Peripheral Blood Hsa-circrna5333-4: A Novel Biomarker For Myasthenia Gravis

ObjectivesCircular RNA（circRNA）is a novel,non-coding,small ribonucleic acid,which widely exists in different tissues of the body.Studies have confirmed the existence of circRNAs specifically expressed in certain diseases（such as tumors,coronary heart disease and type 2 diabetes mellitus）,making them important candidates for novel biomarkers.This study aims to explore whether there are circRNAs specifically expressed in the peripheral blood of myasthenia gravis（MG）and its potential as a biomarker of this disease.methods1.Peripheral blood of three normal control samples and three patients with myasthenia gravis were collected,and the difference in the relative expression of circRNAs in whole blood was detected by microarray analysis of circRNA microarray,and specific circRNAs were screened out.2.Peripheral blood samples from 19 normal control subjects and 20 patients with myasthenia gravis were collected for small independent cohort validation.Real-time fluorescence quantitative nucleic acid amplification detection system（qPCR）was used for validation and ROC curve analysis.3.Further validation of candidate circRNAs in a small independent cohort study（including 19 patients with systemic lupus erythematosus and 20 patients with myasthenia gris）.The real-time fluorescence quantitative nucleic acid amplification detection system（qPCR）was also used for verification,and ROC curve analysis was performed to test the disease-related specificity of selected genes compared with other autoimmune diseases.4.Peripheral blood of 61 normal control samples and 61 patients with myasthenia gravis were collected to verify the candidate circRNAs in a large independent cohort study.ROC curve analysis was performed to determine their diagnostic value,and Pearson correlation coefficient r analysis related to the severity of the disease was performed.Results1.11 circRNAs with significantly differentially expressed circRNAs were screened by microarray analysis.Compared with the normal control group,there were 6 up-regulated relative expression levels（hsa_circ₀102575,hsa_circ₀032119,hsa-circrna3627-24,hsa_circ₀076490,hsa-circRNA5333-4 and hsa_circ₀047056）.5 relative expressions were down-regulated（hsa_circ₀035018,hsa_circ₀035149,hsa_circ₀047502,hsa_circ₀087152 and hsa-circrna16293-1）,P < 0.05.2.In a small cohort study of 19 normal control samples and 20 patients with MG,the relative expression levels of 4 circRNAs showed the same trend as the results of microarray analysis（hsa_circ₀076490,hsa-circRNA5333-4,hsa_circ₀047056 and hsa-circrna16293-1）.Among them,hsa_circ₀076490 and hsa-circRNA5333-4 had the maximum area under the ROC curve（AUC）of 0.782（95%ci: 0.635-0.928）and 0.790（95%ci: 0.632-0.947）respectively,and they were set as candidate biomarkers for MG.3.In a small independent cohort study involving 19 patients with SLE and 20 patients with MG,it was further verified that the relative expression levels of hsa_circ₀076490 and hsa-circRNA5333-4 were consistent with the trend of microarray analysis results.The area under the ROC curve（AUC）was 0.766（95%ci: 0.611-0.920）and 0.790（95%ci: 0.644-0.935）,respectively.4.Hsa-circRNA5333-4 had a higher AUC of 0.864（95% CI = 0.801-0.928）and a higher Pearson correlation coefficient of r = 0.505（P <0.001）with quantitive MG score（qMG）in a large cohort validation trial of 61 normal control samples and 61 MG patients.Chi-square test and t-test showed that hsa-circrna5333-4 was significantly correlated with qMG,gender and acetylcholine receptor antibody levels（P <0.05）.Conclusions1.Hsa-circRNA5333-4 has significant specificity in the peripheral blood of patients with myasthenia gravis,and is positively correlated with moderate severity of the disease,which can be used as a novel biomarker for the diagnosis and monitoring of the disease.2.Hsa-circRNA5333-4 and hsa-miR-4310 and MORF4L2 may be cis-reaction chains,or may be involved in the pathogenesis of myasthenia gravis.