Identification Of Apoptosis-Related Genes And CeRNA Networks In Sevoflurane-Induced Neurotoxicity In Mouse Cerebral Cortex

Objective: Sevoflurane can induce neurotoxicity in mice,which raises concerns about the safety of sevoflurane in clinical applications,and therefore the specific mechanisms of sevoflurane-induced neurotoxicity need to be investigated.It has been reported that the cerebral cortex is the main target region of sevoflurane-induced neurotoxicity,and the activation of inflammatory and apoptotic pathways plays a significant role in this mechanism.Mi RNAs and lnc RNAs are also widely involved in the regulation of related pathways.In this study,we hope to identify gene expression changes related to inflammatory and apoptotic pathways in sevoflurane early exposure-induced neurotoxicity in mouse cerebral cortex and the biological functions and pathways they are involved in by bioinformatics methods,and to construct a triple competitive endogenous RNA(ce RNA)regulatory network consisting of m RNA-mi RNA-lnc RNA based on the mi RNAs and lnc RNAs they target.Methods:1.Gene cluster for apoptosis and IFN(interferon)-associated inflammation were searched through the GSEA database and Pub Med literature.The GEO database was searched to obtain the mouse cortical sequencing dataset required for the analysis following early sevoflurane exposure.2.The sequencing data were cleaned and collated,screened for differential expressed genes(DEGs)using the R package limma,and then intersected with the IFN-associated inflammation and apoptosis gene cluster.IFNassociated inflammation and apoptosis related DEGs were obtained.3.GSEA analysis of overall DEGs after sevoflurane exposure and apoptosisrelated DEGs was performed to understand the signaling pathways and biological functions in which they are primarily involved.4.Construction of PPI networks using apoptosis-related DEGs,visualization and screening of hub modules and hub gene using Cytoscape.5.Use apoptosis-related DEGs to predict the corresponding target mi RNAs and lnc RNAs,and construct ce RNAs that contain a triple regulatory network of m RNA-mi RNA-lnc RNAs after controlled prediction conditions,bibliographic search screening and validation by previous high-throughput sequencing.6.Validation of hub gene expression levels in sequencing data from human embryonic stem cells exposed to sevoflurane.The screening of ce RNA hub nodes and modules was guided based on hub gene screening results.Results:1.The final search yielded 6791 apoptotic related genes and 2906 IFN-related inflammatory genes.Two GEO datasets GSE166607 and GSE148436 were used for subsequent analysis.2.The differential expressed gene analysis of GSE166607 yielded 373 upregulated genes and 298 down-regulated genes in mouse cerebral cortex after sevoflurane exposure.One IFN-related inflammatory genes,IRF9,was obtained after taking intersections with IFN-related inflammatory gene cluster.74 up-regulated genes and 71 down-regulated genes were obtained after taking intersections with the apoptosis-related gene cluster,for a total of 145 genes.IRF9 was also present in the apoptosis-associated differential expressed gene cluster,along with a lack of other evidence of IFN pathway activation,and therefore it was concluded that IFN-related pathways were not significantly activated in this batch of sequenced samples.Further considerations of apoptosis-related differential genes and pathways.3.GSEA analysis of DEGs and apoptosis-associated DEGs showed that both types of DEGs are mainly related through transcriptional and posttranscriptional regulatory pathways such as RNA processing,splicing and binding,more notably in apoptosis-associated DEGs.4.After constructing and visualizing the PPI network,12 hub gene were screened,namely: SPP1,MGST1,ITGA2 B,POLR1A,RALBP1,AK4,P4HA1,GHR,HPX,C3,ITGB7,and RIPK1.5.After construction and visualization of ce RNAs,12 hub genes were evaluated and visualized for expression in GSE166607 and GSE148436,and five genes,GHR,AK4,HPX,P4HA1,and SPP1,were subjected to subsequent analysis,and their associated nodes and linked ce RNA networks were the more plausible ce RNA fraction.Of these,the findings for the AK4-associated pathway are more suitable for generalization to human samples.Conclusion:In the neurotoxicity induced by early exposure to sevoflurane in mice,RNA metabolism was significantly active,and energy metabolism and cytoskeleton related pathways were involved in regulation.The neurotoxicity is mediated through a ce RNA network of GHR,AK4 and RALBP1 as m RNAs,mmu-mi R-15b-5p,mmu-mi R-27b-3p and mmu-mi R-34a-5p as mi RNAs,42 lnc RNAs represented by GAS5,H19 and MALAT1 involved in the regulation of apoptosis-related pathway activity.Among them,the AK4-related pathway has a higher confidence level.