| Objective:Spinal cord injury is one of the common severe diseases in central nervous system with a high incidence and a high disability rate.A systematic and comprehensive understanding of the pathophysiological events in the process of spinal cord injury,as well as the relevant cellular and molecular mechanisms,is crucial for the development of new treatment strategies.Therefore,we established a rat T9 semi-transected model,and performed expression profile sequencing(including circRNAs,long non-coding RNAs,and messenger RNAs)at multiple time points after spinal cord injury in rats,to screen target circRNAs and long non-coding RNAs that play important regulatory roles in a temporal sequence.Then the function and the molecular mechanism of selected circRNAs and long non-coding RNAs in astrocytes were investigated.This dissertation provides a new perspective elucidation of non-coding RNAs' roles in the spinal cord injury.Methods:1.We prepared rat T9 semi-transected models.In order to comprehensively and accurately study the temporal expression of circular RNAs and long non-coding RNAs during spinal cord injury,we harvested samples at 0 h,0.5 h,3 h,6 h,12 h,1 d,3 d,7 d,14 d,21 d and 28 d after T9 hemisection.Tissue samples of a total of 11 time points were collected,stored in liquid nitrogen and sent for commercial full transcriptome sequencing(including messenger RNA,circular RNA and long-chain non-coding RNA sequencing).2.Identification of circular RNAs and long non-coding RNAs.After screening the sequencing data,real-time quantitative PCR was used to verify the expression patterns of the selected circular RNAs and long non-coding RNAs.CircRNA?01477 and LncRNA LOC100909675 were selected for further functional exploration.3.Primary astrocytes derived from spinal cord were cultured in vitro.FISH experiment was used to detect the cellular localization of circRNA?01477 and LncRNA LOC100909675.CircRNA?01477 and LncRNA LOC100909675 si RNA and control si RNA were designed and electrotransfected into P2 generation astrocytes.The CCK-8 method and the Ed U method were used to detect the effect of interference with circRNA?01477 and LncRNA LOC100909675 on the proliferation of astrocytes;the scratch test and the transwell cell experiment were used to detect the effect of interference with circRNA?01477 and LncRNA LOC100909675 on the migration of astrocytes;The flow cytometry was used to detect the influence of interference circRNA?01477 and LncRNA LOC100909675 on astrocyte apoptosis.4.Expression profile analysis and verification after interference with circRNA?01477 and LncRNA LOC100909675.RNA was extracted from the astrocytes with knocked down expression of circRNA?01477 and LncRNA LOC100909675,and m RNA microarray analysis or sequencing analysis was performed.Cluster analysis and protein interaction network analysis were carried out by bioinformatics analysis tools.Genes related to astrocyte proliferation and migration were selected for real-time quantitative PCR verification.5.The influence of circRNA?01477 and LncRNA LOC100909675 on glial scar in vivo.si RNA or construct AAV virus was synthesized for in situ injection at T9 after spinal cord injury.The interference efficiency was detected by real-time quantitative PCR,and the expressions of GFAP and NF200 were detected by immunohistochemistry.The BBB score and TSE fine evaluation system were used to detect the hindlimb motor function of rats.Results:Part 1:1.The results of immunohistochemistry demonstrated that the rat T9 semi-transected model was successfully established.Full transcriptome sequencing results showed that 360 circRNAs were differentially expressed at any time point after spinal cord injury(day 0,1,3,7,14,21,or 28)(P < 0.05).Of these circRNAs,thirty-one were exons(8.61%),thirty-three were intergenic(9.16%),one was intron(0.28%),and two hundred and ninety-five were overlapped(81.95%).Ninety-four percent of the differentially expressed circRNAs decreased from day 3.2.Nineteen circRNAs were selected from the differentially expressed exon types for further study.The circRNAs were characterized by RNase R treatment and DNA sequencing.CircRNA?01477,circRNA?03612,circRNA?26782 and circRNA?17645 were selected for q RT-PCR detection.The results showed that the expression level of the selected circRNAs significantly decreased after spinal cord injury,and the pattern was consistent with RNA-seq results.3.The nuclear and cytoplasmic separation experiments showed that 93.5% of circRNA?01477 molecules were in the cytoplasm and 6.5% were in the nucleus.FISH results showed that 18 s r RNA was expressed in cytoplasm,while circRNA?01477 was expressed in both cytoplasm and nucleus.4.CCK-8 method was used to detect cell viability,and the results showed that interference with the expression of circRNA?01477 significantly reduced the viability of astrocytes.The Ed U staining results showed that interference with the expression of circRNA?01477 significantly inhibited the proliferation of astrocytes.Transwell experiment and scratch experiment were used to verify the effect of knockdown circRNA?01477 on astrocyte migration,and the results showed that interference with circRNA?01477 significantly inhibited cell migration.Flow cytometry results showed that knocking down circRNA?01477 did not induce astrocyte apoptosis.5.The immunohistochemical results demonstrated that reducing the expression of circRNA?01477 in vivo can reduce the expression of GFAP in the spinal cord and reduce the size of the glial scar cavity.6.CircRNA?01477 si RNA was used to treat astrocytes for microarray analysis,and 1528 genes with differential changes were obtained,among which 633 genes were up-regulated and 895 genes were down-regulated.GO analysis of the differential genes showed that chemotaxis was the most enriched ontology in the Biological Process plate.The Cellular Component plate has the most accumulation of extracellular space.The activity of cytokines is the most enriched in the Molecular Function plate.The top enrichment based on the KEGG pathway is the cytokine-cytokine receptor interaction.7.The knockdown of circRNA?01477 significantly reduced the expression levels of IL6 R,Ccnd3,Cxcl12,Cxcl14,Gcnt1,Inhbb1 and Ntrk2 genes.Inhibition of circRNA?01477 increased the level of mi RNA-423-5p,while mi RNA-760 did not change.Overexpression of mi RNA-423-5p mimic in astrocytes was detected by q RT-PCR to detect the expressions of Cxcl12,Cxcl14,Gcnt1 and Ntrk2.The results showed that overexpression of mi RNA-423-5p decreased the expression of Cxcl12 and Cxcl14,while the expression of Gcnt1 and Ntrk2 remained unchanged.Part 2:1.1182 differentially expressed long non-coding RNAs were screened by whole transcriptome sequencing.LncRNA LOC100909675 was gradually up-regulated at each time point after spinal cord injury by q RT-PCR,which was similar to RNA-seq results.The software analysis showed that LOC100909675 could not encode the protein and was a non-coding RNA.2.RACE results showed that the full length of LOC100909675 was 1270 bp.Nuclear and cytoplasmic separation experiments showed that 11.2% of LOC100909675 molecules were in the cytoplasm and 88.8% were in the nucleus.FISH results showed that 18 s r RNA was expressed in cytoplasm,while LOC100909675 was mainly expressed in nucleus.3.CCK-8 method was used to detect cell viability,and the results showed that the expression of interfering LOC100909675 significantly reduced the viability of astrocytes.The Ed U staining results showed that the expression of interfering LOC100909675 significantly inhibited the proliferation of astrocytes.Transwell and scratch experiments showed that the expression of interfering LOC100909675 significantly inhibited the migration of astrocytes.Flow cytometry results showed that LOC100909675 could not induce astrocyte apoptosis.4.Immunohistochemical results showed that the decrease of LOC100109675 expression in vivo could increase the expression of NF200 in the spinal cord and reduce the size of the glial scar cavity.5.The BBB score showed that the hindlimb motor ability of rats injected with AAV9-sh LOC100909675 was observably improved compared with the control group on 7 d,10 d and 14 d.The TSE fine movement evaluation system was further used to evaluate the fine movement of rats.The results showed that the angle change rate of hip joint was 9.7% in the control group and 16.7% in the group injected with AAV9-sh LOC100909675.The rate of angle change of paint joint in control group was 13.1%,and that of AAV9-sh LOC100909675 was 18.9%.In the control group,the angle change rate of the ankle joint was 14.6%,and that of the injection of AAV9-sh LOC100909675 was 30.9%.6.After LOC100909675 si RNA treatment of astrocytes,the expression profiling analysis was performed,and 1457 differentially expressed genes were obtained,of which 705 were up-regulated and 752 were down-regulated.The GO enrichment analysis of the differentially expressed genes revealed that the cell cycle ontology was the most enriched.KEGG enrichment analysis results show that the most significant enrichment is also the cell cycle.q RT-PCR results showed that knockdown of LOC100909675 significantly reduced the expression of cell cycle-related genes Anxa1,Anrkb,Brca1,Bub1,Prc1,Kif11,Foxm1,Ect2,Ccna2,Skp2,Cdk1,Cdc20,and the trend was similar to sequencing.Conclusions:1.The total 360 differentially expressed circRNAs and 1182 differentially expressed LncRNAs were identified,suggesting that non-coding RNAs are involved in the pathophysiological process of spinal cord injury.2.Studies on the function and mechanism of selected circRNA?01477 indicate that the molecule is mainly localized in the cytoplasm,knocking down its expression significantly inhibits the proliferation,migration and wound healing of astrocytes,and has no effect on apoptosis;Knocking down the expression of circRNA?01477 can reduce the size of the glial scar cavity in vivo.The GO and KEGG analyses showed that,circRNA?01477 may be an important regulator of immune inflammation induced by SCI.It is speculated that circRNA?01477 may regulate cell proliferation and migration through the mi RNA-423-5p-Cxcl12/Cxcl14 axis.3.Studies on the function and mechanism of the selected LncRNA LOC100909675 indicate that the molecule is mainly located in the nucleus,knocking down its expression significantly inhibits the proliferation,migration and wound healing of astrocytes,and has no effect on apoptosis.The knockdown of LOC100909675 expression can promote the recovery of hind limb function in rats in vivo.Based on the results of GO and KEGG analyses,LOC100909675 may be a cell cycle regulatory factor. |
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