Mechanism Of MiR-5003 Regulating ETS1-3’UTR To Promote B Cell Differentiation In SLE Patients

Objectives:To screen the significant differential genes in transcriptional level of peripheral venous blood mononuclear cells(PBMC)of patients with systemic lupus erythematosus(SLE),compared with normal controls.To verify the expression level of ETS1,and analyze the correlation between the expression of ETS1 and the distribution of single nucleotide polymorphism(SNP)in ETS1 loci.To explore the mechanism and functional effects of SNP on ETS1 expression in B lymphocytes.Meanwhile,the effects of SNP on different clinical manifestations in SLE patients were explored.This study would provide potential biomarkers for early clinical diagnosis and prognosis of SLE.Methods:Peripheral venous blood was collected from 3 SLE patients and 3 healthy controls.Next generation sequencing(NGS)was adopted to screen for differentially expressed genes(DEGs)significantly in PBMC,then,the enrichment of DEGs were analyzed by GO,KEGG,and Cytoscape.Furtherly,quantitative polymerase chain reaction(qPCR)was used to measure the relative expression of ETS1 in PBMC,B and Th lymphocytes in enlarged samples,and SNaPshot was performed to identify the genotypes.Morever,B lymphocytes purified from PBMCs of SLE patients were infected by lentivirus,constructed with different plasmids either wild-type(wt)C or mutant type(mut)T ETS1 in rs4937333 allele.And the expression of ETS 1 was tested by qPCR and Western Blot.Furthermore,plasmids with different allele were transfected into B lymphocytes isolated from spleens of KM mice.Then,stimulation in vitro with anti-IgM,anti-CD40L,LPS,IL4,respectively or simultaneously,were carried out.Additionally,Flow cytometry(FCM),enzyme-linked immuno sorbent assay(ELISA)were used to identify the activation,secretion,differentiation of B cells and secretion of plasma cells by comparing these two groups.Finally,three professional websites were used to predict the miRNA that might bind to rs4937333.The luciferase reporter gene assay was conducted to identify the binding of the predicted miRNA and the fragment of ETS1 3’UTR containing the rs4937333(C/T)in vitro.And the expression level of ETS1 in cells co-transfected with miR-5003 and the plasmid was measured again by qPCR and Western Blot.Last but not least,we also tested the expression of miR-5003 in Th and B lymphocytes from all samples.Results:A total of 117 up-regulated and 35 down-regulated functional genes were found significantly after PMBC sequenced by RNA-seq.And ETS1 is one of the down-regulated ones.DEGs are mainly enriched in terms related to inflammation and innate immunity,SLE and innate immune pathways.The expanded samples included 66 SLE patients and 42 healthy controls.ETS1 mRNA were reduced significantly in PBMC and B lymphocytes in the case group,comparing with the controls,while no significant was found in Th cells.Meanwhile,it was found that the mutant of rs4937333(C→T)in SLE group was higher than in the control(OR=1.800,95%CI(1.026-3.157),p-value=0.040),after eight SNPs located in the ETS1 3’UTR were sequenced.Additionally,the expression level of ETS1 mRNA in B cells of SLE was associated with five clinical phenotypes significantly,including butterfly erythema,light sensitivity,arthritis,abnormal hematology and positive ANA.We found that both transcriptional and translational levels of ETS1 in primary B lymphocytes isolated from SLE patients transfected with mutant plasmid were reduced significantly compared with that with wt one.After transfection of wt or mut plasmids into B lymphocytes from mouse spleen,no difference was found in expression of activation markers CD23,CD69 and CD86 followed by stimulations in turn.Similarly,significant differences were not observed in IL-6,IL10 and TNFα in the supernatant,either(all of p>0.05).However,the differentiation ability of mut B cells into plasma cells was significantly enhanced after co-stimulation with both anti-IgM and IL4 simultaneously.Furtherly,the secretion of IgM,IgG,IgA and IgE in the supernatant of mut group were also significantly higher than that in the wt one.Lastly,the luciferase reporter gene assay showed that the binding activity between miR-5003-3p and the mutant 3’UTR of ETS1 was enhanced,comparing with wt 3’UTR,which lead to lower expression of ETS1.Consistently,miR-5003 in peripheral venous blood B cells of SLE patients was expressed higher significantly than the healthy controls and Th cells(p<0.001)in clinical specimens.Conclusions:In peripheral blood B lymphocytes of patients with SLE,enhanced binding between miR-5003 and mutant rs4937333 located in 3’UTR of ETS1 promotes the degradation of ETS1 mRNA,reduces the mRNA and protein levels of ETS1,which,as a result,accelerate the differentiation of B lymphocytes into plasmocytes,promote the secretion of immunoglobulins.Eventually,the alteration of ETS1 is associated with several clinical manifestations including butterfly erythema,light sensitivity,arthritis,abnormal hematology and positive ANA.In term of clinically significance,the polymorphism of rs4937333 could affect SLE activity and might contribute to the prognosis of SLE.Moreover,the expression level of miR-5003 in B cells is expected to be a biomarker for both in the early diagnosis of SLE and in the evaluation of disease activity.