Pathway-based And Network Analysis Of The Underlying Mechanism Of BoNT/A Promoting Axonal Regeneration

Objective:By studying the biological function and interaction of(Botulinum toxin A,BoNT/A)BoNT/A targeting genes and its protein-protein interactions network,we revealed the possible molecular mechanism of BoNT/A in promoting axonal regeneration of nerve cells.Methods:(1)Construction of protein-protein interactions(PPI)network for BoNT/A direct protein target.The direct protein target(DPT)and DPT-PPI were identified for BoNT /A by querying the drug database ——Drug Bank and STRING database respectively.(2)Biological function analysis of DPT-PPI.The biological function of DTP-PPI network was analyzed by Web Gestalt(a web-based geneset analysis tool).(3)Identification of DTP-PPI core pathway.The DTP-PPI network was divided into three gene clusters by K-means clustering method,and the biological function of the gene clusters was analyzed respectively.Finally,five related core pathways were identified.(4)Identification of key genes involved in PPI via Steiner Minimal Tree Algorithm.We finished construction of whole human interactome network,and then identified key genes of DTP-PPI in whole human interactome network by Steiner Minimal Tree Algorithm.(5)The dataset(GSE45006)was downloaded from GEO database(Gene Expression Omnibus,http://www.ncbi.nlm.nih.gov/geo).(6)Differentially expressed genes(DEGs)of m RNA was performed using limma package of R software.The screening criteria for DEGs were: P-value< 0.05,log fold-change>1 or log fold-change<1.Results:(1)Two DTPs of BoNT/A,SNAP25 and RhoB,were identified through Drug Bank.Three PPI networks were constructed through string database:SNAP25-PPI,RhoB-PPI,SNAP25 and RhoB-PPI.(2)Main pathways were enriched in SNAP25-PPI(SNAP25-mediated PPI network)related to Synaptic vesicle circulation,SNARE interactions in vesicle transport,metabolic pathways,taurine and low sulfonate metabolism,etc.And RhoB-PPI is mainly involved in the regulation of actin cytoskeleton,neurotrophin signaling pathway,axon guidance and other pathways,etc.(3)It is obvious that these five pathways(regulation of actin cytoskeleton,neurotrophin signaling pathway,axon guidance,adherens junction,focal adhesion)were contained in all three clusters.(4)We extracted 47 Hub genes for RhoB-PPI from the human interactome network by the Steiner minimal tree algorithm.(5)Dataset GSE45006 was downloaded from GEO database.The GSE45006 experimental platform is GPL1355,which includes 20 rat samples with spinal cord injury and 4 control sham samples.There are 4 samples respctively at 4time checkpoints(1 day,3 days,1 week,2 weeks)after SCI.(6)4 time checkpoints of DEGs were obtained,Rho families(RhoA and RhoC)and its subfamilies(CDC42and RAC2)were differentially expression in the four time checkpoints of DEGs.Conclusion:(1)Based on network and pathway analysis of BoNT/A assiociated with RhoB-PPI,it can help us understand the potential mechanism of BoNT/A promoting axonal regeneration.(2)BoNT/A may act on RhoB and RhoA of Rho families,and promote axonal regeneration by inhibiting Rho-ROCK pathway to affect actin skeleton regulation.(3)By analyzing the gene expression profile from rats with spinal cord injury,Rho families(RhoA and RhoC)and their subfamilies(CDC42 and RAC2)after spinal cord injury were persistently differentially expression,which was proved that it was significant for core genes of Hub genes network to participate in the biological process of neural cells after spinal cord injury and RhoA,RhoC are good therapeutic targets for BoNT/A.