The Role And Molecular Mechanism Of ZBTB38 In Targtedly Repairing Secondary Spinal Cord Injury

Spinal cord injury?SCI?is one of the major diseases of the central nervous system?CNS?which commonly appeared in spinal surgery.It can be divided into primary injury?primary stage?and secondary injury?secondary stage?.Secondary injury is a kind of positive regulation at cellular and molecular level caused by the primary injury.It is reversible and controllable.Exploration of the mechanisms of spinal cord injury at the secondary level is pivotal to the understating and treatment of this disease,so reducing the loss caused by the secondary injury of spinal cord is of great importance for clinical study.ZBTB38 encodes a protein called CIBZ,a member of the C2H2 zinc finger protein family that typically contains the BTB domain.The BTB domain is often contained in transcription factors,which regulate the transcription of multiple target genes and participate in several signaling pathways and plays an important role in gene expression regulation,cell differentiation,embryo development,and other biological processes.CIBZ is highly expressed in spinal cord and it functions as a negative regulator in SCI-induced apoptosis.In eukaryotes,endoplasmic reticulum stress?ERS?and autophagy are two important defense and protection mechanisms in the body More studies are needed to understand the mechanisms of ER stress-associated apoptosis in SCI,although ER stress responses seem to divert towards apoptosis pathways in neurons and oligodendrocytes following SCI in the adult rat.Autophagy is a lysosome-dependent degradation pathway highly conserved and widely spread in eukaryotic cells.It is also an important defense and protection mechanism which is different from endoplasmic reticulum?ER?stress of eukaryotic cells.However,whether ZBTB38 plays a role in the signaling pathways of autophagy in central neurons has not been reported.It is proposed that autophagy and apoptosis may share some signaling pathways and participate in the death of spinal cord nerve cells after spinal cord injury.However,which pathways are involved in the autophagy-induced apoptosis remain to be elucidated.In the present study,we demonstrated in vitro that ERS triggered down-regulation of ZBTB38 expression,increased the expression levels of proapoptotic genes,including Bak,Noxa,Puma and Bim,in mitochondria-localization of SH-SY5Y cells.Endoplasmic reticulum stress-related apoptosis mediated by ZBTB38 deletion may be one of the leading causes of secondary injury to traumatic SCI.This data further confirms our previous findings that ZBTB38 negatively regulates apoptosis through the mitochondrial pathway after SCI.When ZBTB38 gene was silenced,the proliferation and viability of neuroblastoma cell line SH-SY5Y was significantly lower than that of the control group.The expression level of autophagy marker protein LC3B in the experimental group was significantly lower than that in the control group,and the expression of p62 was increased.In addition,the immunofluorescence staining of LC3B and the morphology of autophagosomes under electron microscope showed that the autophagy of human bone marrow-derived neuroblastoma cells was obviously inhibited.However,it was observed that eIF2?phosphorylation level?P-eIF2?,an important regulatory element of ERS regulating autophagy?was significantly increased,the results indicate that ZBTB38 may play a role in the regulation of ERS-triggered apoptosis,and it is likely that skipping the induction of ERS directly affects the occurrence of autophagy.To have some insight into the role of ZBTB38 in neuroblastoma development,high throughput RNA sequencing was performed using the human neuroblastoma cell line SH-SY5Y with the deletion of ZBTB38.In the present study,2,438 differentially expressed genes?DEGs?in ZBTB38^-/-SH-SY5Y cells were obtained,83.5%of which was down-regulated functional annotation of the differentially expressed genes in the Kyoto Encyclopedia of Genes and Genomes database revealed that most of the identified genes were enriched in the neurotrophin TRK receptor signaling pathway.In addition,down-regulation of ZBTB38 also promotes apoptosis in neuroblastoma cells by regulating key genes in the p53signaling pathway,combined with gene expression on mRNA levels through qRT-PCR,our study first clarified the down-regulation of ZBTB38 was significantly related to the differential expression of key factor RB1CC1?a factor involved in the biosynthesis of autophagosome in the initial stage?.Expression of RB1CC1 was down-regulated 4.2 times after ZBTB38 knockdown.The results also indicated that the expression of autophagy-related proteins LC3 and p62 was rescued when transfected the vectors overexpressing RB1CC1 in the ZBTB38-downregulated SH-SY5Y cells.Moreover,through this way,autophagy was re-initiated and the proliferation and viability of co-transfected cells were increased significantly.In the traumatic SCI mice,ER stress presented in injured spinal cord induced repression of ZBTB38 expression and triggered ZBTB38-mediated apoptosis.ChIP-QPCR analysis revealed that ATF4,an ER-stress inducible transcription factor,directly activated ZBTB38transcription by binding to the ZBTB38 promoter.However,this binding was significantly reduced following SCI,leading to a sharp decrease in ZBTB38 expression.Restoring ZBTB38 function in injured spinal cord by injection of lentivirus containing ZBTB38 into SCI mice,significantly alleviated secondary damage of spinal cord with decreased ER stress-associated apoptosis and partially recovered spinal cord functions.The expression patterns of RB1CC1 and ZBTB38 were consistent at different stages of secondary spinal cord injury in vivo.At the same time,the biosynthesis of autophagosome as well as the ability of mouse motor neurones was enhanced significantly.These findings demonstrate that restoration of ZBTB38 expression can reduce secondary tissue damage after SCI,and suggest that a therapeutic strategy for targeting ZBTB38 promote functional recovery of spinal cord for patients with SCI.In the later studies,the molecular mechanism of autophagy initiation gene RB1CC1 and neurological disease recovery was explored by regulating the expression changes of ZBTB38 gene,opening up a new strategy for treatment.