The Expression Of Circular RNAs In Peripheral Blood Plasma From RA (Rheumatoid Arthritis) Patients And Its Correlation With The Disease Activity Of RA Patients

Background:Rheumatoid arthritis（RA）is a kind of heterogeneous,systematic and autoimmune disease caused by multifactor.It demonstrates a relatively high incidence,with currently unclear pathogenesis.Therefore it still has no curative therapeutic strategy.The current treatment for RA is to relieve symptoms and to improve the function of affected organ and joints.As a result,this disease will place a heavy mental and economic burden on the patients and their family.There is increasing evidence suggesting that the gene expression regulated by epigenetic mechanisms plays a critical role in RA development.Deep sequencing technique for RNA reveals that circRNA（circular RNA）,a novel non-coding RNA,exerts a key role in regulation of gene expression.CircRNA is a novel class of non-coding RNAs featured by a covalently closed continuous loop.Although it was first discovered early in 1971,research related to circRNA was basically stagnant because of the limited detection methods.Owing to the fast-developing deep-sequencing techniques,the role of circRNA in regulating the expression of protein-coding genes is revealed,which is closely related to many human illnesses.However,research about circRNA in RA is still in the initial stage.The related studies are very limited,especially for circRNA in the plasma of RA patients.Since circRNAs possess a covalently close continuous loop and thus are stabilized in the body,these molecules are quite promising to be novel biomarkers for human diseases.It is therefore worthy of studying on circRNA in RA patients.Objective:CircRNAs would probably participate in the development of RA.The present study was first to screening differential expression of circRNAs in the peripheral blood plasma of RA patients by circRNA microarray compared with healthy controls.RT-PCR（reverse transcription-polymerase chain reaction）was performed to further validate the RA related circRNAs and their relationship with the disease activity of RA patients.We aimed to explore novel biomarkers for RA screening and diagnosis of the different disease activity stages,thereby guiding the therapy for RA patients in the d ifferent disease activity stages.Materials and Methods:1.Recruitment of study subjects:we recruited patients diagnosed with RA in the Rheumatology and Immunology Department and healthy controls from Physical Examination Center of Nanfang Hospital at Southern Medical University.Informed consent was obtained from each participant.Fast blood was obtained from the study subjects and subsequently plasma was separated by centrifugation.2.Screening of circRNAs by circRNA microarray assay:Total RNA was extracted from peripheral blood plasma and RNA quality was assessed.Human circular RNA array 2.0（8 ×15K）was used to detect the circRNA expression profiles in RA patients and healthy controls.Differential expression analysis of circRNA was performed.3.Validation of circRNAs:We detected the potential differential expression circRNAs by RT-PCR method in cobas z480 PCR Amplifier in a larger study samples.4.Analysis of the relationship between the significant circRNAs and the disease activity of RA patients:the correlation between circRNA and clinical stages of RA was performed by spearman rank relational coefficient.Results:1.Numerous circRNAs were found in the plasma of RA patients.Compared with the healthy controls,the expression levels of hsa_circrna₀01653,hsa_circRN A₄0493 5,hsa_circRN A₁00935,hsa_circRNA₀05008,hsa_circRNA₀00407,hsa_circRNA₁04757,hsa_circRNA₀05198,hsa_circRNA₀34642,hsa_circRN A₁03852,hsa_circRNA₁01085,and hsa_circRNA₁00395 were significant higher in the plasma of the RA patients.2.The expression of hsa_circrna₀05008 in the plasma of the patients with rheumatoid arthritis is stable and is higher than that in the healthy controls,and it is statistically significant.3.We found a positive correlation between hsa_circrna₀05008 and the disease activity index including C-reactive protein（CRP）and DAS28 in the patients with rheumatoid arthritis.Conclusions:The expression of circRNA in the plasma of patients with rheumatoid arthritis is different from that in healthy controls.The hsa_circrna₀05008 is stably expressed in RA patients and is higher in the plasma of patients with rheumatoid arthritis than that in healthy controls.Moreover,hsa_circrna₀05008 is positive correlated with C-reactive protein（CRP）and DAS28 in patients with rheumatoid arthritis.Hsa_circrna₀05008 may be used as a potential biomarker for the disease activity of rheumatoid arthritis,which could also guide the treatment for patients with rheumatoid arthritis.