The Effect Of LncRNA GAS5 And MiR-101a-3p On Neuroapoptosis And Inflammation In Spinal Cord Ischemic-reperfusion Injury In Rats

Objective:Spinal cord ischemic-reperfusion injury(SCII)is one of the most serious complications caused by surgery for clinical aortic aneurysms and vascular malformations,and imposes a substantial mental and economic burden on patients'families and society.SCII affects nerve function through many mechanisms,which are complex and not fully understood.It is a big challenge to investigate the pathological mechanisms of SCII and enhance neurological recovery after SCII.In short,the various mechanisms involve SCII,such as lipid peroxidation damage mediated by oxygen free radicals,the inflammatory response,intracellular Ca²⁺overload,glutamate toxicity through excitatory amino acids,apoptosis and autophagy,blood-spinal cord barrier permeability changes,etc.Among these mechanisms,inflammation and apoptosis are very important and of interest to our group.In the central nervous system(CNS),neurons are vulnerable and non-renewable cells.Neuronal damage is associated with lower limb motor dysfunction,and it is challenging to repair nerve function after SCII.Recently,accumulating evidence has indicated that the growth arrest-specific 5(lncRNA GAS5)and miR-101a-3p play an increasingly important role in nervous system diseases.Additionally,it is reported that Matrix metalloproteinases-7(MMP-7)and V-myc myelocytomatosis viral related oncogene(MYCN)can be involved in the occurrence and development of neurological diseases.However,the mechanism of lncRNA GAS5 and miR-101a-3p in SCII remains unknown.In this study,we explored the role of lncRNA GAS5 on SCII and whether it functioned by affecting MMP-7.In addition,we also studied the role of miR-101a-3p on SCII and whether it functioned by affecting MYCN.Methods:1.The SCII model was established by clamping aortic arch for 14 minutes.The RNA expression of lncRNA GAS5 and miR-101a-3p was measured by real-time PCR at 12 h,24 h,36 h,and 48 h.Based on the above findings,we chose 24 h after SCII as the time point of study.The localization of lncRNA GAS5 in gray matter of spinal anterior horn was measured by Fluorescence in situ hybridization(FISH).Significant miRNAs after SCII were analyzed by microarray chip in our previous research.lncRNA GAS5 was knocked down by intrathecal si RNA injection 3 days before the operation.Real-time PCR was used to detect the changes of miR-101a-3p after silencing lncRNA GAS5.The relationship between lncRNA GAS5 and miR-101a-3p was verified by luciferase assays.The Tarlov scoring system and Td T-mediated d UTP Nick-End Labeling(TUNEL)assay were applied to evaluate neurological function and neuronal apoptosis.The effect of SCII on the BSCB was evaluated by Evans blue(EB)staining.2.The MMP-7 protein and RNA levels of different time points were measured by Western blotting and PCR.Based on the above findings,we chose 24 h after SCII as the time point of study.We studied the colocalization of MMP-7 and the different cell types in gray matter of spinal anterior horn by Immunofluorescence staining after SCII.To further evaluate the effect of MMP-7,si-MMP-7 or NC si RNA intrathecally was injected 3 days before the operation.The protein levels of MMP-7,cleaved caspase-3 and IL-1?were measured by Western blotting in Sham group,SCII group,NC group,and si-MMP-7 group.Tarlov scoring system was applied to evaluate neurological function in Sham group,SCII group,NC group,and si-MMP-7 group.According to the results of PCR,Spearman's analysis was used to evaluate the correlation between lncRNA GAS5 and MMP-7.lncRNA GAS5 was knocked down by intrathecal si RNA injection 3 days before the operation.The RNA and protein levels of lncRNA GAS5,MMP-7,the apoptotic factor cleaved caspase-3 and proinflammatory cytokine IL-1?were measured by real-time PCR and Western blotting.Quantitative analysis of MMP-7 and the colocalized neuron cells were measured by Immunofluorescence staining.Tarlov scoring system and TUNEL assay were used to evaluate neurological function and neuronal apoptosis.3.The SCII model was established by clamping aortic arch for 14 minutes.The RNA and protein levels of MYCN were measured by real-time PCR and Western blotting.Based on the above findings,we chose 24 h after SCII as the time point of study.We studied the colocalization of MYCN and the different cell types in gray matter of spinal anterior horn by Immunofluorescence staining after SCII.The relationship between miR-101a-3p and MYCN was elucidated via Target Scan,Western blotting,PCR and luciferase assays.The effect of miR-101a-3p on the relative levels of MYCN,p53,caspase-9 and IL-1?was assessed by intrathecal injection of synthetic miRs.The protein levels of MYCN,p53,caspase-9 and IL-1?were measured by Western blotting in Sham group,SCII group,NC group,mimics group,and inhibitor group.Quantification and co-localization of MYCN and P53 were measured by Immunofluorescence staining.Neurological function and neuronal apoptosis were evaluated by Tarlov scoring system and TUNEL assay.Results:1.The RNA level of lncRNA GAS5 was significantly increased at 24 h after SCII.The results of FISH showed that lncRNA GAS5 was mainly localized in the cytoplasm in gray matter of spinal anterior horn.The RNA level of miR-101a-3p was significantly decreased at 24 h after SCII.The RNA level of miR-101a-3p was significantly increased after silencing lncRNA GAS5 compared with SCII group.However,the luciferase assay results indicated that lncRNA GAS5 could not bind to miR-101a-3p through the 3'-UTR.In the Tarlov scoring system assay,the scores of SCII group were significantly decreased.In the TUNEL assay,the number of apoptotic cells was significantly increased in SCII group.In EB test,the infiltration of EB was significantly increased in SCII group.2.The RNA and protein levels of MMP-7 were significantly increased at 24 h after SCII.In the Tarlov scoring system assay,the scores were significantly decreased in SCII group compared to Sham group,while the scores were significantly increased in si-MMP-7 group compared to SCII group.Intrathecal pretreatment with MMP-7si RNA significantly decreased the protein levels of MMP-7,cleaved caspase-3 and IL-1?.At 24 h after SCII,MMP-7 fluorescent expression significantly increased in SCII group compared with Sham group,and MMP-7 fluorescence mainly overlapped with Neu N fluorescence but not Iba-1 or GFAP.In addition,Spearman's analysis showed a positive correlation.The PCR results showed that the RNA levels of lncRNA GAS5 and MMP-7 were significantly decreased after inhibition of lncRNA GAS5.Consistent with the changes in lncRNA GAS5 levels,the protein expression levels of MMP-7,cleaved caspase-3 and IL-1?increased sharply after SCII and decreased significantly after lncRNA GAS5 knockdown.In the Tarlov scoring system assay,the scores of SCII group were significantly decreased compared to those of Sham group,while the scores of si-lncRNA GAS5 group were significantly increased compared to those of SCII group.In the TUNEL assay,there was a significant decrease in the number of neuronal apoptotic cells in si-lncRNA GAS5 group compared to SCII group after SCII.The immunofluorescence staining results showed SCII increased MMP-7 expression in neurons,and inhibition of lncRNA GAS5reduced MMP-7 expression in neurons.3.The RNA and protein levels of MYCN were significantly increased at 24 h after SCII.MYCN immunoreactivity increased in SCII group compared with Sham group,which was primarily overlapped with Neu N.Then,luciferase assay confirmed that miR-101a-3p directly targeted MYCN.miR-101a-3p mimics decreased the protein and m RNA levels of MYCN as well as the protein levels of p53,caspase-9 and IL-1?.Double-immunofluorescence showed that miR-101a-3p mimics decreased the immunoreactivity of p53 and the number of MYCN-positive double-labeled cells.In the Tarlov scoring system assay,the scores of SCII group were significantly decreased compared to those of Sham group,while the scores of miR-101a-3p mimics group were significantly increased compared to those of SCII group.In the TUNEL assay,there was a significant decrease in the number of neuronal apoptotic cells in miR-101a-3p mimics group compared to SCII group after SCII.Conclusions:Silencing of lncRNA GAS5 can reduce neuroapoptosis and inflammation after SCII by inhibiting MMP-7,and alleviate lower limb motor dysfunction in rats.Overexpression of miR-101a-3p can reduce neuroapoptosis and inflammation after SCII by inhibiting MYCN and p53 signaling pathway,and alleviate lower limb motor dysfunction in rats.However,lncRNA GAS5 can not play a role in SCII by targeting miR-101a-3p.