Screening And Identification Biomarkers Of Circular RNAs For Systemic Lupus Erythematosus

Background Systemic lupus erythematosus(SLE)is a typical autoimmune disease.SLE can lead to abnormal activation of immune cells in patients,producing a large number of autoantibodies and immune complexes,and dysfunction of various cellular immune responses,which eventually lead to damage to the multi-organ system of the body.Previous studies have tended to link SLE with genetics,epigenetics and other genomics studies.The present study revealed that the incidence of SLE is significantly correlated with non-coding RNA,including lnc RNA and mi RNA.Circ RNAs are also members of the non-coding RNA family,and although they have been found for a long time,they have not been studied in depth.In recent years,it has become a star molecule in recent years due to its series of prominent characteristics,such as long half-life and obvious tissue specificity.However,only a few articles have been published on the association between SLE and circ RNAs,and there is a lack of studies on the application of third-generation sequencing technology(RNA-seq)in patients with SLE and normal PBMCs.Objectives First,RNA-seq technology was used to understand the expression of circ RNAs in PBMCs between patients with SLE and normal controls.And preliminary screening of the molecules that may be used as biomarkers for lupus and verified the molecules by a series of experiments.Finally,Circ PTPN22,the molecule with the best pre-experimental results,will continue to preliminarily explore further functional tests and action mechanism.Methods This study uses case control design.First,we collected 4 newly diagnosed SLE patients without any treatment and 3 healthy controls.And 128 differentially expressed circ RNAs(log2FC?1.0)were preliminarily screened by RNA-seq technology.Two up-regulated circ RNAs(circ-LRRK2,circ-PPHLN1)and two down-regulated circ RNAs(circ-PTPN22,circ-MYBL1)were selected for large sample RT-q PCR(Real-time policy polymerase chain reaction)verification in 49 SLE patients and 37 healthy controls.Then,we selected circRNA-circPTPN22 with obvious differences for further analysis,including sanger sequencing,SLEDAI index correlation,experiment indexes before and after treatment,ROC curve,etc.,and predicted the possible biological functions of circ PTPN22,such as targeted mi RNA binding function,ORF frame prediction,IRES binding site prediction,and possible translation of protein sequence prediction.Western blot(WB)western blot assay confirmed that jurkat cells were electroporated with a vector expressing circ PTPN22 and circptpn22-orf,and predicted the possible function of circ PTPN22 and its small translated protein.Subsequently,we constructed circ PTPN22 overexpressed vector using molecular biology techniques,and transfected the plasmid into jurkat cells by electroporation.Finally,western blot(WB)assay confirmed its ability to translate proteins and we predicted the possible function of circ PTPN22 and its small translated proteins.Results In this study,using RNA-seq technology,128 differentially expressed circ RNAs(log2FC?1.0)in PBMCs of SLE patients and healthy control group were preliminarily screened,including 89 up-regulated circ RNAs and 39 down-regulated circ RNAs.Some of the detected circ RNAs were not included in the circbase website.The RT-q PCR trends of the four circ RNAs(circ-LRRK2?circ-PPHLN1?circ-PTPN22?circ-MYBL1) were consistent with the RNA-seq results.Further analysis of circ PTPN22 suggested that circPTPN22 was significantly negatively correlated with the patient's SLEDAI index,that is,the higher the patient's SLEDAI score,the lower the circ PTPN22 expression.In the comparison of patients before and after treatment,the expression of circ PTPN22 in patients after treatment was significantly higher than that before treatment.The ROC curve also indicates that the area under the curve(AUC)of circ PTPN22 is 0.918,suggesting that circ PTPN22 has good diagnostic value.Further biological analysis showed that circ PTPN22 can bind a variety of mi RNAs,and the target genes of these mi RNAs,such as CD28 and IL7,are mostly related to immune regulation.In addition,circ PTPN22 also contains multiple IRES sites and ORF frames,so it is predicted to have protein translation activity.The over expressed circ PTPN22 vector was constructed and electrotransfected into jurkat cells.WB results showed that circ PTPN22 could indeed translate proteins.Conclusions(1)128 circ RNAs were found to be differentially expressed in PBMCs of healthy people and patients with primary SLE;(2)It is confirmed that circ PTPN22 and circ MYBL1 are poorly expressed in SLE patients,while circ-LRRK2 and circ-PPHLN1 are highly expressed in SLE patients;(3)This experiment has proved that circ PTPN22 does exist and has an excellent ability to determine the SLE condition,which is expected to become a new SLE activity marker;(4)Circ PTPN22 does translate a small proteins(circ PTPN22-pro).The function of circ PTPN22 and circ PTPN22-pro was predicted.