Study On The Repair Effect And Related Mechanisms Of LncRNA-Lepr Competitive Binding To MiR-6315 In Spinal Cord Injury

Objectives:Spinal cord injury（SCI）is a serious and disabling disease that severely reduces the quality of life and increases the burden on patients’lives and finances.SCI results in irreversible disruption of neurotransmission,and there is no effective therapy to achieve complete neurological or functional recovery.Competitive endogenous RNA（ceRNA）networks play an important regulatory role in SCI development and repair.This experiment intends to screen the differentially expressed lncRNAs and miRNAs and their target genes in SCI by high-throughput sequencing,detect the role and mechanism of ceRNA network（lncRNA/miRNA/mRNA axis）in SCI,and provide experimental basis for the subsequent search of therapeutic targets for SCI.Methods:1.The SCI model was established,and the success of the rat model was detected by slant plate test,BBB score,footprint detection,spinal cord gross morphology and Nissl staining.2.The spinal cord was removed from the injury center at 1d,3d and 7d after spinal cord injury for high-throughput sequencing（HTS）,and further screened for miRNA-miR-6315,which is associated with SCI repair process.3.The enrichment analysis tool DAVID was used to analyze gene ontology（GO）system and KEGG pathway involving differential mRNA.Smo was predicted to have a binding site for miR-6315 using the Ensembl database and MiRanda software.4.The expression of miR-6315 and Smo after SCI and Glu injury was verified by qRT-q PCR to be consistent with the sequencing results.qRT-PCR and western blot assays were performed to verify the target of miR-6315 on the 3’UTR of the target gene Smo.5.The expression levels of miR-6315 and Smo were measured at 1d,7d,14d and28d after SCI and correlation analysis was performed.6.The possible Smo target was predicted by GENEMANIA database as anti-ferroptosis pathway factor xCT（SLC7A11）.The expression levels of anti-ferroptosis pathway factors xCT,GSH,GPX4 at different time points after SCI were detected by qRT-PCR and Western blot.7.miR-6315 low expression adenovirus was constructed and infected with primary spinal cord neurons and spinal cord tissues.miR-6315 relative expression was detected by qRT-PCR.The groupings in spinal cord neurons and spinal cord tissues were:Normal,Glu,Glu+NC with Glu+miR-6315-si;Sham,SCI,SCI+NC,SCI+miR-6315-si groups,respectively.Neuronal markers（NeuN）,axonal regeneration markers（GAP43）and synaptophysin（SYP）were detected by immunofluorescence staining（IF）in each group,and the migration rate of neurons was detected by scratch test.BBB score,inclined plate test and electrophysiology were used to detect motor function in rats.The expression levels of miR-6315,Smo and anti-ferroptosis pathway factors xCT,GSH,GPX4 were detected by qRT-PCR and Western blot.8.Smo overexpression and low expression adenovirus were constructed and infected with primary spinal cord neurons and spinal cord tissues.qRT-PCR and Western blot were used to detect the relative expression of Smo.The groupings in spinal cord neurons and spinal cord tissues were:Normal group,Glu group,Glu+Smo-NC group,Glu+Smo group,Glu+Smo-NC group and Glu+Smo-si group;Sham group,SCI group,SCI+Smo-NC group,SCI+Smo group,SCI+Smo-NC group and SCI+Smo-si group.Neuronal markers（NeuN）,axonal regeneration markers（GAP43）and synaptophysin（SYP）were detected by immunofluorescence staining（IF）in each group.BBB score,inclined plate test and electrophysiology were used to detect motor function in rats.The expression levels of Smo and anti-ferroptosis pathway factors xCT,GSH,GPX4 were detected by qRT-PCR and Western blot.9.Based on HTS sequencing data,MiRanda software was used to predict the binding sites of differentially expressed lncRNAs with significantly up-regulated miR-6315.lncRNA-Lepr was selected as a potential key lncRNA after comprehensive analysis of Target Scan and MiRanda scores.lncRNA-Lepr was verified by dual luciferase reporter gene assay for the binding relationship between lncRNA-Lepr and miR-6315.The target binding relationship between lncRNA-Lepr and miR-6315 was verified by dual luciferase reporter gene assay.qRT-PCR assay was performed to detect the expression levels of lncRNA-Lepr and correlate with miR-6315 at 1d,7d,14d and 28d after SCI in rats.10.lncRNA overexpression adenovirus was constructed and infected with primary spinal cord neurons and spinal cord tissues.lncRNA relative expression was detected by qRT-PCR.lncRNA relative expression was detected by qRT-PCR.The groupings in spinal cord neurons and spinal cord tissues were:Normal,Glu,Glu+NC and Glu+lncRNA-Lepr groups;Sham,SCI,SCI+NC and SCI+lncRNA-Lepr groups,respectively.Neuronal markers（NeuN）,axonal regeneration markers（GAP43）and synaptophysin（SYP）were detected in each group by immunofluorescence staining（IF）,neuronal mobility by scratch assay,cell viability by CCK8,axonal regeneration by BDA cis-tracking,and ferroptosis by relative expression of ROS,Fe²⁺and MDA.BBB score,inclined plate assay and electrophysiology were used to detect motor function in rats.The expression levels of lncRNA-Lepr,miR-6315,Smo and anti-ferroptosis pathway factors xCT,GSH,GPX4,and ferroptosis related factor ACSL4were detected by qRT-PCR and Western blot.Results:1.The rat SCI model was successfully established,and the hind limbs of rats were completely devoid of motor function,and the local neurons of spinal cord injury were reduced.2.There were five differentially expressed up-regulated miRNAs in the spinal cord at 24 h after SCI,among which miR-6315 was the most significantly differentially expressed miRNA that reached the highest level at 24 h after SCI and gradually decreased at 3d,7d post-SCI.The differentially expressed mRNAs were enriched to the axonal growth cone pathway,in which the axonal growth cone pathway factor Smo had binding sites to miR-6315.3.Dual luciferase reporter assay confirmed that Smo is a direct target of miR-6315.The expression of miR-6315 was negatively correlated with the mRNA of Smo at 1d,7d,14d and 28d after SCI.4.The GENEMANIA database predicts that Smo may regulate the anti-ferroptosis pathway factor xCT.xCT and GPX4 have the lowest mRNA and protein expression at 1d after injury,increasing from 7d after injury and peaking at 28d.The change in GSH expression follows the same trend as xCT and GPX4.xCT/GSH/GPX4 axis is involved in SCI progression process.5.miR-6315 low-expressing adenovirus successfully infected primary spinal cord neurons and spinal cord tissue.In post-injury neurons and spinal cord tissues at1d,14d,28d after SCI,miR-6315 expression was silenced to promote neuronal axon regeneration,survival,synaptophysin production,and neuronal migration,and to improve motor function in rats in the distant period（28d）.The mRNA and protein expression levels of Smo were significantly increased after the silencing of miR-6315,and the mRNA and protein expression of the anti-ferroptosis pathway factors xCT,GSH and GPX4 were also significantly increased.6.Smo low expression and overexpression adenovirus successfully infected primary spinal cord neurons and spinal cord tissue.In post-injury neurons and spinal cord tissues at 1d,14d,28d after SCI,Smo overexpression promoted neuronal axon regeneration,survival and synaptophysin production,and improved motor function in rats in the distant period（28d）.In contrast,Smo overexpression resulted in reduced neuronal number,cell cytosolic atrophy,shortened axon length,and reduced synaptophysin production,exacerbating SCI-induced motor dysfunction.mRNA and protein expression levels of Smo were significantly increased after Smo overexpression,as well as anti-ferroptosis pathway factors xCT,GSH,and GPX4,in contrast to the results of Smo overexpression.7.lncRNA-Lepr was found to have the highest binding rate among all differentially expressed lncRNAs targeting miR-6315 by database binding site prediction.Dual luciferase reporter gene results showed that lncRNA-Lepr could bind directly to miR-6315.Correlation analysis of the expression of lncRNA-Lepr and miR-6315showed a negative correlation in spinal cord tissues at 1d,7d,14d,and 28d after SCI.8.lncRNA-Lepr overexpressing adenovirus successfully infected primary spinal cord neurons and spinal cord tissue.In post-injury neurons and spinal cord tissues at1d,14d,28d after SCI,lncRNA-Lepr overexpression promoted neuronal axon regeneration,survival and synaptophysin production,inhibited Fe²⁺,ROS and MDA production,and improved motor function in rats in the distant period（28d）.lncRNA-Lepr overexpression significantly decreased the mRNA expression of miR-6315,and Smo mRNA and protein expression levels were significantly increased,and anti-ferroptosis pathway factors xCT,GSH and GPX4 were also significantly increased,while ferroptosis-related factor ACSL4 was significantly decreased.Conclusions:1.miR-6315 directly bound to Smo after SCI.miR-6315low-expression could increase the level of Smo,upregulate the expression of xCT,GSH and GPX4,promote neuronal survival and axonal regeneration,and improve the motor function of lower limbs in rats.2.After SCI,Smo overexpression could upregulate the expression of xCT,GSH and GPX4,promote neuronal survival and axonal regeneration,and improve the motor function of rat lower limbs,while Smo low expression could do the opposite.3.lncRNA-Lepr had a competitive inhibitory relationship with miR-6315,and overexpression of lncRNA-Lepr could release Smo by suppressing miR-6315 level,and Smo expression was upregulated to activate xCT/GSH/GPX4 axis,inhibit ferroptosis and promote axon regeneration,thus improving lower limb motor dysfunction in rats.In conclusion,this study identified the lncRNA-Lepr/miR-6315/Smo novel network in SCI.Therefore,the lncRNA-Lepr/miR-6315/Smo axis may be a clinically important candidate target for SCI patients.