Analysis Of Differential Expression Profile Of Enhancer RNAs In Peripheral Blood Mononuclear C Ells From Patients With Systemic Lupus Erythematosus And Preliminary Study On The Regulation Mechanism Of Candidate TCONS＿00034326

Objective:Investigate the distribution and potential biological functions of enhancer RNA(eRNA)in peripheral blood mononuclear cells(PBMCs)of patients with systemic lupus erythematosus(SLE)to look for SLE related differential candidate eRNAs,and further explore the possible regulatory mechanism of candidate eRNAs.Methods:In the first part of this study,the PBMCs of 10 patients with SLE(5 patients with stable condition and 5 patients with active SLE)and 10 healthy controls(HCs)were detected by RNA-Seq.The regulator speculation,differential expression level and pathway were analyzed.The significant upregulation of eRNAs was verified by qRT-PCR in PBMCs samples of 6 SLE patients and 6 HCs.Candidate eRNAs were further identified in a validation cohort consisting of 25 SLE patients and 20 HCs.Bioinformatics methods were used to analyze the possible pathways to explore the possible correlation between candidate eRNAs and the pathogenesis of SLE.In the second part of this study,we first investgated the expression differences of candidate eRNA in different kind of PBMCs,and then preliminarily observe the effect of decreased expression of candidate eRNA by siRNA interference.Results:We reported the eRNAs transcription data of SLE patients and HCs samples,and further identified a series of eRNAs and super enhancers in all 20 samples.In this study,we found that eRNAs are SLE specific and may be mainly driven by SLE specific transcription factors(TFs).Cross analysis of eRNAs expression showed that 13 eRNAs were up-regulated and 23 eRNAs were down-regulated in SLE group.The up-regulated eRNAs are involved in the regulation of SLE related pathways.In addition,qRT-PCR showed that there was only one eRNA,TCONS＿00034326,with significantly up-regulated expression in PBMCs of SLE patients(P<0.05).ROC curve analysis showed that in terms of differentiating SLE patients from HCs,the diagnostic sensitivity and specificity of TCONS＿00034326 was 0.83 and 0.55 respectively,and the AUC was 0.691.The results of bioinformatics pathway analysis show that the target genes of TCONS＿00034326 are significantly enriched in important SLE disease-related signaling pathways,including TNF signaling pathway,immune system,metabolic pathway and NF-κB signaling pathway.The results also show that TCONS＿00034326 and its related master TFs are of great significance in regulating the pathogenesis of SLE.Further investigations showed that the expression of TCONS＿00034326 in PBMCs was present in CD 14+monocytes,CD4+T cells,CD19+B cells,CD8+T cells and CD56+NK cells,with the highest level in CD4+T lymphocytes and CD14+monocytes.Decreased expression of TCONS＿00034326 induced by siRNA interfering leads to the significantly decreased mRNA expression of Ml-related factors CD86,TNF-α,IL-1β,IL-6 and increased mRNA expression of M2-related factors IL-10,CCL18,CD206,CLEC10A respectively.Conclusion:This study first described the expression profile of eRNAs in peripheral blood mononuclear cells of SLE patients and HCs,and found the most differentially expressed eRNA(TCONS＿00034326)between SLE patients and HCs.TCONS＿00034326 is associated with super enhancer(chr1:89238617-89240917),suggesting that it plays an important role in regulating the gene expression of SLE.The target genes and master TFs of TCONS＿00034326 are involved in SLE-related pathological pathways including not only TNF,but also interferon α/β and y signaling.TCONS＿00034326 may play a regulatory role in the pathogenesis of SLE by promoting M1 polarization of macrophages.