Bioinformatic Analysis Of Non-coding Expression In Rotator Cuff Tendinopathy

Background and objective:Rotator cuff(RC)tendinopathy is a common musculoskeletal disorder in the shoulder,which is one of the major causes contributing to shoulder pain and RC tear.This disease brings great pain and financial burden to athletes and ordinary people.Despite th e advances in understanding RC tendinopathy and innovations in surgical techniques,conservative treatments have limited efficacy and the RC re-tear rate after surgical repair is still high due to a poor understanding of the occurrence mechanism of RC tendinopathy.Therefore,potential factors that trigger RC tendinopathy and its therapeutic targets for clinical work should be identified.With the help of the RNA-sequence(RNA-Seq)technique,the potential function of non-coding RNAs(ncRNA)represented by long non-coding RNAs(lncRNA)and circular RNAs(circRNA)have been continuously explored in the occurrence and development of various of diseases.In this study,we subjected samples obtained from patients with RC tendinopathy and normal tendons to ncRNA and mRNA profiling by RNA-Seq in order to identity potential roles and mechanisms of lncRNAs,circRNAs and mRNAs in RC tendinopathy.Method:Five RC tendinopathy tissue and five normal tendons were identified through MRI,arthroscopic and histological staining.RNA-Seq was performed to identify lncRNAs and mRNAs differentially expressed among these identified tendons.Gene Ontology(GO),Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway,competing endogenous RNA(ceRNA),co-expression network construction,Protein-Protein Interaction(PPI)network and Gene Set Enrichment Analysis(GSEA)were used to identify the potential functions of these RNAs.Three lnc RNAs and three m RNAs were validated by quantitative reverse transcription polymerase chain reaction(PCR).The previous RNA-seq data was used for analysis.The bioinformatic analysis was performed based on the DE circRNA and their parental genes,including GO,KEGG and ceRNA network construction.Results:RC tendinopathy showed uneven thickness of tendon,collagen fiber curling and heterogeneity high signal in T2-weighted coronal magnetic resonance imaging when compared with normal tendon.The RC tendinopathy samples exhibited synovial hyperplasia,and they were light yellow when compared with normal tendon tissues in arthroscopy.When dragged with arthroscopic forcep,the pathological tendon appeared stiff and less elastic.Histologically,in RC tendinopathy,the collagen had lost its normal architecture and showed uneven thickness.There was a cluster of capillaries among fibers and the nucleus were round.Stainable mucin was present in the ground substance between discrete fiber bundles.Through the RNA-Seq,419 differentially expressed lncRNAs and 1541 m RNAs were identified using criteria with fold change of > 2 and P of < 0.01.The GO enrichment analysis showed the differentially expressed genes were enriched in FATZ binding,skeletal muscle thin filament assembly,muscle filament sliding,actin-myosin filament sliding and structural muscle constituent.The KEGG analysis showed genes were enriched in citrate cycle,renin-angiotensin system,sphingolipid signaling pathway,glyoxylate and dicarboxylate metabolism,B cell receptor signaling pathway and p53 signaling pathway.The ceRNA network showed the interaction of differentially expressed RNAs,comprising 139 lncRNAs,126 mRNAs,and 35 miRNAs.And the most linked RNAs were hsa-miR-1273g-3p,NONHSAT224676.1 and ARHGAP6.PPI analysis showed revealed several hub genes including KLHL41 and TNNT3.GSEA analysis showed the three most significantly enriched gene sets positively correlated with RC tendinopathy were complement activation,immunoglobulin complex and immune response mediated by circulating immunoglobulin.The three most significantly enriched gene sets negatively correlated with RC tendinopathy were DNA packing,DNA packing complex,and chromatin assembly or disassembly.NONHSAT209114.1,ENST00000577806,NONHSAT168464.1,PLK2,TMEM214,and IGF2 were validated by PCR,show the same trend with RNA-Seq and steady expression in RC tendinopathy.In circRNA profiling analysis,94 differentially expressed circ RNAs were identified using criteria with fold change of > 2 and P of < 0.01.GO analysis show the genes were mostly enriched in response to cAMP.KEGG analysis showed that most genes were enriched in ECM-receptor interaction,protein digestion and absorption,cell cycle,B cell receptor signaling pathway,Fox O signaling pathway,NF-kappa B signaling pathway and T cell receptor signaling pathway.The ceRNA network showed the interaction of differentially expressed RNAs,comprising 28 circRNAs,46 mRNAs and 13 miRNAs.And circRNA.8951-has-miR-6089-DNMT3 B had the most sum max energy.Summary:1 The lncRNA,Circrna and mRNA expression profile are significantly changed in RC tendinopathy.2.The outcomes of this study provide valuable insights into the mechanism of RC tendinopathy and suggest a series of potential targets for the diagnosis and treatment,paving the way to better treatment and prevention of this disorder.3.The differentially expressed RNA confirmed by q RT-PCR,including NONHSAT209114.1,ENST00000577806,NONHSAT168464.1,PLK2,TMEM214,and IGF2,may have potential roles in RC tendinopathy occurrence.4.The ceRNA regulation axis of circRNA.8951-has-mi R-6089-DNMT3B may be involved in the occurrence of RC tendinopathy.