Li Zhenyun, Gao Yuan, Zhang Bianfang, Dong Wei, Xi Yuling, Li Yan, Cui Junwei
Department of Tuberculosis, The First Affiliated Hospital of Xinxiang Medical University, Weihui, 453100, Henan, China.
Clinical Pharmacy Office, The First Affiliated Hospital of Xinxiang Medical University, Weihui, 453100, Henan, China.
Sci Rep. 2024 Mar 4;14(1):5233. doi: 10.1038/s41598-024-55493-9.
Macrophages act as the first immune defense line of the host against Mycobacterium tuberculosis (Mtb). A previous study showed that circRNA_SLC8A1 was significantly upregulated in Mtb-infected macrophages, but its regulatory mechanism in anti-tuberculosis infection is unclear. Therefore, this study aimed to investigate the role of circRNA_SLC8A1 in the anti-tuberculosis activity of macrophages. We showed that circRNA_SLC8A1 was upregulated in tuberculosis patients. Moreover, the binding sites of miR-20b-5p on circRNA_SLC8A1 and Sequestosome 1 (SQSTM1/p62) mRNA were predicted by StarBase and verified by the double luciferase reporter gene assay. Next, we found that miR-20b-5p expression was decreased, while SQSTM1 protein expression was increased in a time- and dose-dependent manner in the human macrophage U937 in response to Mtb infection. Furthermore, circRNA_SLC8A1 overexpression vector (circRNA_SLC8A1) or shRNA (sh-circRNA_SLC8A1) and/or miR-20b-5p mimic or inhibitor and/or SQSTM1 overexpression vector (SQSTM1) or small interfering RNA (si-SQSTM1) or its corresponding control were transfected into Mtb-infected macrophages. Results showed that overexpression of circRNA_SLC8A1 or miR-20b-5p inhibitor promoted the secretion of pro-inflammatory factors IL-1β, IL-6, and TNF-α, increased Nitric Oxide (NO) content and inducible nitric oxide synthase (iNOS) expression, inhibited Reactive oxygen species (ROS) production. Cleaved-caspase-3 protein expression, and cell apoptosis, and promoted Mtb survival. Silencing SQSTM1 inhibited secretion of pro-inflammatory factors and activation of the NF-κB pathway. Overexpression of miR-20b-5p blocked the promoting of circ-SLC8A1 on SQSTM1 protein expression. In summary, circRNA_SLC8A1 sponged miR-20b-5p to upregulate SQSTM1/p62 expression and promoted Mtb survival in macrophages through the NF-κB signaling pathway.
巨噬细胞作为宿主抵御结核分枝杆菌（Mtb）的第一道免疫防线。先前的一项研究表明，circRNA_SLC8A1在Mtb感染的巨噬细胞中显著上调，但其在抗结核感染中的调控机制尚不清楚。因此，本研究旨在探讨circRNA_SLC8A1在巨噬细胞抗结核活性中的作用。我们发现circRNA_SLC8A1在结核病患者中上调。此外，通过StarBase预测了miR-20b-5p在circRNA_SLC8A1和聚集体蛋白1（SQSTM1/p62）mRNA上的结合位点，并通过双荧光素酶报告基因检测进行了验证。接下来，我们发现，在人巨噬细胞U937中，响应Mtb感染，miR-20b-5p表达降低，而SQSTM1蛋白表达呈时间和剂量依赖性增加。此外，将circRNA_SLC8A1过表达载体（circRNA_SLC8A1）或短发夹RNA（sh-circRNA_SLC8A1）和/或miR-20b-5p模拟物或抑制剂和/或SQSTM1过表达载体（SQSTM1）或小干扰RNA（si-SQSTM1）或其相应对照转染到Mtb感染的巨噬细胞中。结果表明，circRNA_SLC8A1或miR-20b-5p抑制剂的过表达促进了促炎因子IL-1β、IL-6和TNF-α的分泌，增加了一氧化氮（NO）含量和诱导型一氧化氮合酶（iNOS）表达，抑制了活性氧（ROS）产生、裂解的半胱天冬酶-3蛋白表达和细胞凋亡，并促进了Mtb存活。沉默SQSTM1抑制了促炎因子的分泌和NF-κB途径的激活。miR-20b-5p的过表达阻断了circ-SLC8A1对SQSTM1蛋白表达的促进作用。总之，circRNA_SLC8A1通过海绵吸附miR-20b-5p上调SQSTM1/p62表达，并通过NF-κB信号通路促进巨噬细胞中Mtb的存活。
