Differential Expression And Diagnostic Significance Of Circular RNAs In The Peripheral Whole Blood Of Systemic Lupus Erythematosus Patients With Renal Damage

Background:Systemic lupus erythematosus?SLE?is an autoimmune disease characterized by the production of autoantibodies,binding with self-antigens to form immune complexes which deposit in various organs and potentially causing various organs or systems damage.SLE patients with renal involvement?SLE+RI?is that SLE with different pathological types of immunity damage in kidney,and has apparent kidney damage of clinical manifestations,affecting up to 70%SLE,and about 10-30%of SLE patients with renal involvement will evolve to renal failure,however,the early symptoms of SLE+RI are often insidious and cannot be reversed once they develop renal failure,so early diagnosis of SLE+RI is of great significance.Circular RNAs?CircRNAs?are a type of closed circular non-coding RNAs,which often show tissue and developmental stage-specific expression.Because circRNAs do not have 5' or 3' ends,they can resist RNase digestion and more stable than most linear RNAs.Increasing studies reveals that circRNAs not only are involved in the progress of many diseases,such as atherosclerotic vascular disease risk,neurological disorders,prion diseases,cancer and autoimmune disease,but can also use as molecular biomarkers in the diagnosis and prognosis of many diseases.However,little is known about peripheral blood circRNAs in SLE+RI.Objective:This study screened and identified differentially expressed circRNAs in SLE+RI.Then to explore its value to evaluate the diagnosis and the efficacy of treatment of SLE+RI,in order to provide new valuable biomarkers for the diagnosis and treatment of SLE+RI patients.Method:CircRNAs expression in peripheral blood from 3 SLE+RI patients,3 SLE patients without renal involvement?SLE-RI?and 3 healthy controls?HC?were performed by microarray.Eleven the most elevated circRNAs in SLE+RI?hsa_circ₀046995,hsa_circ₀082626,hsa_circ₀082689,hsa_circ₀082688,hsa_circ₀001093,hsa_circ₀002715,hsa_circ₀004156,hsa_circ₀031482,hsa_circ₀022383,hsa_circ₀008675 and hsa_circ₀027070?were selected for clinical validation utilizing real time-quantitative polymerase chain reaction?qRT-PCR?in SLE+RI,SLE-RI,HC,neprhritis without SLE?NWS?and rheumatoid arthritis?RA?.The diagnostic value of these circRNAs for SLE+RI was evaluated by receiver operating characteristic?ROC?curve.A clinical follow-up was evaluated in newly diagnosed SLE+RI patients to investigate the level change of these circRNAs after treatment.Finally,the miRanda software and miRNA response element?MRE?were used for comparison analysis to predict the potential miRNA targets of circRNA molecules.Results:1.The result of microarray analysis show that compared with HC group,4235 circRNAs?2197 of them upregulated?and 1566 circRNAs?753 of them upregulated?were dysregulated in SLE-RI group and SLE+RI group,respectively,and 828 circRNAs?234 of them upregulated?were differentially expressed in both SLE-RI group and SLE+RI group.2.Validation of circRNA expression:This study validated eleven circRNAs that were most significantly up-regulated in the SLE+RI group?compared with the SLE-RI and HC groups?as candidated circRNAs?including hsa_circ₀046995,hsa_circ₀082626,hsa_circ₀082689,hsa_circ₀082688,hsa_circ₀001093,hsa_circ₀002715,hsa_circ₀004156,hsa_circ₀031482,hsa_circ₀022383,hsa_circ₀008675 and hsa_circ₀027070?.Then we detected the expression levels via qRT-PCR in an independent set of samples from 77 SLE patients?37 SLE+RI patients and 40 SLE-RI patients?and 33 HC.Consistent with the microarray data,the average expression levels of hsa_circ₀082626,hsa_circ₀082689,hsa_circ₀082688,hsa_circ₀002715,hsa_circ₀004156,hsa_circ₀008675 in the peripheral blood of patients with SLE were significantly increased than those of the HC.Moreover,we analyzed the expression differences of these dysregulated circRNAs between SLE+RI patients,SLE-RI patients and HC.The data suggested that the expression level of hsa_circ₀082689,hsa_circ₀082688 and hsa_circ₀008675 were significantly higher in SLE+RI patients than in SLE-RI and HC,while the expression level of hsa_circ₀004156,hsa_circ₀002715 and hsa_circ₀082626 did not show any remarkable differences between SLE+RI patients and SLE-RI patients.3.The identification of candidate circRNA between SLE+RI and other diseases:we collected 38 RA patients and 24 NWS patients.Using qRT-PCR detected the expression level of hsa_circ₀082689,hsa_circ₀082688 and hsa_circ₀008675 in peripheral blood from 37 SLE+RI patients,38 RA patients,24 NWS patients.And we found that the expression level of hsa_circ₀082689,hsa_circ₀082688 and hsa_circ₀008675 in peripheral blood from SLE+RI patients were significantly higher compared to RA patients and NWS.4.Correlation analysis results showed that the level of hsa_circ₀082688,hsa_circ₀082689 and hsa_circ₀008675 were associated with C4,anti-dsDNA,anti-nucleosome.The level of hsa_circ₀008675 was associated with C3.And,the level of hsa_circ₀082688 and hsa_circ₀008675 were associated with treatment.5.ROC curve analysis suggested that hsa_circ₀082688-hsa_circ₀008675 had significant value in the diagnosis of new-onset SLE+RI patients from controls?new-onset SLE-RI patients,RA patients,NWS patients and HC?with area under the curve of 0.925,sensitivity of 79.17%and specificity of 96.64%.6.CircRNA-miRNA interaction analysis:To confirm the function of hsa_circ₀082689,hsa_circ₀082688 and hsa_circ₀008675,potential miRNA targets of the circRNAs were predicted by aligning with the miRNA response elements?MREs?using miRanda software.Bioinformatics identified that hsa-miR-5003-3p,hsa-miR-4277,hsa-miR-506-3p and hsa-miR-4298 might be potential common targets of hsa_circRNA₀082689 and hsa_circRNA₀082688.In addition,hsa-miR-3192-3p and hsa-miR-5579-5p may be potential other targets of hsa_circRNA₀082689 and hsa_circRNA₀082688,respectively.hsa_circRNA₀008675 was indicated to potentially bind hsa-miR-3074-5p,hsa-miR-6875-5p,hsa-miR-5001-5p,hsa-miR-5581-5p and hsa-miR-6842-3p.Conclusions:1.The expression profile of circRNA in peripheral whole blood of SLE+RI patients suggested that circRNAmay play a role in the development of SLE+RI patients2.This study suggests that peripheral blood hsa_circ₀082688-hsa_circ₀008675 level in SLE+RI patients is upregulated and can effectively distinguish SLE-RI,RA,NWS patients and HC,may also serve as a potential biomarker for SLE+RI patient diagnosis and treatment.