Comprehensive Microarray Analysis Of Circular RNA Expression Profile And The Screening And Identification Of Potential Biomarkers For Systemic Lupus Erythematosus

AimsSystemic lupus erythematosus（SLE）is a typical chronic systemic autoimmune disease（AID）that occurs in adult women and is significantly more common in women than men（about 9:1）.The course of the disease is repeatedly and the clinical manifestations are complex and diverse.Multiple tissues and organs were involved,resulted in autoimmune reactions,produced a large number of autoantibodies and immune complexes,deposited in the vascular wall of tissues and organs,activated complement system,generating vasculitis,Cause more tissues and organs damage.The etiology and pathogenesis of SLE are still not clearly,and generally considered to be related to family inheritance,environment,hormones,and immunity.Although the rational use of glucocorticoids,immunosuppressive agents and biological agents can significantly alleviate the condition and prognosis of patients with SLE,the treatment status of the disease is still not satisfactory.At the same time,recurrence is also a serious problem in the course of treatment.At present,the laboratory diagnosis and disease judgment of SLE mainly depend on the detection of autoantibodies,such as ANA,anti-ds-DNA,anti-Sm and anti-phospholipid antibodies,while,these autoantibodies have the defects of poor sensitivity or specificity,which greatly limits their clinical value.Therefore,it is urgently to establish new technologies and discover new biomarkers to solve these problems.Circular RNA is a new type of RNA that is different from the traditional linear RNA.It has a closed loop structure and exists in a large number of eukaryotic transcriptomes.Originally,circRNAs were considered as a class of low-abundance RNA which were misinterrupted by exon transcripts and were considered as interfering products.More and more studies have found that circRNAs are involved in the development of many diseases,including autoimmune diseases.Therefore,looking for differentially expressed circRNAs of SLE patients and exploring the relationship between the expression level of circRNA and the pathogenesis of SLE may help elucidate the pathogenesis of SLE and provide a theoretical basis for the diagnosis and targeted therapy of SLE.Methods1.In this project,a case-control study was conducted to collect differentially expressed circRNAs in plasma using gene chip technology by collecting blood samples from 6 paired SLE patients and normal healthy controls,then,the preliminary validation phase,the target circRNAs were verified in twenty-four paired plasma samples by qRT-PCR.2.The bioinformatics analysis of the significantly differentially expressed circRNAs was performed by using TargetScan,miRbase,miRanda,GO and KEGG databases,the prediction of target genes and signal pathway analysis were performed.3.Expanding the sample size to verify the expression of hsa_circ₄00011 and hsa_circ₁00226 in 43 patients with SLE,40 healthy controls and 36 patients with other autoimmune diseases through qRT-PCR technology,to analyze their correlations between the laboratory indicators and evaluate their diagnostic efficacy.4.PBMCs in peripheral blood of SLE patients and healthy controls were isolated,the expression levels of hsa_circ₄00011 and hsa_circ₁00226 in PBMCs were detected and their diagnostic efficacy were evaluated.Meanwhile,the miRNAs corresponding to hsa_circ₄00011 and hsa_circ₁00226 predicted by bioinformatics were also detected in PBMCs by qRT-PCR.Results1.The screeing results of circRNA microarray showed that 207 differentially expressed circRNAs were detected in the plasma of SLE patients,among which 113 genes were up-regulated and 94 genes were down-regulated.Selected five up-regulated（hsa_circRNA₄00011,hsa_circRNA₁04807,hsa_circRNA₁01471,hsa_circRNA₁02584,hsa_circRNA₁02571）and 3 down-regulated（hsa_circRNA₁00226,hsa_circRNA₁00775,hsa_circRNA₁01889）circRNAs with larger fold difference and smaller P value from the microarray analysis.Preliminary verification were performed,the results showed that hsa_circ₁01471,hsa_circ₁02584 and hsa_circ₄00011 were up-regulated,hsa_circ₁00226 was down-regulated,all results of them were consistent with the results of gene chip.2.The in-depth bioinformatics analysis of circRNAs with significantly differentially expressed genes was performed to predict the target genes.The interaction between circRNA and miRNA-mRNA networks was constructed by GO analysis and KEGG Pathway analysis.It was found that hsa_circRNA₁00226 may play an important role in renal cell carcinoma renal cell carcinoma pathways which is associated with SLE disease.hsa_circRNA₁00226 may affect the expression of target gene Ets-1 by inhibiting miRNA levels,therefore affect the development of SLE disease.3.Expanding the sample size to verify the expression of hsa_circ₄00011 and hsa_circ₁00226 in plasma.The results showed that comparing with normal controls and other autoimmune diseases controls,the expression level of hsa_circ₄00011 was significantly increased in plasma of SLE patients（SLE vs.Normal control:P =0.016;SLE vs Disease control:P = 0.014）,and the expression level of hsa_circ₁00226 was significantly reduced in plasma of SLE patients（P = 0.007;SLE vs Disease control:P = 0.043）.Evaluation of their diagnostic efficacy showed that both of the areas under the ROC curve were between 0.5 and 0.7,and the disease recognition ability were lower.The combined AUC of hsa_circ₄00011 and ANA was 0.925,the combined AUC of hsa_circ₁00226 and ANA was 0.931,while the AUC of the three combined diagnosis was 0.921,which was higher than that of AUC when diagnosed with a single index.The results suggested that hsa_circ₄00011 and hsa_circ₁00226 can improve the diagnostic performance.The correlation between the expression levels of circRNAs and the laboratory indicators of SLE patients showed that,the expression of hsa_circ₁00226 was significantly different between elevated serum creatinine group and non-elevated serum creatinine group（Zelevated serum creatinine=-2.307,Pelevated serum creatinine =0.021）.No correlation was found between hsa_circ₄00011 and hsa_circ₁00226 and other disease-related clinical indicators.There was no significant difference in the expression of hsa_circ₄00011 and hsa_circ₁00226 between stable and active SLE patients.4.The expression of hsa_circ₄00011 and hsa_circ₁00226 in PBMC of SLE patients detected by qRT-PCR showed that,comparing with normal healthy controls,the expression of hsa_circ₄00011was significantly increased,while the expression of hsa_circ₁00226 was significantly reduced,both the differences were statistically significant（P400011=0.026 and P100226=0.000）.The area under the ROC curve of hsa_circ₄00011 was 0.624,which showed a lower diagnostic efficiency.The area under the ROC curve of hsa_circ₁00226 was 0.738,which was between 0.7 and 0.9,and had a good diagnostic efficacy.The combined diagnosis of the AUC was 0.769,that slightly higher than single diagnosis,which can improve the diagnostic performance.In addition,the expression of miRNAs corresponding with hsa_circ₄00011 in PBMC showed that:hsa-miR-296-3p,hsa-miR-146b-3p and hsa-miR-181d-3p were significantly higher than that of normal healthy controls,however,it is not consistent with the prediction of bioinformatics analysis,and this indicates that hsa_circ₄00011 may not have a competitive adsorption relationship with these miRNAs;the expression of miRNAs corresponding with hsa_circ₁00226 in PBMC showed that:hsa-miR-138-5p,hsa-miR-145-3p and hsa-miR-875-3p were significantly higher than that of normal healthy controls,and it is consistent with the prediction of bioinformatics analysis.It is speculated that the pathway of hsa_circ₁00226/miR-875-3p/Ets-1 may play an important role in the pathogenesis of SLE.ConclusionOur results indicate that there are differentially expressed circRNAs in plasma and PBMC of patients with SLE.The expression level of hsa_circ₄00011 is highly increased and the expression level of hsa_circ₁00226 is significantly reduced,both of them can be serve as the potential biomarkers for SLE and may be related to the pathogenesis of SLE.Hsa_circ₁00226 may be involved in the renal cell carcinoma signaling pathway which is associated with the pathogenesis of SLE,and affects the expression of target gene Ets-1 through competitive adsorption of miR-875-3p,thereby affects the occurrence and development of SLE disease.