The Function And Regulatory Mechanism Of Sox4 In Spinal Cord Injury

BackgroundSpinal cord injury(SCI)is a series of cascade reactions such as local compression,rupture,ischemia and inflammation of the spinal cord caused by various trauma or non-traumatic factors.There are sensory,motor and autonomic dysfunction,which are clinically disabled and have a high mortality rate.At present,the treatment of spinal cord injury mainly intervenes through mechanisms such as inhibiting local inflammatory response,improving local microenvironment,promoting nerve regeneration and reducing the scope of lesions.The curative effect is dissatisfied,and there is also a lack of standard,standardized and effective treatment methods.Clarifying the molecular mechanism of the occurrence and development of SCI and providing new targets for clinical treatment are urgent problems to be solved.Sox(Sry-related high mobility group box)genes are homologous to the Sry gene located on the Y chromosome of male animals,which have a highly conserved sequence,and are expressed in many animals.The proteins encoded by the Sox genes play a key role in the developmental process.Studies have shown that SOX proteins play an important role in the central nervous system,natural immune system,tumor immunity and other aspects.In SCI,the research on Sox family genes is still in its infancy,and the role of Sox genes in the pathophysiological process of SCI need to be further explored.Sox genes as a clinical intervention target need to be tested in clinical practice.Sox4 gene is an important member of the Sox family of genes.Previous studies have shown that Sox4 gene has important functions in neurodevelopment and immune regulation.However,in the research fields of microglia function regulation and the occurrence and development of SCI,there is no research report on Sox4 gene.ObjectivesThrough previous preliminary experiments used q PCR method,we found that Sox4 gene showed low expression in SCI tissues.The purpose of this study is in the vitro level to further explore the expression of Sox4 gene in SCI,and to analyze the effect of its expression on the pathophysiological process of SCI,to study the function of Sox4 gene in SCI and to preliminarily explain the regulatory mechanism of Sox4 gene in SCI.Materials and methods1.Construction of lipopolysaccharide LPS-induced SCI in vitro cell model BV2 cell lineWe cultured BV2 microglia in a suitable medium to obtain stable expression,intervened BV2 microglia with different concentrations of LPS,and used CCK-8 assay to detect the effect of LPS on cell proliferation,flow cytometry to detect the effect of LPS on apoptosis and ELISA to detect the effect of LPS on the expression of cellular inflammatory factors.2.Effects of Sox4 gene on the biology of microglia and regulation of inflammatory factor expression after SCIThe expression of Sox4 gene in SCI in vitro cell model BV2 microglia cell line was detected by q PCR method,and the stable cell line with Sox4 gene overexpression was further constructed and screened according to the results.CCK-8 assay,flow cytometry and ELISA were used to detect the effects of Sox4 gene overexpression on the proliferation,apoptosis and inflammatory factor expression of microglial cells.3.A preliminary study on the regulatory mechanism of Sox4 on SCI progressionThe representative biological processes and signaling pathways of DEGs were constructed through the STRING database and Cytoscape.And the functional enrichment analysis of GO and KEGG was performed on DEGs.Besides,the activation maps of IL-17 signaling pathway and TNF signaling pathway were constructed to explore the role of Sox4 gene in their signaling pathways.Furthermore,we performed chromatin immunoprecipitation experiments on LPS-treated Sox4-overexpressing BV2 cells and LPS-treated control BV2 cells to further investigate the underlying mechanism of Sox4 gene.Results1.Construction of lipopolysaccharide LPS-induced SCI in vitro cell model BV2 cell lineThe results showed that LPS inhibited the proliferation of BV2 microglia cells and promoted their apoptosis,while LPS promoted the expression of inflammatory factors in BV2 microglia cells.We successfully constructed the LPS-induced SCI in vitro cell model BV2 microglia cell line.2.Effects of Sox4 gene on the biology of microglia and regulation of inflammatory factor expression after SCIUsing q PCR method,it was found that Sox4 gene showed low expression in SCI in vitro cell model BV2 microglial cell line.A stable cell line with Sox4 overexpression was successfully constructed,and it was found that Sox4 overexpression promoted the proliferation of BV2 microglial cells,inhibited their apoptosis,and inhibited the expression of inflammatory factors.3.A preliminary study on the regulatory mechanism of Sox4 on SCI progressionWe constructed the representative biological processes and signaling pathways of DEGs through the STRING database and Cytoscape,and found that IL-17 and TNF signaling pathways were highly correlated with DEGs.We performed GO and KEGG functional enrichment analysis on DEGs,and constructed an activation map of IL-17 and TNF signaling pathways.It was found that the expression of inflammatory genes was significantly down-regulated after the IL-17 pathway and TNF pathway were inhibited.Prompting us,Sox4 overexpression may suppress inflammation in an in vitro SCI model through IL-17 and TNF signaling pathways.In addition,through chromatin immunoprecipitation experiments,SOCS3,Edn1 and Fos genes were found to be DEGs with significant binding peaks associated with inflammation.Socs3 and Edn1 were identified as DEGs associated with the TNF signaling pathway,while the Fos gene was a common gene in the IL-17 and TNF signaling pathways.Socs3,Edn1 and Fos genes are expected to become new clinical therapeutic targets for SCI.Conclusions1.The LPS-induced SCI in vitro cell model BV2 microglial cell line was successfully constructed.2.Sox4 overexpression promotes BV2 microglia proliferation,inhibits their apoptosis,and inhibits the expression of inflammatory factors.It prompts us that Sox4 plays an important role in the occurrence and development of SCI,which may provide new targets and ideas for the treatment of SCI.3.Sox4 overexpression may inhibit inflammation in SCI models in vitro through IL-17 and TNF signaling pathways.Socs3,Edn1 and Fos genes are significantly associated with inflammation and are expected to become new clinical therapeutic targets for SCI.