Transcriptome analysis of LPS-stimulated BMDMs pretreated with Ctrl MO or Regnase-1-targeting MOs (Reg1-MOs)

Regnase-1 plays essential roles in restricting inflammation by acting as a RNase degrading mRNAs involved in immune reactions via the recognition of stem-loop structures in the 3’untranslated regions (UTRs). Dysregulated expression of Regnase-1 is implicated in the pathogenesis of inflammatory and autoimmune diseases in mice and humans. Here we developed a novel therapeutic strategy to suppress inflammatory responses by blocking Regnase-1 self-regulation, which was enabled by the simultaneous use of two antisense phosphorodiamidate morpholino oligonucleotides (MOs) to alter the binding affinity of Regnase-1 towards the stem-loop structures present in its 3’UTR. The Regnase-1-targeting MOs successfully stabilized Regnase-1 mRNA expression. Furthermore, increasing the abundance of Regnase-1 by MO treatment effectively reduced multiple pro-inflammatory transcripts that were controlled by Regnase-1 in BMDMs. Collectively, these data suggested that MO-mediated disruption of the Regnase-1 self-regulation pathway is an attractive therapeutic strategy to enhance Regnase-1 abundance, which could provide clinical benefits for treating inflammatory diseases through the suppression of inflammation. Bome marrow cells were isolated from wild-type B6 animals, and were cultured in macrophage growth medium for 7 days (exchange with fresh macrophage growth medium on D4). BMDMs were transfected with Ctrl MO (2 µM) or Reg1-MOs (2 µM) together with Endoporter for 24 hours, followed by stimulation with LPS for 4 hours.