WITHDRAWN: miR-99a-3p Targeting EIF4EBP1 Affects B Lymphocytes Function Through Autophagy and Aggravates SLE Disease Progression

Abstract Background:Overactivation of immune cells plays a key role in the pathogenesis of systemic lupus erythematosus (SLE). The regulation of immune cells by miRNA is a research hotspot.In this study, the second-generation high-throughput sequencing found that the expression of miR-99a-3p in SLE decreased, but the specific mechanism is still unclear.The purpose of this study is to explore the potential target genes, target cells of miR-99a-3p and their potential mechanisms affecting the progression of SLE.Methods: Isolate PBMC from healthy individulas and SLE patients, transfect Ball-1, Jurkat, THP-1 and K562 cells with miR-99a-3p agomir and antagomir,detect miR-99a-3p, predict target genes and autophagy pathway mRNA and protein expression by RT-qPCR and Western blotting.CCK-8 method detects cell proliferation, PI method detects cell cycle, flow cytometry detects cell apoptosis, and luciferase reporter gene experiment determines miR-99a-3p target genes.With C57BL/6J mice as control,construct miR-99a-3p overexpression and interference model based on MRL/lpr mice,ELISA detects plasma ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS.Pathological analysis of HE staining and C3 immunofluorescence(IF) deposition in mouse kidney tissue,Immunohistochemistry(IHC) detects changes in target genes and pathway proteins in kidney tissue,isolate B cells to verify the differential expression of miR-99a-3p, target genes, pathway mRNA and protein.Results: Compared with healthy individulas, miR-99a-3p in SLE was down-regulated, while EIF4EBP1, LC3II, LAMP-2A mRNA and protein expression were up-regulated.After Ball-1 was transfected with miR-99a-3p agomir, cell proliferation decreased and apoptosis increased.After transfected with miR-99a-3p antagomir, the effect was opposite;Luciferase reporter gene detection proved that miR-99a-3p directly targets EIF4EBP1. Rescue experiments confirmed the interaction model between miR-99a-3p and EIF4EBP1. Clinical, in vitro, and in vivo experiments further confirmed that miR-99a-3p agomir can reduce the expression of EIF4EBP1, LC3-Ⅱ, and LAMP-2A, while miR-99a-3p antagomir had the opposite effect.In vivo experiment antagomir group mice serum ANA, dsDNA, IgE, IgM, IL-6, IL-10, BLyS were higher than those in the MRL/lpr group, EIF4EBP1, LC3-Ⅱ, LAMP-2A mRNA, protein and IHC levels also increased, and the urinary protein and C3 IF deposition of mice in the antagomir group were increased, and the related indexes of mice in the agomir group were lower than those in the MRL/lpr group.Conclusion:The expression of miR-99a-3p in SLE PBMC was down-regulated. Up-regulation of miR-99a-3p by transfection can protect B cells from autophagy mediated by EIF4EBP1.The down-regulation of miR-99a-3p induces autophagy by regulating the autophagy signaling pathway mediated by EIF4EBP1 in SLE B cells. These results indicate that miR-99a-3p and EIF4EBP1 may be potential targets of SLE.