| Objective:To identify the aberrantly expressed lncRNAs and mRNAs in the thoracic spinal cord(T8-12)following ischemia/reperfusion-induced acute kidney injury(Model group)and normal kidney rats(Control group)by High throughput sequencing,find lncRNAs and mRNAs involved in the pathogenesis of acute renal injury,and analyze the biological function of differential expression genes;To verify the differentially expressed lncRNA TCONS00058568 and its predicted target gene of P2X7 receptor(P2X7R)in the lower thoracic spinal cord(T8-12).In vivo,exploration the effect of the targeting up-regulation IncRNA TCNS00058568 on the progression of acute renal injury induced by ischemia-reperfusion,and investgation its related molecular mechanism.Methods:1.The expression of IncRNA and mRNA in the lower thoracic spinal cord(T8-12)of acute renal injury(AKI)rats(n=3)induced by ischemia-reperfusion(I/R)and normal kidney rats(n=3)was detected by high throughput sequencing.Sequencing data were statistically analyzed and determined the differential expression profile of lncRNAs and mRNAs in the thoracic spinal cord(T8-12).The differential expression of IncRNAs and mRNAs were tested by qRT-PCR.GO function enrichment and KEGG pathway enrichment were analyzed for differential expression genes,and predicted their molecular biological function.2.The selected IncRNA TCONS00058568 was perfomed for subsequent functional experiments.To verify lncRNA TCONS00058568 expression and localization in the lower thoracic spinal cord(T8-12)from AKI rats and control rats.The different expression of P2X7 receptor(P2X7R)that was IncRNA TCONS00058568 target gene was detected by qRT-PCR between the two groups.The stable microglia cell line(BV2)expressing IncRNATCONS00058568 was established by lentivirus infection.and the expression change of P2X7R was confirmed by qRT-PCR.The effect of lncRNA TCONS00058568 on cell proliferation was detected by CCK-8 assay.3.The animals were divided into normal control group,Lv-Mock group and Lv-TCONS00058568 group,and each group was divided into sham group and ischemia-reperfusion group(I/R group).The Overexpression of Lv-TCONS00058568 and Lv-Mock was injected into the thoracic spinal cord T9 segment.The model of renal ischemia-reperfusion were established after 1 weeks of virus injection into rats.After 24 hours of reperfusion,the rats were sacrificed to test the renal function,observe renal histopathology morphology,and detect apoptosis and pro-inflammatory factor.Rat’s spinal cord stably overexpressing lncRNA TCONS00058568 was confirmed by qRT-PCR.The change of P2X7R expression in the spinal cord and the protein phosphorylation in PI3K/Akt signaling pathway were examined by western-blot.Results:1.Compared with control group,lncRNAs and mRNAs were extensively abnormal differential expression in the lower thoracic spinal cord tissues of rats with acute kidney injury through the high-throughput sequencing.2.Through bioinformatics analysis,we found that the abnormal expression of lncRNAs may be involved in the occurrence and development of acute renal injury,which may be participate in regulating renal function through the MAPK,PI3K/Akt signaling pathway or RASS system.3.The P2X7 receptor expression in microglia cell line which stably overexpressing lncRNA TCONS00058568 was reduced,and CCK-8 assay indicated the overexpression of lncRNA TCONS00058568 promoted the proliferation rate of microglia cell line.4.Comparing with the Lv-Mock group,the levels of serum creatinine,urea nitrogen and renal tissue injury score were significantly reduced in overexpression IncRNA TCONS00058568 group.Also,the number of tunel-positive cells in renal tubular cells,the level of Bax and Caspase-3 protein and proinflammatory factors IL-6 and TNF-α levels in the overexpression + I/R group were significantly lower than in Lv-Mock + I/R group.5.In vivo experiments,we found that lncRNA TCONS00058568 dramatically decreased the levels of P2X7R mRNA and protein expression in spinal cord tissue,and the level of phosphorylated Akt protein also decreased significantly.Conclusion:The abnormally expressed lnCRNAs/mRNAs in the lower thoracic spinal cord tissue from acute renal injury are systematically screened.They could play an important role in the development and progression of acute renal injury.Targeting upregulation the lncRNA Tcons00058568 in spinal cord downregulated P2X7 receptor expression,which may be mediated by PI3K/Akt signaling pathway to improve renal injury.LncRNA Tcons00058568 can be regarded as the potential therapeutic target for ischemia-reperfusion induced acute renal injury. |
