Ultraviolet B Exposure Induced The DNA Hypomethylation In CD4~+ T Cells Of SLE Patients And Its Underlying Mechanisms

Systemic lupus erythematosus(SLE)is the prototype of autoimmune disease.It is an autoimmune disease characterized by multiple autoantibodies,clinical manifestations,and systemic damage.At present,the etiology and pathogenesis are still unclear.Genetic factors,environmental factors,infection and so on are considered to be the main factors of the disease.In recent years,more and more studies have confirmed that environmental factors can play an important role in the pathogenesis of SLE by influencing epigenetic mechanisms.Therefore,the in-depth exploration of the mechanism of environmental factors involving in SLE pathogenesis will provide important theoretical basis for improving SLE treatment strategy.Among environmental factors,drug exposure and ultraviolet exposure have attracted much attention.Previous studies have shown that environmental factors can affect the level of DNA methylation.In the field on SLE,DNA methylation,a major mechanism of epigenetics,has been shown to be associated with the progression of the disease.In recent years,people have attempted to explore the mechanisms of environmental factors inducing SLE pathogenesis and disease progression from the perspective of DNA methylation.In this field,our research team have also launched earlier research work.We first found that SLE patients had lower levels of DNA methylation compared with healthy volunteers.Then,peripheral blood mononuclear cells(Peripheral blood mononuclear cell,PBMC)of SLE patients and healthy volunteers under different doses of ultraviolet B(UVB,wave length: 290-320)radiation were showed lower DNA methylation level.In recent years,studies have gradually found that mi R-410 and Gadd45 a are related to the DNA methylation of SLE.Meanwhile,we also found mi R-410 has potential binding sites with Gadd45 a through bioinformatics software analysis,suggesting its certain regulation relationship.Therefore,on the basis of our preliminary studies and literature reports,the present experiments aimed to explore the influence of UVB exposure on the DNA methylation of CD4~+ T cells in patients with SLE and its potential mechanisms.The findings will provide new strategies for the treatment of SLE and shed light on a theoretical basis for the pathogenesis of SLE.Part Ⅰ Influence of ultraviolet B radiation on DNA methylation in CD4~+ T cells of SLE patientsObjective To investigate the effect of ultraviolet B(UVB)irradiation on the DNA methylation level of CD4~+ T cells in patients with systemic lupus erythematosus(SLE)and its underlying mechanism.Methods We collected blood samples from 33 patients with SLE and 12 healthy subjects.The CD4~+ T cells were isolated and purified and then exposed to ultraviolet light at different doses(0,50 and 100 m J/cm2).The level of DNA methylation in CD4~+ T cells of each group was detected by ELISA using DNA Methylation Quantification Ultra Kit before and after ultraviolet irradiation;real-time quantitative PCR and Western blotting were used respectively to examine the expression levels of DNA methyltransferases(DNMT1 and DNMT3A)m RNA and protein.Meanwhile,according to the SLEDAI score,SLE patients were divided into two groups: SLE disease activity group(n =22 cases,SLEDAI score > 4)and SLE group(n =11 cases,SLEDAI score ≤ 4),for subsequent subgroup analysis.Results The levels of DNA methylation and DNMT3 A m RNA in SLE patients were significantly decreased compared with that in healthy subjects at baseline.After different dosages of ultraviolet irradiation(0,50 and 100 m J/cm2),DNA methylation levels of CD4~+ T cells were all reduced in a dose-dependent manner in three subgroups.Additionally,100 m J/cm2 ultraviolet irradiation in active SLE group contributed to a significant decrease both of DNA methylation and DNMT3 A m RNA levels in CD4~+ T cells.UVB exposure had no significant effects on expression levels of DNMT1 m RNA and protein and DNMT3 A protein.Conclusion UVB decreases DNA methylation level of CD4~+ T cells in SLE patients probably via inhibiting DNMT3 A m RNA expression level,which needs to be further exploration.Part Ⅱ The mechanism of Mi R-410 regulating Gadd45 a on DNA hypomethylation in CD4~+ T cells induced by UVB exposure in patients with SLEObjective In the first part of the study,we have found that UVB irradiation could cause the decrease of DNA methylation level in CD4~+ T cells of SLE patients.On this basis,this part of study was aimed to further explore the underlying mechanism that may be related to mi R-410 and Gadd45 a.Methods We continued to collect blood samples from 20 SLE patients and 10 healthy subjects.First,the CD4~+ T cells were isolated and purified,then ultraviolet irradiation was administered at different doses(0,50 and 100 m J/cm2).The level of DNA methylation in CD4~+ T cells of each group was detected by ELISA using DNA Methylation Quantification Ultra Kit before and after ultraviolet irradiation;real-time quantitative PCR was employed to examine the expression levels of mi R-410 and Gadd45 a m RNA;western blotting was used to examine the expression level of Gadd45 a protein;regulation of the relationship between mi R-410 and Gadd45 a the dual luciferase reporter assay;then,we upregulated the mi R-410 expression using gene transfection method,to detect its influence on the expression of DNA methylation and Gadd45 a after ultraviolet irradiation in CD4~+ T cells of SLE patients.Results At baseline,compared with healthy controls,DNA methylation and mi R-410 levels were significantly decreased and Gadd45 a levels were remarkably increased in CD4~+ T cells of SLE patients.While after receiving different doses(0,50 and 100 m J/cm2)of UVB irradiation,DNA methylation and mi R-410 levels were decreased and Gadd45 a levels were increased both significantly in CD4~+ T cells of SLE patients,with the changes showing in UVB dose dependent manner,especially in the 100 m J/cm2.Dual luciferase reporter gene assay confirmed that mi R-410 negatively regulate the expression of Gadd45 a.Moreover,after the expression of mi R-410 was up-regulated by gene transfection,the expression level of Gadd45 can be reduced,eventually partially reversed the DNA hypermethylation induced by ultraviolet exposure.Conclusion Mi R-410 can negatively regulate Gadd45 a and participate in the DNA hypomethylation of CD4~+ T cells in SLE patients exposed to UVB radiation.