Institute of Microbiology and Immunology, National Yang-Ming University, Taipei City, Taiwan.
Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei City, Taiwan; VYM Genome Research Center, National Yang-Ming University, Taipei City, Taiwan.
Immunol Lett. 2020 Oct;226:22-30. doi: 10.1016/j.imlet.2020.06.015. Epub 2020 Jul 2.
MicroRNA-122 (miR-122) is the most abundant miRNA in the liver and it plays an important role in regulating liver metabolism and tumor formation. Previous studies also reveal an anti-inflammatory function of miR-122; however, relatively little is known about the mechanisms by which miR-122 suppresses inflammation. This study aims to search the effect of miR-122 on proinflammatory chemokines/cytokines production in mice.
Quantitative real-time PCR, Western blot analysis, and ELISA were performed to examine gene expression. TargetScan, miRanda, and microT v3.0 were used to search for possible miR-122 target sites in the 3'-untranslated regions (3'-UTR) of candidate genes. Luciferase reporter assay and site-directed mutagenesis were applied to verify miR-122 target sequences. LPS was applied to peritoneal macrophages and mice to evaluate inflammatory response.
The expression of proinflammatory chemokines, including Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10, and Relb in the livers of miR-122 knockout (KO) mice was increased. We identified Relb as a direct miR-122 target. Overexpressing RelB in the mouse liver increased the expression of Ccl2, Ccl4, Ccl20, Cxcl2, and Cxcl10. Peritoneal macrophages from miR-122 KO mice had a higher level of RelB, and they showed a stronger NF-κB activation and more TNF-α and IL-6 secretion after LPS stimulation. Overexpression of RelB in a macrophage cell line augmented LPS-induced TNF-α and IL-6 production. miR-122 KO mice showed a greatly increased mortality rate and generated a stronger and lasting inflammatory response to LPS.
Deletion of miR-122 caused an upregulation of proinflammatory chemokines and RelB in the liver. Increased RelB may contribute to increases in these chemokine in the liver. Intriguingly, deletion of miR-122 also enhanced the sensitivity of macrophages and mice to LPS. Our results reveal that reducing RelB expression is a new mechanism by which miR-122 regulates inflammation.
miR-122（miR-122）是肝脏中最丰富的 miRNA，在调节肝脏代谢和肿瘤形成中发挥重要作用。先前的研究还揭示了 miR-122 的抗炎功能；然而，关于 miR-122 抑制炎症的机制知之甚少。本研究旨在探讨 miR-122 对小鼠促炎细胞因子/趋化因子产生的影响。
采用定量实时 PCR、Western blot 分析和 ELISA 检测基因表达。使用 TargetScan、miRanda 和 microT v3.0 在候选基因的 3'非翻译区（3'UTR）中搜索可能的 miR-122 靶位。应用荧光素酶报告基因检测和定点突变验证 miR-122 靶序列。应用 LPS 处理腹腔巨噬细胞和小鼠以评估炎症反应。
miR-122 敲除（KO）小鼠肝脏中促炎趋化因子，包括 Ccl2、Ccl4、Ccl20、Cxcl2 和 Cxcl10，以及 Relb 的表达增加。我们鉴定 Relb 为 miR-122 的直接靶标。在小鼠肝脏中过表达 Relb 会增加 Ccl2、Ccl4、Ccl20、Cxcl2 和 Cxcl10 的表达。miR-122 KO 小鼠的腹腔巨噬细胞中 Relb 水平更高，在 LPS 刺激后 NF-κB 激活更强，TNF-α 和 IL-6 分泌更多。巨噬细胞系中 RelB 的过表达增强了 LPS 诱导的 TNF-α 和 IL-6 产生。miR-122 KO 小鼠的死亡率大大增加，对 LPS 的炎症反应更强且持续时间更长。
miR-122 的缺失导致肝脏中促炎趋化因子和 Relb 的上调。Relb 的增加可能导致肝脏中这些趋化因子的增加。有趣的是，miR-122 的缺失也增强了巨噬细胞和小鼠对 LPS 的敏感性。我们的结果表明，降低 Relb 表达是 miR-122 调节炎症的一种新机制。
