Aberrant Dysregulated Circular RNA In Peripheral Blood Mononuclear Cells Of Patients With Rheumatoid Arthritis

As a common chronic autoimmune disease,rheumatoid arthritis?RA?is characterized by joint swelling and joint tenderness.Circular RNA?circRNA?a novel type of non-coding RNA,is characterized by a covalently closed continuous loop without5'and 3'polarities and has been shown to be involved in several types of diseases.However,the profiles of circRNA in patients with RA have not been explored by RNA sequencing?RNA-seq?to evaluate the diagnostic value of RA.Therefore,our study was designed to analyze the expression of circRNA in peripheral blood mononuclear cells?PBMCs?with RA patients by RNA-seq.Then we screened out molecular markers that can assist in the diagnosis of RA,thus laying the foundation for further investigation of RA pathogenesis.Methods Approximately 5 ml of peripheral blood was collected from each participant using ethylenediaminetetraacetic acid?EDTA?anticoagulant tubes.Then,the PBMCs were freshly isolated from anticoagulation blood by density gradient centrifugation using human peripheral blood lymphocyte separation fluid.The PBMCs were frozen in TRIzol reagent and then were sent to Shanghai Lie Bing Biological Technology Co.LTD.They constructed a cDNA library of RNA samples using VAHTSTM Total RNA-seq?H/M/R?.The cDNA library was paired and sequenced on IIIumina HiSeq TM2500 to detect differentially expressed circRNA and mRNA in PBMCs.GO-significant enrichment analysis was performed on differentially expressed circRNA based on GO?Gene ontology?database;Pathway significant enrichment analysis was performed on differentially expressed mRNA based on KEGG?Kyoto Encyclopedia of Gene and Genomes?database.The circRNA-miRNA-mRNA molecular regulatory network was constructed with the dysregulated circRNA as the core,showing the interaction between them.Two up-regulated circRNA and two down-regulated circRNA were selected from circRNA with significant differential expression to be validated by real-time fluorescence quantitative PCR?qRT-PCR?in 32 patients with RA and 20 healthy controls.Then the receiver operating characteristic curve?ROC?was used to analyze the diagnostic value of circRNA.The target circRNA hsa_circ₀000396 was localized by RNA fluorescence in situ hybridization?FISH?.And using flow cytometric sorting and qRT-PCR,we analyzed the relative expression level of hsa_circ₀000396 in CD3⁺T,CD19⁺B and CD56⁺NK cells of RA patients compared with healthy controls.Results RNA-seq results showed that there were 71 significant differentially expressed circRNAs between RA patients and healthy controls,of which 41 were up-regulated and30 were down-regulated.There were 1078 significantly differentially expressed mRNAs in RA patients and healthy controls,and among them,662 were up-regulated and 416were down-regulated.GO enrichment analysis of differentially expressed circRNAs showed that negative regulation of the nucleotide-binding oligomerization domain containing 2?NOD2?signaling pathway was the most noteworthy enriched category in biological processes?BP?;poly?A?-specific ribonuclease activity was the most significantly enriched term in molecular function?MF?;cytoplasm and cytoplasmic side of mitochondrial outer membrane were first two significantly enriched category in cell composition?CC?.KEGG pathway analysis of differentially expressed mRNAs revealed that differential mRNAs were mainly enriched in the TNF signaling pathway and the NF-kappaB signaling pathway.The circRNA-miRNA-mRNA network shows that differentially expressed circRNAs regulate the function of downstream target genes by interacting with miRNA.The results of qRT-PCR showed that the expression levels of hsa_circ₀000396 and hsa_circ₀130438 were significantly lower in the RA group than the healthy group?P<0.05?,which was consistent with the results of RNA-seq.However,the expression levels of hsa_circ₀004442 and hsa_circ₀025887 between the RA group and healthy group were no significant differences and the data on these circRNA were not validated in RNA-seq?P>0.05?.Then hsa_circ₀000396 and hsa_circ₀130438 were further subjected to ROC curve analyses to determine their diagnostic values for RA patients.The results showed that the AUC of hsa_circ₀000396 for diagnosing RA was0.809,and the AUC of hsa_circ₀130438 for diagnosing RA was 0.774.Therefore,hsa_circ₀000396 has greater potential as a molecular marker for RA diagnosis.Using RNA FISH assay,we demonstrated that hsa_circ₀000396 predominately localized in the cytoplasm.The qRT-PCR results showed that the expression of hsa_circ₀000396 in CD3⁺T cell and CD19⁺B cell was significantly decreased in RA patients compared with healthy controls,while the expression levels of hsa_circ₀000396 in CD56⁺NK cell was not significantly different between the two groups.Conclusion Our RNA-seq identified aberrant dysregulated circRNA and mRNA were present in RA group compared to healthy control group,and the pathways involved in these differentially expressed genes were mostly relevant to inflammation and transcriptional activity.Hsa_circ₀000396 is expected to be a molecular marker for the diagnosis of RA,and may be involved in the regulation of RA by T cell and B cell,and then provide a new research direction for the diagnosis and treatment for RA.