Department of Vascular Biology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
Department of Gastroenterology and Hematology, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori, 036-8562, Japan.
Inflammation. 2019 Dec;42(6):2095-2104. doi: 10.1007/s10753-019-01073-3.
The molecular mechanisms of innate immunity are closely associated with the development of non-alcoholic fatty liver disease (NAFLD). TNF-α is a key cytokine involved in the pathogenesis of metabolic inflammation like NAFLD. Melanoma differentiation-associated gene 5 (MDA5) is a member of the intracellular RNA helicase family proteins that play a pivotal role in an antiviral immune response. Previous studies have demonstrated that TNF-α induces the expression of MDA5 in some types of cells. However, the correlation between TNF-α and the expression of MDA5 in hepatocytes remains unknown. In the present study, we used two human hepatocellular carcinoma cell lines, HuH-7 and HLE, and examined the expression of MDA5 in these cells upon stimulation with TNF-α. The expression of MDA5 induced by TNF-α was analyzed by quantitative real-time RT-PCR and western blotting. Next, RNA interference against MDA5 was performed and the expressions of CXCL10 and STAT1 were examined. We found that the expression of MDA5 had increased upon stimulation with TNF-α in a concentration-dependent manner. Gene silencing against MDA5 suppressed the expression of TNF-α-induced CXCL10 in both cells. In HLE cells, gene silencing of MDA5 impaired STAT1 phosphorylation 24 h after stimulation with TNF-α. On the other hand, TNF-α-induced STAT1 phosphorylation was not detected in HuH-7 cells. These results indicated that MDA5 positively modulated the TNF-α-induced expression of CXCL10 in both STAT1-dependent and -independent manner and may be associated with metabolic inflammation in the liver.
先天免疫的分子机制与非酒精性脂肪性肝病 (NAFLD) 的发展密切相关。TNF-α 是一种关键的细胞因子，参与代谢性炎症如 NAFLD 的发病机制。黑色素瘤分化相关基因 5 (MDA5) 是细胞内 RNA 解旋酶家族蛋白的成员，在抗病毒免疫反应中发挥关键作用。先前的研究表明，TNF-α 在某些类型的细胞中诱导 MDA5 的表达。然而，TNF-α 与肝细胞中 MDA5 的表达之间的相关性尚不清楚。在本研究中，我们使用了两种人肝癌细胞系 HuH-7 和 HLE，并检测了 TNF-α 刺激这些细胞时 MDA5 的表达。通过定量实时 RT-PCR 和 Western blot 分析 MDA5 的表达。接下来，对 MDA5 进行 RNA 干扰，并检测 CXCL10 和 STAT1 的表达。我们发现，MDA5 的表达在 TNF-α 刺激下呈浓度依赖性增加。MDA5 的基因沉默抑制了两种细胞中 TNF-α 诱导的 CXCL10 的表达。在 HLE 细胞中，MDA5 的基因沉默抑制了 TNF-α 刺激 24 小时后 STAT1 的磷酸化。另一方面，在 HuH-7 细胞中未检测到 TNF-α 诱导的 STAT1 磷酸化。这些结果表明，MDA5 以 STAT1 依赖和非依赖的方式正向调节 TNF-α 诱导的 CXCL10 表达，可能与肝脏的代谢性炎症有关。
