MicroRNA-326 is abnormally expressed in autoimmune diseases, but its roles in autoimmune hepatitis (AIH) are unknown. In this study, we aimed to investigate the effect of miR-326 on AIH and the underlying mechanism. Concanavalin A was administrated to induce AIH in mice and the expression levels of miR-326 and TET2 was evaluated by qRT-PCR and western blot, respectively. The percentages of Th17 and Treg cells were evaluated by flow cytometry and their marker proteins were determined by western blot and ELISA. The mitochondrial membrane potential (MMP) and ROS level were tested with the JC-1 kit and DCFH-DA assay. The binding relationships between miR-326 and TET2 were verified by dual-luciferase reporter assay. The liver tissues were stained by the HE staining. In vitro, AML12 cells were cocultured with mouse CD4+T cells. The expression levels of pyroptosis-related proteins were assessed by western blot. Concanavalin A triggered AIH and enhanced the expression level of miR-326 in mice. It increased both Th17/Treg ratio and the levels of their marker proteins. The expression of TET2 was decreased in AIH mice. Knockdown of miR-326 could decrease the levels of pyroptosis-related proteins, the ROS level and increase MMP. In mouse CD4+T cells, miR-326 sponged TET2 to release IL-17A. Coculture of AML12 cells with isolated CD4+T cells from miR-326 knockdown AIH mice could relieve pyroptosis. Knockdown of miR-326 exerted anti-pyroptosis effects via suppressing TET2 and downstream NF-κB signaling to dampen AIH. We highlighted a therapeutic target in AIH.

中文翻译：

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MiR-326 海绵 TET2 引发 Th17/Treg 分化失衡，加剧刀豆蛋白 A 诱导的自身免疫性肝炎中肝细胞焦亡

MicroRNA-326在自身免疫性疾病中异常表达，但其在自身免疫性肝炎（AIH）中的作用尚不清楚。在本研究中，我们旨在探讨miR-326对AIH的影响及其潜在机制。给予伴刀豆球蛋白 A 诱导小鼠 AIH，并分别通过 qRT-PCR 和蛋白质印迹评估 miR-326 和 TET2 的表达水平。通过流式细胞仪评估 Th17 和 Treg 细胞的百分比，并通过蛋白质印迹和 ELISA 测定其标记蛋白。使用 JC-1 试剂盒和 DCFH-DA 测定法测试线粒体膜电位 (MMP) 和 ROS 水平。通过双荧光素酶报告基因测定验证了 miR-326 和 TET2 之间的结合关系。肝组织进行HE染色。在体外，AML12 细胞与小鼠 CD4+T 细胞共培养。通过蛋白质印迹评估细胞焦亡相关蛋白的表达水平。 Concanavalin A 触发小鼠 AIH 并增强 miR-326 的表达水平。它增加了 Th17/Treg 比率及其标记蛋白的水平。 AIH 小鼠中 TET2 的表达降低。敲除 miR-326 可以降低细胞焦亡相关蛋白的水平、ROS 水平并增加 MMP。在小鼠 CD4+T 细胞中，miR-326 海绵 TET2 释​​放 IL-17A。将 AML12 细胞与来自 miR-326 敲除 AIH 小鼠的分离 CD4+T 细胞共培养可以缓解细胞焦亡。敲除 miR-326 通过抑制 TET2 和下游 NF-κB 信号传导来抑制 AIH，从而发挥抗焦亡作用。我们强调了 AIH 的一个治疗靶点。