Circular RNA hsa_circ_0099188 Regulates Inducible Nitric Oxide Synthase and Chemokine Transcription in Macrophages by Targeting the hsa-miR-381-3p/PPP3CA and hsa-miR-381-3p/KLF4 Pathways in Response to 4,4'-Methylene Diphenyl Diisocyanate-Glutathione Conjugate exposure.

Workplace exposure to 4,4'-methylene diphenyl diisocyanate (MDI), the most used monomeric diisocyanate, can lead to the development of occupational asthma (OA). However, the molecular mechanisms by which MDI induces OA remain poorly understood. Previous studies have shown that exposure to MDI or MDI-glutathione (GSH) conjugate reduces the levels of endogenous human (hsa)/murine (mmu)-microRNA(miR)-206/381-3p, triggering the activation of calcineurin/nuclear factor of activated T cells (NFAT)/inducible nitric oxide synthase (NOS2) regulatory axis and Krüppel-Like Factor 4 (KLF4)/chemokine pathways in macrophages. Circular RNAs (circRNAs) play important roles on miR and miR-mediated functions in the cells. CircRNA hsa_circ_0008726 is induced by MDI-glutathione (GSH) to downregulate endogenous hsa-miR-206-3p in macrophages; however, the MDI-GSH mediated circRNA response to downregulate hsa-miR-381-3p is currently unknown. The expression of previously identified candidate circRNAs that bind hsa-miR-381-3p were analyzed in differentiated/enhanced THP-1 macrophages treated with MDI-GSH conjugates using RT-qPCR. MDI-GSH exposure induces endogenous hsa_circ_0099188 and its host gene thyrotropin-releasing hormone-degrading ectoenzyme (TRHDE); however, other candidate circRNAs were neither detected nor altered. RNA immunoprecipitation (RIP) experiments confirmed the binding of hsa-miR-381-3p to hsa_circ_0099188. Further experiments demonstrate that modulating hsa_circ_0099188 expression through siRNAs or overexpression plasmids alter the levels of endogenous hsa-miR-381-3p, PPP3CA, and KLF4, as well as NOS2 and M2 macrophage-associated markers and chemokine transcripts. These findings suggest that MDI/MDI-GSH exposure leads to the downregulation of hsa-miR-381-3p by inducing the expression of hsa_circ_0099188/TRHDE, thereby enhancing the regulatory effects of hsa-miR-381-3p in macrophages.