MicroRNA-494-3p alleviates inflammatory response in sepsis by targeting TLR6

H.-F. Wang, Y. Li, Y.-Q. Wang, H.-J. Li, L. Dou

Department of Intensive Care Unit, Tianjin First Center Hospital, Tianjin, China. doulint@163.com

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• Infectious Diseases

OBJECTIVE: To clarify whether microRNA-494-3p could exert an anti-inflammation effect by suppressing the expression of toll-like receptor 6 (TLR6), thus inhibiting the development of sepsis.
PATIENTS AND METHODS: Plasma levels of microRNA-494-3p and TLR6 in sepsis patients and healthy controls were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Diagnostic potential of microRNA-494-3p in sepsis was evaluated by receiver operating characteristic (ROC) curve. In vitro macrophage inflammation model was established by lipopolysaccharides (LPS) induction in RAW264.7 cells. Expression levels of microRNA-494-3p, TLR6 and tumor necrosis factor-α (TNF-α) in LPS-induced RAW264.7 cells were observed. After transfection of microRNA-494-3p mimics in LPS-induced RAW264.7 cells, mRNA and protein levels of TNF-α were determined by qRT-PCR and Western blot, respectively. Meanwhile, cytoplasmic and nuclear fractions of nuclear factor-kappa B (NF-κB) p65 were respectively extracted for evaluating nuclear translocation of NF-κB p65 by Western blot analysis. Dual-luciferase reporter gene assay was performed to verify the binding between microRNA-494-3p and TLR6. Finally, rescue experiments were carried out to elucidate whether microRNA-494-3p attenuated sepsis-induced inflammation through degrading TLR6.
RESULTS: Plasma level of microRNA-494-3p in sepsis patients was markedly lower than healthy controls, while plasma level of TLR6 was conversely higher in sepsis patients. With the prolongation of LPS induction in RAW264.7 cells, expression levels of TLR6 and TNF-α gradually increased, whereas microRNA-494-3p expression decreased. Transfection of microRNA-494-3p mimics in RAW264.7 cells reduced TNF-α level, and inhibited nuclear translocation of NF-κB p65. TLR6 was found to be a target gene of microRNA-494-3p, and its expression was markedly downregulated by microRNA-494-3p overexpression. Finally, we proved that the inhibitory effects of microRNA-494-3p on TNF-α level and nuclear translocation of NF-κB p65 were reversed by TLR6.
CONCLUSIONS: High expression of microRNA-494-3p attenuated sepsis-induced inflammatory response by degrading TLR6.

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