Experimental Study Of MiRNAs In Osborne's Ligament Of Cubital Tunnel Syndrome

?Objective?Cubital tunnel syndrome(CuTS),also known as delayed ulnar neuritis,is the second most common peripheral nerve compression disease.Hypertrophy of Osborne's ligament,which is caused by elbow trauma and chronic fatigue,is an important cause of ulnar nerve compression in CuTS.In order to further validate the related pathways and provide new ideas for the research of pathogenesis and etiology of CuTS,here we used microarray to detect the microRNAs(miRNAs)expression difference of Osborne's ligament and made bioinformatics analysis.?Methods?1.Three Osborne's ligament and three control tendinous tissue samples came from CuTS patients who underwent operation of anterior subcutaneous transposition in Tianjin Medical University General Hospital during August 2014 to November 2014.General RNA was extracted and a microarray assay was performed.Results of miRNAs expression were partially validated by qRT-PCR.2.miRWalk2.0 software was used to predict putative target genes.Relation network diagram of miRNAs and their corresponding target genes was made by Cytoscape3.0 software.GO enrichment and KEGG pathway enrichment analysis were performed by DAVID6.7 software and Bioconductor gene annotation software.Cluster3.0 analysis of miRNAs and pathways were analyzed by Cluster3.0 software.3.Human skin fibroblast cell lines(HSF)were constructed and transfected with miR-146b-5p overexpression vector(miR-146b-5p mimic)and antisense interference vector(anti-miR-146b-5p).Fibrosis markers,COL1A1,COL3A1,Fibronectin(FN),SMAD4 and TGFB1 were detected by qRT-PCR and Western-Blot to determine whether miR-146b-5p affects fibrosis through the TGF-?/SMAD4 pathway.?Results?70 differentially expressed miRNAs were found in Osborne's ligament(P<0.05),3 up-regulated miRNAs(hsa-miR-422 a,hsa-miR-7855-5p,hsa-miR-1343-3p),and 67 down-regulated miRNAs(hsa-miR-196a-5p,hsa-miR-146b-5p,hsa-miR-297,hsa-miR-21-3p,hsa-miR-595,hsa-miR-663 b,hsa-miR-615-5p,hsa-miR-185-3p and others).The expression patterns of hsa-miR-1343-3p,hsa-miR-21-3p and hsa-miR-146-5p detected by qRT-PCR were consistent with the microarray data.A total of 1804 predicted target genes of miRNAs were found by miRWalk2.0 software.Target gene analysis revealed that most of these target genes were regulated by 7 miRNAs(hsa-miR-146b-5p,hsa-miR-21-3p,hsa-miR-185-3p,hsa-miR-615-5p,hsa-miR-659-3p,hsa-miR-663 a and hsa-miR-760).Cluster analysis by Cluster3.0 software showed that hsa-miR-146b-5p,hsa-miR-196a-5p and hsa-miR-659-3p clustered functionally,hsa-miR-21-3p and hsa-miR-297 clustered functionally.Target gene analysis revealed that WNT2,ILK,TGFB1,IL6,FGF1 and others were high frequency target genes of the differentially expressed miRNAs,and they took part in protein metabolism process,cell growth regulation,cell cycle,cell division,cell metabolism,signal transduction and other biological processes.KEGG pathway analysis indicated that the target genes were mainly concentrated in inadherens junction,focal adhesion,axon guidance,lysine degradation,other glycan degradation,cell adhesion molecule(CAMs),mitogen activated protein kinase(MAPK)signaling pathway,retrograde endocannabinoid signaling,glycosaminoglycan biosynthesis-heparan sulfate/heparin,neurotrophin signaling pathway and other 8 pathways.Cluster analysis by Cluster3.0 software showed that focal adhesion and ErbB signaling pathway clustered functionally,adherens junction and CAMs clustered functionally.MAPK and focal adhesion signaling pathways were closely related with the predicted target genes.qRT-PCR and Western-Blot validated that the fibrosis markers,COL1A1,COL3A1,FN,SMAD4 and TGFB1 were all down-expressed in miR-146b-5p mimic groups,and were all up-expressed in anti-miR-146b-5p groups.?Conclusions?By analyzing the expression of miRNAs between degenerative proliferative Osborne's ligaments and control tendon tissues in CuTS patients,the differentially expressed miRNAs were found,and their target genes as well as potential signal pathways were obtained,which provided clues for the pathogenesis about hypertrophy of Osborne's ligament in CuTS.