Study On The Roles Of LncRNA-TBIAT In Oxidative Stress And Apoptosis After Traumatic Brain Injury

BackgroundTraumatic brain injury（TBI）,a mainly lethal and highly debilitating disease,has been increasing as a global public health problem.TBI includes primary brain injury and secondary brain injury.Secondary brain injury is related to the change of protein coding gene expression and the regulation of various pathophysiological processes after TBI,including neuroexcitotoxicity,oxidative stress,inflammation,and apoptosis.Although the study on the pathophysiology of TBI has made rapid progress,its potential mechanism is stillunclear.The clinical treatment is relatively simple,and the prognosis is always poor.Genomics research is one of the important signs of precision medicine for TBI.With the development of genomic sequencing technology,long noncoding RNA（lncRNA）has been found to be able to bind DNA,RNA and proteins.It plays an important role in many central nervous system diseases such as glioma,neurodegenerative diseases and ischemic stroke.However,the research on lncRNA and TBI is currently in its infancy,and the exactmechanism of lncRNA in the occurrence and development of TBI is stillunclear.The study of lncRNA and TBI could further elucidate the pathophysiological mechanism of secondary brain injury from the perspective of genomics,and also contribute to the development of specific targeted drugs.Objectives1.Screening differentially altered lncRNAs,mRNAs and miRNAs following TBI and performing bioinformatics analysis;2.To construct the cell model of oxidative stress and clarify the expression of specific lncRNAs in vitro;3.To clarify the mechanism of lncRNA-TBIAT on regulating oxidative stress and apoptosis of neurons.Methods1.Altered expression of lncRNAs,mRNAs and miRNAs in cortex tissue in TBI ratsThere were 16 sprague-dawley rats that were randomly divided into TBI group（n=8）and control group（n=8）.TBI model of rats was established by fluid percussion device.The left cortical tissue of rats was collected at 6h post-injury.Four samples of TBI group and four samples of control group were collected respectively for high-throughput sequencing.The left samples were stored in a-80℃refrigerator for q PCR verification.The total RNA was extracted from the left cortex of rats.After RNA library construction,purification,the expression was sequenced using Illumina sequencer.Sequence data and genome was compared to identify lncRNA,mRNA and miRNA expression profiles.According to the screening criteria of P value less than 0.05 and fold change more than 2,the difference analysis was carried out,and the function was described by GO and KEGG enrichment analysis.The reliability of sequencing data was validated by quantitative real-time PCR.LncRNAs-mRNAs coexpression network,lncRNAs-miRNAs-mRNAs regulatory network and lncRNAs-TF（transcription factor）regulatory network were constructed to comprehensively analyze the roles of differentially expressed lncRNAs in TBI.2.Apoptosis changes after TBI and expression of specific lncRNAs in cell model of oxidative stressWestern blot was used to detect the changes of apoptotic related proteins after TBI.The rat neural cell lines（PC-12,Manassas,VA,USA）were induced by H₂O₂ to construct the cell model in vitro.The cell model were divided into 100,200,and 400μmol/L group.CCK-8 was used to screen the appropriate concentration of H₂O₂.The levels of CAT,ROS and SOD were detected by the kit after 0,3,6,12,24 hours.After determining the appropriate concentration and time,the expression level of specific lncRNAs in oxidative stress cell model was detected by q PCR.The NONRATT017220.2 was named as lncRNA-TBIAT.The Target Scan database and miRTar Base database were used to predict the possible regulated miRNAs for further mechanism verification3.Study on the mechanism of lncRNA-TBIAT in the regulation of oxidative stress and cell apoptosis in PC-12 cellsPC-12 cells were used for cell transfection and were divided into normal control group（NC group）,negative control group（si NC group）and siRNA-lncRNA-TBIAT group（si group）.The plasmid was prepared before transfection and siRNA-lncRNA-TBIAT was transfected when the cell growth reached 60%fusion.The q PCR was used to verify the interference effect of siRNA-lncRNA-TBIAT,and CCK-8was used to detect the effect on cell activity after knockdown of lncRNA-TBIAT.Apoptosis was detected by flow cytometry.ROS was detected by fluorescence microscopy,and CAT and SOD were detected by kits.Whether lncRNA-TBIAT could sponge miR-124-3p was detected by double luciferase report and AGO2 experiment.The effects of miR-124-3p inhibitor and mimic on lncRNA-TBIAT expression regulation,cell activity,apoptosis and oxidative stress were detected after knockdown of lncRNA-TBIAT in vitro.The double luciferase report experiment was used to verify the targeted binding of miR-124-3p and Pim-1 gene,and western blot was used to detect the regulatory effect of mir-124-3p on the expression of Pim-1 protein.Results1.Altered expression of lncRNAs,mRNAs and miRNAs in cortex tissue in TBI ratsAccording to the screening criteria of P<0.05 and the fold change more than 2,the number of differentially expressed lncRNAs was 464,among which the number of up-regulated and down-regulated lncRNAs were 249 and 215 respectively.The number of differentially expressed mRNAs was 537,and the number of up-regulated and down-regulated genes was 467 and 70,respectively.The number of differentially expressed miRNAs was 25,and the number of up-regulated and down-regulated genes was 5 and 20,respectively.Six lncRNAs,mRNA and miRNA were selected for qPCR analysis,and the expression tendency was consistent with the sequencing results.The GO and KEGG pathway analysis for differentially expressed genes could be related to JAK-STAT signaling pathway,oxidative stress,inflammatory response and other pathophysiological processes.According to the differentially expressed lncRNAs,miRNAs and mRNA,lncRNAs-mRNAs coexpression network,lncRNAs-miRNAs-mRNAs regulatory network and lncRNAs-TF regulatory network were constructed,which indicated that the differentially expressed lncRNAs could play different roles in TBI in multiple ways.2.Apoptosis changes after TBI and expression of specific lncRNAs in cell model of oxidative stressAccording to the Western blot results at different times after TBI,the expression level of Caspase-1,Caspase-3,cleaved caspase-3 and Bax showed a time-dependent increase.H₂O₂in 200μmol/L concentration showed a significant effect on thecell activity without excessive injury at 24 h.Therefore,200μmol/LH₂O₂ was selected to construct the oxidative stress cell model.The levels of CAT and SOD decreased,and the fluorescence intensity of ROS increased significantly,indicating that the degree of oxidative damage increased.The expression of lncRNA such as lncRNANONRATT017220.2（named as lncRNA-TBIAT）was detected by q PCR,which was consistent with the sequencing results.The targeted gene of lncRNA-TBIAT was Pim-1.According to the GOand KEGG enrichment results,JAK-STATsignaling pathway was related.Therefore,lncRNA-TBIAT was predictedto be related to oxidative stress following TBI.According to the prediction of Targetscan and miRTar Basedatabase,miR-124-3p could be the sponging miRNA regulated by lncRNA-TBIATand Pim-1.3.LncRNA-TBIATmay regulate oxidative stress and cell apoptosis through miR-124-3p/Pim-1After knockdown of lncRNA-TBIAT,the expression level of lncRNA-TBIAT in siRNA-lncRNA-TBIATgroup was significantly decreased.The cell activity was significantly reduced.The cell apoptosis rate was significantly increased.The ROS level was significantly increased,and the activities of CAT and SOD were decreased.Double Luciferase Report and AGO2 experiment confirmed that lncRNA-TBIAT could sponge miR-124-3p.After knockdown of lncRNA-TBIAT,miR-124-3p inhibitor group showed the cell activity increased,the apoptosis rate decreased,and the level of oxidative stress decreased.However,the miR-124-3pimic group showed the cell activity decreased,the rate of apoptosis increased,and the level of oxidative stress increased.After overexpression of miR-124-3p,the expression level of miR-124-3p increased significantly with Pim-1 decreased significantly.The target binding of miR-124-3p and Pim-1 was verified by double fluorescence experiment.Western blot confirmed that miR-124-3p overexpression inhibited the expression level of Pim-1 protein in PC-12cells.ConclusionsThe expression profiles of lncRNAs,mRNAs and miRNAs were significantly altered.In this study,we screened and identified lncRNA-TBIAT for mechanism validation.The inhibition of lncRNA-TBIAT could result in the decrease of cell activity,the aggravation of oxidative stress and the increase of apoptosis rate in oxidative stress cell model.LncRNA-TBIAT may regulate the oxidative stress and cell apoptosis through miR-124-3p/Pim-1.