Analysis Of Differential Expression Of CircRNA In Muscular Atrophy And Fibrosis Of Facial And Neck Cranial Nerve Injury And Preliminary Exploration Of Its Mechanism

PurposesIn this project,a 12-week-old male C57BL/6 mouse sternocleidomastoid muscle was selected to construct a facial and neck peripheral nerve injury model to explore the role of circular RNA(circ RNA)in the process of skeletal muscle atrophy and fibrosis after accessory nerve injury.The potential function and regulation effect of sclerotherapy provide new ideas for finding new targets to delay denervated skeletal muscle atrophy and fibrosis.Methods1.Masson staining to observe skeletal muscle morphology,using high-throughput transcriptome sequencing technology(RNA-seq)to compare 4 time points and control mice at 1 week,2 weeks,3 weeks,and 4 weeks after denervation injury circ RNA expression profile,focusing on the analysis of the differential expression and characteristics of circ RNA in denervated sternocleidomastoid muscle.2.Construct the differential expression circ RNA-mi RNA(micro RNA,micro RNA)network of 4 groups of denervated groups.Using bioinformatics,GO and KEGG enrichment analysis of differential circ RNA parent genes.3.Immunofluorescence method was used to detect the expression of fibrosis-related connective tissue growth factor(CTGF)in denervated skeletal muscle;then the mfuzz software was used to perform time series trend cluster analysis in the differentially expressed circ RNA obtained from sequencing to find the CTGF gene expression trend Consistent circ RNA,mi Randa software is used to predict the mi RNA binding site of these circ RNAs,and draw a circ RNA-mi RNA interaction network diagram.Real-time fluorescent quantitative PCR(real time-PCR,RT-PCR)was used to verify the differential expression of circ RNA.Results1、This study first verified the success of the skeletal muscle fibrosis model by Masson staining.A total of 3090 circ RNAs were identified by RNA-seq sequencing technology,and differentially expressed circ RNAs were screened at each time point.In this study,a total of 1386 differentially expressed circ RNAs in denervated skeletal muscle were identified,of which 686 were up-regulated and 700 were down-regulated.They were differentially expressed at different times of denervation.Compared with the control group,differentially expressed circ RNA was involved in denervation injury1 There were 83,138,314,and 151 increases in weeks,2,3,and 4 weeks,and 103,149,204,and 244 downward adjustments,respectively.2、Gene ontology(GO)functional annotation analysis in bioinformatics analysis shows that these differentially expressed circ RNAs may have corresponding biological functions at different stages of skeletal muscle injury.The early stage of injury may be related to muscle injury,muscle regeneration,and muscle injury.The formation of tubes is related to functions such as development;the later stage of injury is closely related to biological processes such as muscle cell hypertrophy and programmed muscle cell apoptosis.The kyoto encyclopedia of genes and genomes(KEGG)enrichment analysis shows that these functions may play a regulatory role in the early stage of denervation injury through focal adhesion,c GMP-PKG signaling pathway,vascular smooth muscle contraction,and MAPK signaling pathway In the late stage of injury,it plays a regulatory role through fibrosis-related TGF-β signaling pathway,Apelin signaling pathway,Fox O and other signaling pathways.3、The immunofluorescence method showed that the skeletal muscle CTGF of normal mice had basically no expression.The expression of CTGF was present after 1week of denervation,and the expression of CTGF increased significantly after 2 weeks of denervation.The expression of CTGF was weakened at 3 weeks and 4 weeks of denervation.Time-series trend cluster analysis found that mmu＿circ＿0000372 、mmu＿circ＿0001529、 mmu＿circ＿0014982.etc are molecules consistent with CTGF gene expression trends;RT-q PCR experiments verified that mmu＿circ＿0001529 and mmu＿circ＿0000372 were up-regulated in denervated skeletal muscle,which was consistent with the sequencing results.ConclusionSignificant changes have occurred in the expression profile of circ RNA after denervation injury.These differentially expressed circ RNAs and their potential target molecules may be related to the overall process of denervation and skeletal muscle atrophy and fibrosis.After denervation injury,the expression of mmu＿circ＿0001529 is up-regulated,which may promote the expression of CTGF through the action of mi R-30 c,and regulate the muscle atrophy and fibrosis of facial and neck cranial nerve injury.