The Mechanism Of Utx/Top2β Axis In Spinal Cord Injury Recovery By Modulating Senescence Of Spinal Cord Microvascular Endothelial Cells

Background:Spinal Cord Injury（SCI）is a severe traumatic disease of the central nervous system,leading to severe disability and even death.Microvascular destruction after spinal cord injury is one of the important pathological changes after SCI.The injury of spinal microvascular endothelial cells increases permeability of blood spinal cord barrier,triggers vascular immune inflammatory response,all of those severely inhibits angiogenesis post SCI.Histone 3 lysine demethylase（Utx）is a histone demethylase of H3K27me2/3.Our previously study have demonstrated that Utx is highly expressed in spinal cord of SCI mice,knockout Utx in microvascular endothelial cells significantly increase vascular angiogenesis,promoting neurological recovery after SCI,while the mechanism is unclear.Recent studies have shown that senescent vascular endothelial cells cause vascular regeneration depression by releasing angioinhibitory factors.Meanwhile,it has been reported the methylation status of histone H3K27 are related to cell senescence,and Utx is highly expressed in senescent human embryonic fibroblasts.However,whether SCMECs senescence is involved in Utx mediated angiogenesis after SCI and its specific mechanism needs to be further explored.Aim:We set to explore whether SCMECs senescence is involved in Utx mediated cell proliferation and angiogenesis post SCI;and further explore its specific mechanism through high-throughput sequencing technology.Method:Modified Allen’s weight drop apparatus was used to establish contusion SCI model.Firstly,Forty female wild-type C57 mice were selected as the research object,and 5 mice in SCI control group（SHAM group）and 35 mice in the SCI group,respectively.The SCI group was divided into 3,5,7 and 14-day groups,with 5 mice in each group.Spinal cord specimens were collected at corresponding time（the excess mice were used as replacement for mice that died unexpectedly）.Spinal cord of mice in each group was collected for immunofluorescence staining（Utx,senescence marker（p16）and endothelial cell marker（CD31））.Subsequently,female wild type C57 mice were further divided to 2-month-old group and 12-month-old group with five mice in each group.Spinal cord of mice in each group was collected for immunofluorescence staining（Utx,p16 and CD31）.Then,Utx^-/-mice and Utx^f/fmice were constructed by Cre/lox P technique.Spinal cord of Utx^-/-and Utx^f/fmice after SCI mice were collected for Utx,p16 and CD31staining.The primary SCMVECs of Utx^-/-and Utx^f/fmice were extracted by enzyme digestion method,and the cell purity was determined by flow cytometry.Then,western blotting,q-PCR and immunofluorescence were used to confirm Utx expression in SCMVECs.The senescence and proliferation of the SCMVECs s were detected by Sa-β-Gal staining,Ki67 staining and CCK-8.Finally,RNA-seq and Chip-seq（chromatin immunoprecipitation sequencing）were conducted to further explore potential senescence-related target genes of Utx,and Ch IP-q PCR and western blotting were used to further verify the target genes.Results:（1）Compared with sham group,the expression of Utx and cell senescence marker p16 in SCMVECs significantly increased in a time-dependent manner（day 3,5 and 7）after SCI,and reached the highest expression on day 7 after SCI.The expression of Utx and p16decreased on day 14 compared with day 7（P<0.05）.（2）Compared with2-month-old mice,both Utx and p16 staining was significantly increased in the spinal cord of 12-month-old mice（P<0.05）.（3）Compared with Utx^f/f,Utx expression in SCMVEC from Utx^-/-mice was significantly decreased,and p16,the senescence marker,was also significantly decreased（P<0.05）.（4）Primary SCMVECs from Utx^f/fand Utx^-/-mice was successfully extracted.Compared with the SCMVECs from Utx^f/f,Utxknockout in SCMVECs significantly decreased cell senescence（p16expression and Sa-β-Gal staining,P<0.05）,while cell proliferation was also significantly increased（Ki67 staining and CCK-8 assay,P<0.05）.（5）Primary SCMVECs from Utx^f/fand Utx^-/-mice was extracted for RNA-seq.With the threshold of fold change≥1.2 times,p-value≤0.05,and intra-group FPKM≥0.5,a total of 426 differential genes,with 286 genes were up-regulated and 176 genes were down-regulated were identified in Utx^-/-when compared to Utx^f/f.Upregulated differential genes are mainly enriched in lipoteichoic acid pathway,NF-kappa B signaling pathway,protein phosphorylation pathway,and cell senescence and other pathways;Downregulated differential genes are mainly enriched in cell division,cellular metabolic process,cell cycle and other pathway.（6）SCMVEC from Utx^f/fmice was successfully extracted for Ch IP-seq.A total of 4391peak were found to bind to Utx protein,among which 39%（1724）binding sites located in intergenic region,15%（653）in upstream sequence and 18%（774）in promoter region.The exon region was 2%（100）and the intron was 26%（1140）.Genes bound to Utx in promoter region are mainly enriched in hepatocellular carcinoma,viral carcinogenesis,cell senescence,cell cycle,Wnt signaling pathway,Hipo signaling pathway,TGF-βand other signaling pathways.（7）774 genes with binding site at promoter region screened by Ch IP-seq and 462differentially expressed genes identified by RNA-seq were intersected,and a total of 16 differential expressed genes shared by Ch IP-seq and RNA-seq were selected.Next,senescence pathway related genes,Top2βand Rbl1,were selected from the 16 differential expressed genes after GO annotation.（8）Previous literature has reported that the binding rate of Utx and Rbl1 is extremely low（0.031%）,so we next mainly focused on Top2βas the potential target of Utx.Ch IP-q PCR and western blotting were conducted and verified the mutual binding between Utx and Top2βpromoter,and also demonstrated that Utx knockout caused increased expression of Top2βat both RNA and protein level.Conclusion:Utx binds to Top2βpromoters mediates SCMVECs senescence and participates in vascular proliferation and repair after SCI.This study further elucidates the new mechanism of Utx knockout promoting neural function recovery after SCI,providing a new target and theoretical basis for SCI therapy.