Objective

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.

Methods

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.

Results

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.

Conclusions

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.

中文翻译：

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在miR-122基因敲除小鼠中RelB的上调有助于肝脏和巨噬细胞中促炎性趋化因子/细胞因子水平的升高。

目的

MicroRNA-122（miR-122）是肝脏中含量最丰富的miRNA，在调节肝脏代谢和肿瘤形成中起着重要作用。先前的研究还揭示了miR-122的抗炎功能；但是，关于miR-122抑制炎症的机制知之甚少。这项研究旨在寻找miR-122对小鼠促炎性趋化因子/细胞因子产生的影响。

方法

进行定量实时PCR，蛋白质印迹分析和ELISA来检查基因表达。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靶位点。荧光素酶报告基因测定和定点诱变被应用于验证miR-122靶序列。将LPS应用于腹膜巨噬细胞和小鼠以评估炎症反应。

结果

miR-122基因敲除（KO）小鼠肝脏中促炎性趋化因子（包括Ccl2，Ccl4，Ccl20，Cxcl2和Cxcl10和Relb）的表达增加。我们确定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。来自miR-122 KO小鼠的腹膜巨噬细胞具有更高的RelB水平，并且在LPS刺激后显示出更强的NF-κB活化和更多的TNF-α和IL-6分泌。RelB在巨噬细胞细胞系中的过表达增加了LPS诱导的TNF-α和IL-6的产生。miR-122 KO小鼠表现出大大提高的死亡率，并产生了对LPS的更强且持久的炎症反应。

结论

miR-122的缺失导致肝脏中促炎性趋化因子和RelB的上调。RelB的增加可能有助于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调节炎症的新机制。