The Role Of Antiphospholipid Antibodies In Lupus Nephritis And Identification Of A New Diagnostic Biomarker LncRNA For Systemic Lupus Erythematosus

Part Ⅰ The role of antiphospholipid antibodies in patients with proliferative and membranous lupus nephritisObjective:Anti-phospholipid antibodies(aPLs)are a collective term for a group of autoantibodies that target phospholipids and(or)phospholipid binding proteins.In the past,it was considered that antiphospholipid syndrome nephropathy(APSN)is the main form of kidney damage for antiphospholipid antibody,which is essentially non-inflammatory thrombotic vascular disease,but the role of APLs in immune related inflammatory injury of glomerulus has not been fully studied.Lupus nephritis(LN)is a typical autoimmune complex nephritis.This study took it as the research object to explore whether APL plays a role in autoimmune inflammatory injury.Methods:In patients who completed renal biopsy from July 2014 to December 2017 were selected.The following data were obtained in the same period before renal biopsy:including general characteristics of patients;urine test indexes;biochemical indexes such as serum creatinine;immune indexes such as serum complement C3;serum IgG,IgA,IgM APL;anti-inflammatory activity β2-GPI antibody and lupus anticoagulant;SLE disease activity index(SLEDAI).Compare the general and clinical characteristics of patients with proliferative LN(types Ⅲ and Ⅳ)and non proliferative LN(type Ⅴ);the positive rates of various aPL antibodies in patients with proliferative and non proliferative lupus nephritis;and the clinical and pathological characteristics of patients with proliferative LN with or without APL antibody.Results:A total of 75 cases of proliferative LN and 31 cases of membranous LN were analyzed.It was found that the level of serum creatinine and SLEDAI score in proliferative LN group were higher,the incidence of hematuria was higher(P<0.001),the detection rate of ACL antibody between the two groups(45.3%vs 22.7%,P=0.029)and the detection rate of IgG ACL antibody(40%vs 12.9%,P=0.007)In patients with proliferative LN,the levels of serum complement C3 and C4 were lower(P=0.010 and P=0.021)and the positive rate of complement Clq deposition in renal tissue was higher(P=0.003).Multiple linear regression analysis showed that aCL antibody was a factor influencing the immunofluorescence intensity of complement C1q deposition in renal tissue pathology(P<0.001).Conclusion:Our results show that anticardiolipin(ACL)antibodies,especially IgG ACL antibodies,may play a role in proliferative LN renal injury,which may involve the activation of classical complement pathway.Part Ⅱ TCONS＿00483150 is a potential new diagnostic biomarker for SLEPurpose:Long non-coding RNA(lncRNA)is a type of non-coding RNA with a length of more than 200 nt,with a high degree of heterogeneity and versatility.Differentially expressed lncRNAs are involved in the pathophysiological response of a variety of autoimmune diseases.In the first part of the study,we found that aPLs play a certain role in the autoimmune-related inflammatory damage of the glomerulus.The process may involve the classical pathway of complement activation.To further explore SLE-related lncRNAs,and to identify and analyze the possible correlations between candidate differentially expressed lncRNAs and clinical features such as aPLs and hypocomplementemia,explore their role in SLE,and look for potential new diagnostic biomarkers for SLE,from the perspective of lncRNA to explore new mechanisms regulating the occurrence and development of SLE.Methods:From January 2018 to July 2019,36 patients with systemic lupus erythematosus(cohort of discovery period,training period and verification period)were enrolled in the First Affiliated Hospital of Wenzhou Medical University;From June 2018 to June 2019,enrolled 64 SLE patients and 50 RA patients served as cohorts in the external verification phase.Collect peripheral blood leukocytes(PBMCs)from patients for NGS analysis during the discovery phase,and for qRT-PCR analysis during the training,validation,and external validation phases.NGS is carried out in the discovery stage,and the RNA integrity factor of one microgram of total RNA is greater than 7 for NGS library preparation.We performed gene ontology enrichment analysis on NGS results,used miRanda software to predict the association between lncRNA and target mRNA,analyzed differentially expressed lncRNAs through MiRTarget2,catRAPID omics and Bedtools/blast/Pearson,and analyzed the expression level of lncRNAs by fluorescence quantitative qRT-PCR.And study their prognostic value and potential functions.Results:This study included a total of 244 participants,divided into the following four phases of cohort:discovery phase(n=20),training phase(n=10),verification phase(n=42)and external verification phase(n=172).In the discovery phase,we first performed next-generation sequencing(NGS)on the PBMCs of 10 SLE patients(5 patients with stable SLE and 5 patients with active SLE)and 10 HCs.A total of 12705 lncRNAs were detected,of which 143 There were significant differential expressions of lncRNAs in SLE patients but not HCs patients(adj-p value<0.05,fold change≥2),of which 97 lncRNAs were up-regulated and 46 were down-regulated.Cluster screening was performed to identify lncRNAs related to SLE status.The results showed that 19 lncRNAs were significantly up-regulated and 12 lncRNAs were significantly down-regulated.Through the qRT-PCR analysis of the training cohort(5 SLE patients and 5 HCs samples),the expression levels of 12 lncRNAs from these PBMCs were evaluated.The results showed that the expression trends of 9 lncRNAs were similar to the sequencing results,of which only TCONS＿00483150(P=0.002)and TCONS＿00026072(P=0.022)in the training cohort,the expression of SLE patients was significantly reduced.The last two candidate lncRNAs,TCONS＿00483150 and TCONS＿00026072,were further evaluated on 42 subjects in the validation phase(8 patients with stable SLE,13 patients with active SLE and 21 HCs),and there were significant differences of TCONS＿00483150 in the expression of SLE patients and HCs in the stable or active phase in the validation phase.The expression of TCONS＿00026072 was only significantly different between the active SLE patients and HCs,but it was not obvious between the stable SLE group and HCs,that is why we excluded TCONS＿00026072 in the final verification phase.Finally,this study independently evaluated the diagnostic effect of TCONS＿00483150 in the external validation phase(172 additional subjects;64 SLE patients,58 HCs,and 50 RA patients).The results showed that TCONS＿00483150 can be used to distinguish SLE from HCs,rheumatoid arthritis,active/stable SLE and HCs.The diagnostic sensitivity and specificity of TCONS＿00483150 used to distinguish SLE patients from HCs are 61.90 and 95.00 respectively.The results showed that TCONS＿00483150 can be used as a diagnostic biomarker for SLE.Further analysis with clinical characteristics showed that the low expression of TCONS＿00483150 was significantly related to anti-Rib-P antibody positive and low complement C3 levels,but not related to aPLs.Bioinformatics analysis showed that TCONS＿00483150 interacts with HNRNPA1,FUS and ELAVL1.The abnormal expression of TCONS＿00483150 may be mainly related to the processing,splicing,transport,translation and metabolism of RNA and protein in SLE patients.Conclusion:This study provides an overview of the scope of the transcriptome for abnormally expressed lncRNAs in SLE patients.Through a four-stage cohort study that is progressive,the results showed that TCONS＿004831500 is a potential new diagnostic biomarker for SLE.This study provides new insights for the exploration of the pathogenesis of SLE.