RESUMO
Toll-like receptors (TLRs) are crucial players in immune recognition and regulation, with aberrant activation leading to autoimmune, chronic inflammatory, and infectious diseases. MicroRNAs (miRNAs) have been shown to regulate gene expression at transcriptional and post-transcriptional levels. While miRNA-mediated regulation of TLR signaling has been studied in mammals, the underlying mechanisms of TLR-miRNA interactions in molluscs remain unclear. In a previous study, one of the TLR genes potentially targeted by miRNAs was identified and named McTLR-like1. McTLR-like1 was later found to be targeted by miRNA Mc-novel_miR_196 through bioinformatic prediction. In this study, we aim to experimentally determine the interaction between McTLR-like1 and Mc-novel_miR_196, as well as their functional role in the innate immune response of molluscs. The results showed that the expression of Mc-novel_miR_196 was suppressed, while the expression of McTLR-like1 was enhanced in M. coruscus hemocytes treated with lipopolysaccharide (LPS). Moreover, in vitro assays demonstrated that Mc-novel_miR_196 directly targets the 5' UTR of McTLR-like1 and leads to the down-regulation of proinflammatory cytokines in hemocytes. In addition, co-transfection experiments confirmed that Mc-novel_miR_196 inhibits McTLR-like1 and inhibits the expression of proinflammatory cytokines. The Tunel assay also showed that Mc-novel_miR_196 inhibited apoptosis in hemocytes induced by LPS. Our findings suggest that microRNA Mc-novel_miR_196 acts as a regulator of innate immunity in M. coruscus by targeting McTLR-like1 and inhibiting inflammatory response and apoptosis. These results provide further insights into the complex molecular mechanisms underlying TLR signaling in molluscs.
Assuntos
MicroRNAs , Mytilus , Animais , MicroRNAs/genética , Lipopolissacarídeos/farmacologia , Imunidade Inata/genética , Citocinas , Apoptose , MamíferosRESUMO
Toll-like receptors (TLRs) mediated signaling plays a vital role in activating innate and adaptive immunity. Although TLR mediated signaling has been comprehensively investigated in mammalian species, the mechanisms underlying TLR signaling in molluscs remain obscure. In the present study, a novel TLR isoform namely McTLR-like1 was identified in the thick shell mussel Mytilus coruscus. McTLR-like1 was highly expressed in molluscan immune-related tissues, and its transcriptional levels in hemocytes were significantly increased when challenged by V. alginolyticus. McTLR-like1 activated nuclear factor κB (NF-κB) and strengthened the transcription and phosphorylation of NF-κB subunit P65 in mammalian cells. Upon the silencing of McTLR-like1, the mRNA expression levels of pro-inflammatory cytokines were down-regulated, and the animals exhibited higher levels of resistance when challenged with V. alginolyticus. McMyD88a mRNA expression was also downregulated alongside McTLR-like1. Furthermore, GST-pull down assays revealed a visible affinity between McTLR-like1 and McMyD88a. Collectively, these results demonstrated that the newly identified gene affiliated to the molluscan TLR family and plays a role in the TLR-mediated activation of inflammatory response via its affinity with MyD88. The present study enhances our knowledge of TLR signaling mechanisms in molluscs and provides new insights into the evolution of TLRs.