Background/Purpose: Systemic lupus erythematosus (SLE) is a complex autoimmune disease caused by genetic and epigenetic mechanisms. MicroRNAs (miRNAs) are small non-coding RNAs, post-transcriptionally regulate the stability of target genes via base-pairing with mRNA 3′-untranslated regions (3′-UTRs), and have been reported to be implicated in the development of autoimmune diseases. To identify candidate miRNA and its target in unbiased fashion, we conducted the RNA sequencing using CD4+ T cells from spleen of MRL/lpr (MRL) lupus model mice and C57BL/6J (B6) mice as a control. Here we newly determined candidate disease-related miRNA, miR-200a-3p, which was significantly downregulated in MRL mice. Since its target genes, CtBP and ZEB were also upregulated in MRL mice and play an important role to suppress IL-2 as a transcriptional co-suppressor complex, we examined the role of miR200-3p in increased expression of CtBP2/ZEB and subsequent defects of IL-2 production in lupus T cells.

Methods: Functional analyses were performed by transfection of miR-200a-3p mimics in EL4 mouse T cell line. The expression levels of miR-200a-3p and its target gene were examined using TaqMan Quantitative PCR (qPCR). The transcript and protein level of IL-2 under the stimulation with phorbol 12-myristate 13-acetate (PMA) (10ng/ml) and ionomycin (1uM) was examined by qPCR and ELISA respectively. Promoter activity was evaluated with luciferase plasmid containing IL-2 promoter region. Specific binding of CtBP/ZEB to IL-2 sequence that negatively regulates IL-2 expression was evaluated by electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) assay.

Results: We confirmed the lower expression of miR-200a-3p (p<0.0001) as well as the higher expression of CtBP2 (p<0.05) in MRL mice compared with B6 mice using qPCR. The expression level of ZEB2 was also tended to decreased (p=0.56) while ZEB1 (p<0.01) was downregulated in MRL mice by qPCR. In EL4 cells with miR-200a-3p overexpression, the expression level of ZEB and CtBP2 mRNA was significantly downregulated, while IL-2 mRNA level was upregulated by q-PCR. IL-2 concentration in the supernatant from stimulated EL4 cells was also higher under the treatment of miR-200a-3p by ELISA. IL-2 promoter activity was elevated by miR-200a-3p overexpression by luciferase assay. EMSA demonstrated that specific binding of ZEB1, ZEB2 and CtBP2 to IL-2 gene was decreased after miR-200a-3p overexpression. Finally, ChIP assay revealed that the complex of CtBP2 in regulatory sequence of IL-2 was augmented in MRL mice compared with B6 mice, while the complex was downregulated in EL4 with miR-200a-3p overexpression.

Conclusion:  Collectively, IL-2 production was elevated after miR-200a-3p overexpression by targeting ZEB1, ZEB2 and CtBP2. Our data suggest that downregulation of miR-200a-3p causes IL-2 suppression through ZEB1, ZEB2 and CtBP2 in SLE T cells, which could involve the lupus pathogenesis by dysregulation of T cells.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/down-regulation-of-microrna-200a-3p-targeting-c-terminal-binding-protein-2-ctbp2-is-involved-in-hypoproduction-of-il-2-in-sle-derived-t-cells/