of part I to find the Tnxa-psl related mRNAs,some of which were verified by qRT-PCR.6.SiRNA was transferred to the neurons to knock down the expression of IncRNA Tnxa-psl.The expression of Vip,Chga,nefm and Nefl were detected by qRT-PCR.7.By using RegRNA2.0 bioinformatics software,the Tnxa-psl-targeting miRNAs were predicted,and then these predicted miRNAs were further validated to be available for targeting itga5 mRNA by using Targetscan database.Result:1.The differentially expressed IncRNAs of each group were screened out by the RNA-Seq of OGD/R neurons.2.When the Tnxa-psl expression was inhibited,cell viability was increased and neuronal apoptosis was decreased.3.Coding and non coding co-expression network analysis and qRT-PCR validation showed that Tnxa-psl was associated with some mRNAs.4.Inhibition of Tnxa-ps1 can reverse the changes of mRNAs expression after OGD/R,including Vip,Chga,nefin,and Nefl.5.Bioinformatics analysis showed that there were 13 potential miRNA targetinf for Tnxa-psl including no-miR-326,rno-miR-329,rno-miR-330,rno-miR-298,rno-miR-224,rno-miR-483,rno-miR-423,rno-miR-484,rno-miR-667,rno-miR-3541,rno-miR-3547,rno-miR-1188-5p and rno-miR-702-5p.itga5 mRNA 3'UTR existed 2 rno-miR-484 binding sites,locating on the position of 427-434 and 780-786 respectively.Conclusion:In this study,the RNA-seq technique was used to sequence the neurons in the OGD/R model and to screen the differentially expressed lncRNAs.Knocking down the expression of lncRNA Tnxa-ps1 could promote the survival of the neurons by inhibiting the cellular apoptosis.The expression of Tnxa-psl was highly correlated with the change of the specific genomes.Downregulation of Tnxa-ps1 reversed the expression of 4 mRNAs after OGD/R,indicating that Tnxa-psl has a regulatory effect on the selected genes.Tnxa-ps1 might functioned as a ceRNA to participate in OGD/R-induced neurons apoptosis via competitive binding with rno-miR-484 to regluate Itga5expression.In conclusion,our data reveals that lncRNAs may be involved in the pathophysiology of cerebral ischemia/reperfusion.