Expression Characteristics Of Adenosine Deaminases Acting On RNA-1 In Systemic Lupus Erythematosus And Its Correlation With Serum IFN-α

Background:Systemic lupus erythematosus(SLE)is a multifactor systemic autoimmune disease,which can affect many organs such as skin,joint,central nervous system or kidney.Although hormone,environment and genetic factors have been found to be related to SLE,the pathogenesis of SLE still needs further exploration.Studies have shown that blood samples from SLE patients have abnormally high RNA editing levels,some of which affect protein translation and may produce new autoantigens.RNA adenosine deaminases acting on RNA(ADAR)is an A to I editing enzyme that can convert adenine to hypoxanthine.There are three kinds of ADAR genes in mammals,among which ADAR1 is the first recognized and most widely expressed form,and it is one of the deaminases with editing enzyme activity in mammalian cells.It has two subtypes,ADAR1 P150(interferon-inducible)and ADAR1 P110(tissue-expressing).Recent studies have found that mutations in the ADAR1 gene are associated with the pathogenesis of a variety of autoimmune diseases,including Aicardi-Goutieres Syndrome(AGS)and Dyschromatosis Symmetrica Hereditaria(DSH),both of which show some phenotypes similar to SLE.For example,AGS is characterized by increased production of type I interferon,and the severe autoimmune performance of AGS patients emphasizes the importance of ADAR1 in maintaining the immune balance in the body.Nikolaos and other researchers also confirmed that there was a high level of ADAR1 in the synovial tissue of all rheumatoid arthritis patients and in the blood of active patients.After clinical treatment,the level of ADAR1 in patients decreased.In addition,many researchers conducted in vitro experiments on HeLa cells,MCF7 cells and HEK293T cells,and found that changes in IFN-a levels can regulate the expression of ADAR1.Recently,a number of popular studies have shown that knocking out ADAR1 can reduce the survival ability of some cancer cells,make tumor cells sensitive to immunotherapy,and can overcome the resistance to blocking checkpoints.For some tumor cells that can produce IFN themselves,they are more susceptible to the loss of ADAR1.In the immune system of SLE,the level of serum IFN-I is abnormally high(mainly IFN-α)and is involved in the pathogenesis,but the correlation between IFN-I and ADAR1 expression is not clear,which is worth further exploration.Researchers have demonstrated that the expression levels of ADAR1 p150 are up-regulated in peripheral blood mononuclear cells(PBMCs),T cells,B cells and natural killer(NK)cells in patients with SLE.However,at present,there is no study on the specific changes of ADAR1 expression in PBMCs of patients with different disease activities and their correlation with serum IFN-α levels at home and abroad.This study has been expanded on this basis.Objectives:In this study,we used qRT-PCR to determine ADAR1 mRNA expression levels in PBMCs and renal tissues of healthy controls and SLE patients,and determined serum IFN-α levels using Elisa.It aims to explore the following issues:1.The specific changes of ADAR1 expression levels in PBMCs of SLE patients with different disease activities and their correlation with serum IFN-a levels.2.The expression characteristics of ADAR1 in renal tissue of patients with Lupus Nephritis(LN).3.The correlation between the expression levels of ADAR1 in PBMCs and clinical indexes in SLE patients.4.The mechanism of action between ADAR1 and serum IFN-α in patients and its role in SLEMethods:In this study,human PBMCs,renal tissues and serum IFN-α were selected as research objects.1.Collection of PBMCs and serum samples:Twenty-five milliliters of peripheral venous blood was collected under fasting conditions from SLE patients(n=30)and healthy volunteers(n=30)early on in the day,and transferred five milliliters to dry vacuum blood collection tubes and twenty milliliters to EDTA anticoagulation tubes.Isolation of PBMCs from blood in anticoagulation tubes using Ficoll-Hypaque density gradient centrifugation method,and separation of serum in vacuum tube by low speed centrifugation.Finally,the collected samples were cryopreserved at-80℃ for subsequent experiments.2.Collection of renal tissue samples:Collecting 30 renal tissue samples of LN patients who have been punctured and diagnosed clearly at the Department of Nephrology of our hospital.The renal tissue samples of 10 cases in the control group were collected from the patients who underwent nephrectomy at the Department of Urology of our hospital.The experimental samples were collected from a site at a distance>5 cm from the cancer tissue.The glomerular pathology showed normal and no clinical features of renal insufficiency.3.Determination of ADAR1 levels:We used qRT-PCR to determine ADAR1 mRNA expression levels in PBMCs and kidney tissues,and Western Blot method was used to detect the expression levels of ADAR1 protein in vitro experiments.4.Determination of serum IFN-αlevels:Elisa technology was used to detect serum IFN-α expression levels5.Immunohistochemistry:The kidney tissues of 20 LN patients(class Ⅲ,n=5;class Ⅳ,n=5;class V,n=5;class Ⅲ+Ⅴ,n=5)and 5 controls were analyzed by immunohistochemistry,in order to show the positive levels of ADAR1 expression in renal tissue and its cell localization characteristics.6.Serum IgG purification:Collecting the serum of SLE patients,and using the ProteinIso(?)Protein G Resin to isolate and purify the serum IgG for in vitro experiments.7.Cell experiments in vitro:PBMCs were extracted from the same healthy volunteer using the abovementioned method and cultured,cells were subjected to the following treatment:(1)Adding IgG(1.5mg/mL)purified from the serum of SLE patients to the culture medium and co-culture with different concentrations of IFN-αfor 24 hours;(2)In another experimental group,only different concentrations of IFN-α were added to the medium and co-cultured with PBMC cells for 24 hours.8.The expression of ADAR1 in PBMCs collected from SLE patients with varying degrees of the disease and controls were compared,and their correlation with serum IFN-α expression levels were analyzed.The expression characteristics of ADAR1 in kidney tissues of LN patients and controls were analyzed.9.Collecting demographic,clinical,and renal histopathology data of SLE patients and controls for correlation analysis.Result:1.Expression characteristics of ADAR1 in PBMCs:(1)ADAR1 expression was significantly higher in PBMCs of SLE patients(n=30)than in those of healthy controls(n=30)(P<0.05);(2)SLE patients were divided into three groups:NSLE(SLEDAI 0～4,n=6),LSLE(SLEDAI 5～9,n=12),and SSLE(SLEDAI≥10,n=12)according to SLEDAI score,the ADAR1 expression levels in the SSLE group were significantly higher than that in the NSLE group(P<0.05)and the LSLE group(P<0.05);(3)Based on the effect of the disease on the kidneys,the patients were divided into the SLE#group(＃:SLE patient group without the kidney involved,n=17)and LN group(lupus nephritis group,n=13).Compared with healthy controls group,the expression level of ADAR1 was higher in the LN group(P<0.05).2.Expression characteristics of ADAR1 in kidney tissue:(1)qRT-PCR experiments showed:There was no significant difference in ADAR1 expression levels of renal tissue between healthy controls and LN patients(P>0.05);(2)Immunohistochemical experiments showed:There was no significant difference in the ADAR1 cell positive rate between the healthy controls(n=5),LN patients(n=20),and different pathological subgroups(class Ⅲ,n=5;class Ⅳ,n=5;class Ⅴ,n=5;classⅢ+Ⅳ,n=5)(P>0.05).The positive expression rate of ADAR1 in renal tubular cells was higher than that in glomerular cells in both healthy controls and patients with LN(P<0.05).3.The relationship between the ADAR1 expression levels in PBMCs and the serum IFN-α levels of SLE patients:When serum IFN-a levels in SLE patients was<260.0 pg/mL,there was a positive correlation between ADAR1 expression in PBMCs and IFN-α levels(P<0.05),and ADAR1 p150 and serum IFN-αconcentration formed a curve.The ADAR1p150 levels peaked when serum IFN-αlevels reached 100.0 pg/mL.However,no such changes in correlation were observed in healthy controls.4.The expression characteristics of ADAR1 in vitro PBMCs co-cultured with IFN-α and serum IgG from SLE patients are similar to that of SLE patients.In the PBMCs co cultured with IFN-α only,the above correlation changes were not observed.In addition,when IFN-α<200U,the main component of ADAR1 is AD AR1 p150 subtype,while when IFN-α>250U,the expression level of ADAR1 p110 exceeds ADAR1 p150,which becomes the main component of ADAR1.5.We observed a positive correlation between ADAR1 expression in PBMCs of SLE patients and the SLEDAI score(P<0.01)and the total 24-hour urine total protein(P<0.01),negatively correlated with serum total protein(P<0.01)and albumin levels(P<0.05).Conclusion:1.The inhibitory mechanism of ADAR1 on the expression of Interferon-Stimulated Genes(ISGs)in PBMCs of SLE patients may be impaired.2.Serum IgG of SLE patients can affect the process of IFN-a regulating ADAR1 expression in PBMCs.In addition,the main subtypes of ADAR1 are different with different IFN-a levels.3.ADAR1 in PBMCs of SLE patients may become a potential biomarker to evaluate disease activity.4.This study provides new clues for further exploring the pathogenesis of SLE and provides a theoretical basis for the development of targeted drugs.