Preliminary Study On LncRNA Expression Profiling Of Synovial Fluid-Derived Exosomes In Patients With Rheumatoid Arthritis

Background:Rheumatoid arthritis（RA）is an autoimmune disease characterized by synovitis and progressive destruction of articular cartilage and bone caused by immune disorders and inflammation.However,the specific mechanism has not been clarified,which call for further exploration of pathogenesis and biomarkers of RA.It has been reported that the activation of abnormal immune cells and the secretion of immune molecules in the synovial fluid may be closely related to the occurrence and development of RA.Exosome,a type of extracellular vesicles secreted by cells,performs biologic functions by transporting RNA to target cells.Long-chain non-coding RNA（lncRNA）is a kind of RNA with a length of more than 200 nucleotides,which plays an important role in the processes of cell differentiation,inflammation,immune response,etc.There are no relevant reports that focus on lncRNA expression profiling of synovial fluid-derived exosomes in patients with RA yet.Objective:To identify the lncRNA expression profile of synovial fluid-derived exosomes in RA patients,and to carry out bioinformatics analysis on target genes of differentially expressed lncRNAs.Methods:In this study,the synovial fluid samples were collected from 9 RA,11 gout,and 10 osteoarthritis（OA）patients,among which RA was the experimental group and gout and OA were the control group.Exosomes were separated from synovial fluid by traditional ultracentrifugation.Total RNA was extracted from exosomes by using HiPure Liquid RNA/miRNA kits.The lncRNAs expression levels of synovial fluid-derived exosomes was detected by lncRNA sequencing.Clustering heatmap was drawn to reflect the relative levels of lncRNAs.Differentially expressed lncRNAs in RA were screened,and the enrichment analysis of their target genes were analyzed by Gene Ontology and Kyoto encyclopedia of genes and genomes.Real time quantitative reverse transcription PCR（qRT-PCR）was conducted to detect the lncRNAs expression levels of synovial fluid-derived exosomes,and the correlation between lncRNAs expression levels and clinical indicators was analyzed.Results:A total of 1067 differentially expressed lncRNAs were detected in synovial fluid-derived exosomes of RA patients.347 differentially expressed lncRNAs were found in RA,compared with OA,of which 236 were up-regulated（including ENST00000430787.1 and ENST00000451781.1）and 111 were down-regulated（including NR₁45469.1 and ENST00000552334.1）.Functional analysis of the differentially expressed lncRNAs target genes revealed that they were mainly enriched in "T cell activation","apoptosis" and "mTOR signaling pathway".805 differentially expressed lncRNAs were found in RA,compared with gout,of which 479 were up-regulated（including ENST00000562852.1 and ENST00000420522.1）and 326 were down-regulated（including ENST00000590368.1 and ENST00000433344.1）.Functional analysis of the differentially expressed lncRNAs target genes revealed that they were mainly enriched in"positive regulation of autophagy","leukocyte transendothelial migration" and "ferroptosis".85 specially expressed lncRNAs were found in RA,compared with OA and gout,of which 65 were up-regulated（including ENST00000430787.1 and ENST00000420522.1）and 20 were down-regulated（including NR₁45462.1 and NR₁45461.1）.Functional analysis of the specially expressed lncRNAs target genes revealed that they were mainly enriched in "autophagy","rheumatoid arthritis disease pathway" and "mTOR signaling pathway".qRT-PCR results showed that the level of ENST00000433825.1 was increased in RA when comapared to OA and gout,and the level of ENST00000449119.1 in RA was decreased when compared to gout.qRT-PCR results verified that the trends of the differentially expressed lncRNAs between groups were consistent with that in the lncRNA sequencing.Correlation analysis showed that the levels of ENST00000433825.1 and ENST00000449119.1 in RA patients were positively correlated with the levels of C-reactive protein（CRP）and erythrocyte sedimentation rate,and were negatively correlated with rheumatoid factor and anti-cyclic citrulline polypeptide antibody,among which ENST00000433825.1 was most significantly correlated with CRP（P<0.001）.Conclusions:Compared with OA and gout,the study revealed the differentially expressed lncRNA expression characteristics of synovial fluid-derived exosomes in patients with RA.Biological information analysis indicated that differentially expressed lncRNAs performs their biological function such as "autophagy,mTOR signaling pathway" by regulating the target genes.Correlation analysis showed that ENST00000433825.1 was significantly positively correlated with CRP in RA patients,suggesting that ENST00000433825.1 may be potential biomarkers of RA,which provides new insight for exploring the pathogenesis of RA.