The State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Front Cell Infect Microbiol. 2020 Mar 17;10:108. doi: 10.3389/fcimb.2020.00108. eCollection 2020.
() can survive in the hostile microenvironment of cells by escaping host surveillance, but the molecular mechanisms are far from being fully understood. MicroRNAs might be involved in regulation of this intracellular process. By RNAseq of -infected PMA-differentiated THP-1 macrophages, we previously discovered down-regulation of miR-378d during infection. This study aimed to investigate the roles of miR-378d in infection of THP-1 cells by using a miR-378d mimic and inhibitor. First, infection was confirmed to decrease miR-378d expression in THP-1 and Raw 264.7 macrophages. Then, it was demonstrated that miR-378d mimic promoted, while its inhibitor decreased, survival in THP-1 cells. Further, the miR-378d mimic suppressed, while its inhibitor enhanced the protein production of IL-1β, TNF-α, IL-6, and Rab10 expression. By using siRNA of Rab10 (siRab10) to knock-down the Rab10 gene in THP-1 with or without miR-378d inhibitor transfection, Rab10 was determined to be a miR-378d target during infection. In addition, a dual luciferase reporter assay with the Rab10 wild-type sequence and mutant for miR-378d binding sites confirmed Rab10 as the target of miR-378d associated with infection. The involvement of four signal pathways NF-κB, P38, JNK, and ERK in miR-378d regulation was determined by detecting the effect of their respective inhibitors on miR-378d expression, and miR-378d inhibitor on activation of these four signal pathways. As a result, activation of the NF-κB signaling pathway was associated with the down-regulation of miR-378d. In conclusion, during infection of macrophages, miR-378d was down-regulated and functioned on decreasing intracellular survival by targeting Rab10 and the process was regulated by activation of the NF-κB and induction of pro-inflammatory cytokines IL-1β, TNF-α, IL-6. These findings shed light on further understanding the defense mechanisms in macrophages against infection.
() 可以通过逃避宿主监测在细胞的恶劣微环境中存活，但分子机制远未完全了解。microRNA 可能参与调节这一细胞内过程。通过对 PMA 分化的 THP-1 巨噬细胞中感染的 RNAseq，我们之前发现 miR-378d 在感染过程中下调。本研究旨在通过使用 miR-378d 模拟物和抑制剂来研究 miR-378d 在 THP-1 细胞感染中的作用。首先，证实感染降低了 THP-1 和 Raw 264.7 巨噬细胞中的 miR-378d 表达。然后，证明 miR-378d 模拟物促进，而其抑制剂减少，在 THP-1 细胞中的存活。此外，miR-378d 模拟物抑制，而其抑制剂增强了 IL-1β、TNF-α、IL-6 和 Rab10 表达的蛋白产生。通过使用 Rab10 的 siRNA（siRab10）敲低转染或不转染 miR-378d 抑制剂的 THP-1 中的 Rab10 基因，确定 Rab10 是感染期间 miR-378d 的靶标。此外，带有 miR-378d 结合位点野生型序列和突变体的双荧光素酶报告基因测定证实 Rab10 是与感染相关的 miR-378d 的靶标。通过检测各自抑制剂对 miR-378d 表达的影响以及 miR-378d 抑制剂对这四个信号通路的激活，确定了 miR-378d 调节中涉及的四个信号通路 NF-κB、P38、JNK 和 ERK。结果表明，NF-κB 信号通路的激活与 miR-378d 的下调有关。总之，在巨噬细胞感染期间，miR-378d 下调，并通过靶向 Rab10 降低细胞内存活，这一过程受 NF-κB 激活和诱导促炎细胞因子 IL-1β、TNF-α、IL-6 的诱导而调节。这些发现为进一步了解巨噬细胞抵抗感染的防御机制提供了线索。
