The Role Of Long Non-coding RNA In The Abnormal Activation Of Type Ⅰ Interferon Pathway In Systemic Lupus Erythematosus

Objective:Systemic lupus erythematosus(SLE)is an autoimmune disease that affects multiple organs and leads to the production of multiple autoantibodies.Long non-coding RNAs(lncRNAs)have recently been found to be closely associated with a variety of autoimmune diseases,but our understanding of systemic lupus erythematosus(SLE)-associated lncRNAs remains limited.1.This study aimed to explore the mechanism of long non-coding RNA linc00513 regulating the involvement of type I interferon pathway in systemic lupus erythematosus.2.Immune cell subpopulations are stimulated by IFN,and long non-coding RNAs that specifically express and respond to IFN stimulation in each cell subgroup are screened as biomarkers for detecting SLE disease progression.Methods:1.RNA-Seq sequencing analysis of SLE patients,combined with GWAS database to screen lncRNA with lupus susceptibility locus.2.Luciferase assay to detect whether lupus susceptible sites affect the expression of linc00513.3.Transfection and stimulation of Hela cells to study the function of linc00513,GO analysis of the gene types regulated by lncRNA,to determine the signaling pathway regulated by lncRNA.4.The expression level of linc00513 was knocked down by ASO and other methods.Western Blot and flow imaging were used to detect the effect of linc00513 on the phosphorylation of STAT1 and STAT2 and their nuclear status after IFN stimulation.5.The relationship between the degree of disease activity and the expression level of linc00513 was analyzed by SLEDAI score and linc00513 expression level.6.The healthy human immune cells were divided into five sub-populations:CD14~+,CD19~+,CD4~+,CD56~+and CD8~+.After IFN stimulation,transcriptome analysis was performed.The fold change>2,the total FPKM value was greater than 100 as the standard screening that specifically expresses and responds to IFN stimulation as a candidate lncRNA for detecting biomarkers of disease progression.RESULTS:lncRNAlinc00513 in the lupus-associated region was screened,and lupus susceptibility sites(rs205764 and rs547311)were present in the promoter region.Determining the G allele of rs205764 and the A allele of rs547311 enhanced the transcriptional activity of the linc00513 promoter.RT-PCR results showed that linc00513 promoted the expression of genes downstream of the IFN pathway.Western Blot experiments demonstrated that linc00513 positively regulates the phosphorylation of key transcription factors STAT1 and STAT2 on the IFN pathway.At the same time,flow imaging results showed that linc00513 also affected p-STAT1into the nucleus.The SLEDAI score showed a significant positive correlation between the expression level of linc00513 and the IFN score.Through the expression of lncRNA in different cell subpopulations and the fold change after IFN stimulation,lncRNA with selective expression and specific response to IFN stimulation in each subgroup was screened as a candidate gene for subsequent screening biomarkers.Conclusion:The first part of the study revealed that linc00513 plays a regulatory role in the pathogenesis of lupus by affecting the expression of genes downstream of the IFN signaling pathway.In the second part,according to the screening strategy,several lncRNAs were selected as candidates for biomarkers for detecting SLE disease progression.All in all,the study provides new strategies for detecting SLE disease progression in the future.