Screening And Validation Of Differentially Expressed Circular RNA In Plasma Of Systemic Lupus Erythematosus

Background Systemic lupus erythematosus?SLE?is one of autoimmune disease that affects multiple systems and multiple organs throughout the body.Studies have shown that the incidence of SLE related to genetic factors,including coding genes and non-coding genes.Studies have found only coded genes play an important role in the development of the disease,with little attention to non-coding genes,especially non-coding RNAs?nc RNAs?.Nc RNAs include micro RNA?mi RNA?,circular RNAs?circ RNAs?and long noncoding RNAs?lnc RNAs?,which can be used as the novel class of biomarkers for several diseases.Circ RNAs are endogenous closed loop nc RNAs and consists of the RNA sequence?5'?of upstream and the pre-m RNA of downstream?3'?reverse shear formation.The functions of circ RNAs mainly involved in the regulation of target genes,gene transcription and protein production.Due to the remarkably stability and high conserved of circ RNAs,they are more promising as a biomarker for clinical diagnosis and prognosis than mi RNAs and lnc RNAs.Studies have shown that circ RNAs play an important role in the development and progression of many human diseases.Moreover,circ RNAs can be stably present in plasma and used as a biomarker for early screening of diseases,such as cancer,type II diabetes.Recent study have shown that a large number of abnormally expressed circ RNAs exist in peripheral blood mononuclear cells?PBMCs?and plasma of SLE patients and found that abnormally expressed circ RNAs may become biomarkers of SLE diagnosis.However,up to date,the research on circ RNAs in SLE patients is still limited.Objectives First,we use high-throughput circ RNAs microarray technology to explore the dysregulated expression circ RNAs in plasma of SLE patients.And then we verified the differently expressed circ RNAs and evaluated the diagnostic value of dysregulated circ RNAs.Finally,we explored potential competing endogenous RNA?ce RNA?for differentially expressed circ RNAs.Methods This study uses case control design.Firstly,four SLE patients and four healthy controls were collected respectively.The circ RNAs expression profiles were detected by circ RNAs microarray to screen the differentially expressed circ RNAs.Secondly,we expand the sample size to verification.Additional plasma samples were collected from 122 SLE patients and 102 healthy controls,meanwhile,PBMCs samples from 26 SLE patients and 26 healthy controls were collected from the above cases and controls.The expression levels of three differentially expressed candidate circ RNAs were validated by quantitative reverse transcription polymerase chain reaction?q RT-PCR?.And then we explored the correlation of expression levels of the differentially expressed circ RNAs with major clinical parameters of SLE patients.This study also evaluated the single differential expression of circ RNAs as diagnostic value of SLE biomarkers using receiver operating characteristic?ROC?curve analysis,and then a discrimination model of circ RNAs was constructed to predict the risk of diagnosis with SLE patients by stepwise Logistic regression.Finally,we used Target Scan and mi Randa software to predict differentially expressed circ RNAs potential ce RNA based on the results of q RT-PCR validation of circ RNAs in PBMCs.Results?1?A total of 4 763 circ RNAs were screened out by circ RNAs sequencing,and 112 circ RNAs were found to be statistically significant?fold change>1.5,P<0.05?including 53 up-regulated circ RNAs and 59 down-regulated circ RNAs.Then three candidate circ RNAs were selected for further validation.?2?In plasma,the results of q RT-PCR showed that the expression levels of hsa_circ RNA₄07176 and hsa_circ RNA₀01308 in patients with SLE were lower than those in healthy controls?Z=-2.563,P=0.010;Z=-4.169,P<0.001?.The clinical correlation analysis showed that the expression level of hsa_circ RNA₀01308 in SLE patients was significantly correlated with anti-sj?gren's syndrome-related antigen A?anti-SSA?and C reactive protein?CRP??Z=-2.089,P=0.037;rs=-0.210,P=0.023?.ROC curve analysis showed that the area under the curves?AUC?of hsa_circ RNA₄07176 and hsa_circ RNA₀01308 were 0.599 and 0.662 respectively,and the AUC of the two-circ RNA panel was 0.665.In PBMCs,the expression levels of hsa_circ RNA₄07176,hsa_circ RNA₄06567 and hsa_circ RNA₀01308 in SLE patients were significantly lower than those in healthy controls?Z=-3.788,P<0.001;Z=-3.020,P=0.003;Z=-2.745,P=0.006?.Clinical analysis showed that there was a higher expression of hsa_circ RNA₀01308 level in patients with leukopenia than patients without?Z=-2.333,P=0.020?.The AUC of hsa_circ RNA₄07176,hsa_circ RNA₄06567 and hsa_circ RNA₀01308 were 0.806,0.744 and 0.722,respectively,and the AUC of three-circ RNA panel as 0.855.?3?The results showed that hsa_circ RNA₄07176,hsa_circ RNA₄06567 and hsa_circ RNA₀01308 have 6,4 and 138 potential ce RNA,respectively,which can bind 141,323 and 62 different mi RNAs,respectively.Conclusions?1?The expression levels of hsa_circ RNA₄07176 and hsa_circ RNA₀01308 in plasma of SLE patients were significantly lower than those in healthy controls.The expression levels of hsa_circ RNA₄07176,hsa_circ RNA₀01308 and hsa_circ RNA₄06567 in PBMCs of SLE patients were significantly lower than those of healthy controls.?2?Hsa_circ RNA₄07176 and hsa_circ RNA₀01308 in PBMCs are more promising as biomarkers for identifying patients with SLE and healthy controls than the corresponding circ RNA in plasma.?3?Hsa_circ RNA₄07176,hsa_circ RNA₀01308 and hsa_circ RNA₄06567 are likely to act as ce RNA and affect the regulation of mi RNAs on target genes.