Department of Intensive Care Unit, Tianjin First Center Hospital, Tianjin, China.
Eur Rev Med Pharmacol Sci. 2019 Apr;23(7):2971-2977. doi: 10.26355/eurrev_201904_17578.
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.
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.
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.
High expression of microRNA-494-3p attenuated sepsis-induced inflammatory response by degrading TLR6.
通过抑制 Toll 样受体 6（TLR6）的表达来阐明 microRNA-494-3p 是否具有抗炎作用，从而抑制脓毒症的发展。
采用实时定量聚合酶链反应（qRT-PCR）检测脓毒症患者和健康对照者血浆中 microRNA-494-3p 和 TLR6 的水平。通过接受者操作特征（ROC）曲线评估 microRNA-494-3p 在脓毒症中的诊断潜力。通过脂多糖（LPS）诱导 RAW264.7 细胞建立体外巨噬细胞炎症模型。观察 LPS 诱导的 RAW264.7 细胞中 microRNA-494-3p、TLR6 和肿瘤坏死因子-α（TNF-α）的表达水平。在 LPS 诱导的 RAW264.7 细胞中转染 microRNA-494-3p 模拟物后，通过 qRT-PCR 和 Western blot 分别测定 TNF-α 的 mRNA 和蛋白水平。同时，提取核因子-κB（NF-κB）p65 的细胞质和核部分，通过 Western blot 分析评估 NF-κB p65 的核转位。通过双荧光素酶报告基因检测验证 microRNA-494-3p 与 TLR6 的结合。最后，进行挽救实验以阐明 microRNA-494-3p 是否通过降解 TLR6 减轻脓毒症引起的炎症。
脓毒症患者血浆中 microRNA-494-3p 的水平明显低于健康对照组，而脓毒症患者血浆中 TLR6 的水平则相反升高。随着 LPS 诱导 RAW264.7 细胞时间的延长，TLR6 和 TNF-α 的表达水平逐渐升高，而 microRNA-494-3p 的表达水平降低。在 RAW264.7 细胞中转染 microRNA-494-3p 模拟物可降低 TNF-α 水平，并抑制 NF-κB p65 的核转位。TLR6 被发现是 microRNA-494-3p 的靶基因，microRNA-494-3p 过表达可显著下调其表达。最后，我们证明了 microRNA-494-3p 对 TNF-α 水平和 NF-κB p65 核转位的抑制作用可被 TLR6 逆转。
高表达 microRNA-494-3p 通过降解 TLR6 减轻脓毒症引起的炎症反应。
