Identification Of Circular RNAs Hsa＿circ＿0044235 And Hsa＿circ＿0068367 As Novel Biomarkers For Systemic Lupus Erythematosus

Objective:Systemic lupus erythematosus(SLE)is a typical autoimmune disease.Although genetic,environmental,epigenetic and hormonal factors have been reported to participate in the initiation and progression of SLE,the exact pathogenesis of SLE is not clear.Circular RNA(Circular RNA,circRNA)is a new endogenous species widely distributed in the cytoplasm of eukaryotic cells and is discovered after micro RNA(miRNA)and long noncoding RNA(LncRNA).Recent studies have shown that circRNAs have multiple biological functions and participate in the development of various diseases,which have emerged as novel biomarkers for disease diagnosis.However,the expression profiles and clinical significance of circRNAs in peripheral blood mononuclear cells(PBMC)from systemic lupus erythematosus(SLE)remain unclear.Therefore,to explore the abnormal expression of circRNAs in PBMCs of SLE patients and the value on the pathogenesis and diagnosis of SLE can provide new theoretical basis for the diagnosis and treatment of SLE.Method:1.In the present study,the expression profile of circRNAs in PBMC of 4 newly diagnosed SLE patients and 4 healthy controls(HC)with matching gender and age was detected by using microarray analysis.2.Reverse transcription quantitative polymerase chain reaction(qRT-PCR)was used to verify the screened differentially expressed circRNA(hsa＿circ＿0068367,hsa＿circ＿0037274,and hsa＿circ＿0044235)in 30 SLE patients and 20 HC.3.The role of hsa＿circ＿0044235 and hsa＿circ＿0068367 was screened by ROC curve analysis in the diagnosis of SLE.4.Double-validated tests were performed to test PBMC levels and diagnostic evaluation on 45 differential SLE patients,30 rheumatoid arthritis(RA)patients,and38 HC.5.Target Scan and miRanda’s miRNA target prediction software was used to predict the miRNA interacting with hsa＿circ＿0044235 and hsa＿circ＿0068367,verified the expression of the interacting miRNA.Results:1.A of 1,603 circRNAs were identified to be significantly aberrantly expressed in PBMC from patients with SLE of which 838 were up-regulated and 765 were down-regulated.2.The three differentially expressed circRNAs selected(hsa＿circ＿0068367,hsa＿circ＿0037274,and hsa＿circ＿0044235)were verified by qRT-PCR.Compared with HC,the average expression levels of hsa＿circ＿0044235 and hsa＿circ＿0068367in PBMC of SLE patients were significantly decreased(P <0.0001;P = 0.0015),which was consistent with the chip results,while the expression of hsa＿circ＿0037274was not significantly different(P = 0.3137).3.Receiver operating characteristic curve analysis suggested that hsa＿circ＿0044235 and hsa＿circ＿0068367 were significant for SLE diagnosis.The highest area under the curve(AUC)was hsa＿circ＿0044235 [AUC =0.873;95%confidence interval(CI),0.778 ? 0.967;P<0.0001;sensitivity=70.00%,specificity=100.00%],followed by hsa＿circ＿0068367(AUC =0.768;95% CI,0.629?0.907;P=0.0014;sensitivity =73.33%;specificity =80.00%).The combined AUC of hsa＿circ＿0044235 and hsa＿circ＿0068367(AUC=0.876;95% CI,0.778 ? 0.967;P<0.0001;sensitivity =70.00%;specificity =100.00%)was identical to the individual hsa＿circ＿0044235 AUC value(AUC =0.873).4.Furthermore,the diagnostic potential of hsa＿circ＿0044235 and hsa＿circ＿0068367 for SLE was validated in an independent validation set with 45 patients with SLE,38 HC and 30 patients with rheumatoid arthritis.Compared with HC and RA,hsa＿circ＿0044235 and hsa＿circ＿0068367 in SLE patients were decreased significantly(P <0.0001;P <0.0001);In addition,ROC curves from the patients with SLE and HC indicated that the AUC values of hsa＿circ＿0044235,hsa＿circ＿0068367and hsa＿circ＿0044235-hsa＿circ＿0068367 were 0.861,0.707 and 0.860,respectively.the AUC based on hsa＿circ＿0044235 was 0.825,hsa＿circ＿0068367 was0.893 and the combination of hsa＿circ＿0044235 and hsa＿circ＿0068367 was 0.903.5.The miRNAs interacting with hsa＿circ＿0044235 were hsa-miR-892 a,hsa-mi R-135b-5 and hsa-mi R-135a-5p.qRT-PCR detection of these miRNAs found that the level of hsa-mi R-892 a in PBMC of SLE patients were significantly increased(P = 0.0002),and hsa-mi R-135b-5 and hsa-mi R-135a-5p levels were not significantly abnormal(P> 0.0500);the miRNA interacting with hsa＿circ＿0068367 was hsa-mi R-136-5p,hsa-mi R-501-5p,hsa-mi R-5696;qRT-PCR detection of these miRNAs found that the hsa-mi R-136-5p level of PBMC in SLE patients was significantly reduced(P = 0.0123),and there was no significant difference in hsa-miR-501-5p level(P = 0.8766),and hsa-mi R-5696 expression was not detected in SLE patients and HC..Conclusions:The present study suggested that the dysregulation of circRNAs may serve a role in SLE pathogenesis,and that the levels of hsa＿circ＿0044235 and hsa＿circ＿0068367 in PBMC have potential as biomarkers for SLE diagnosis.hsa＿circ＿0044235 affects the expression of target genes through competitive adsorption of mi R-875-3p,which in turn affects the occurrence and development of SLE disease.