Liu Zhuohui, Lu Tao, Liu Shumin, Zhang Fan, Yang Jinxiong, Dai Shumin, Ruan Biao, Long Ruiqing
Department of Otolaryngology, The First Affiliated Hospital of Kunming Medical University, Kunming, Yunnan 650032, P.R. China.
Exp Ther Med. 2021 Dec;22(6):1360. doi: 10.3892/etm.2021.10795. Epub 2021 Sep 24.
Acute otitis media (AOM) is a common infectious disease in children that is accompanied by signs and symptoms of middle ear inflammation and infection. Previous studies have shown that the long non-coding (lnc)RNA nuclear-enriched abundant transcript 1(NEAT1) participates in various inflammatory conditions and plays an important regulatory role. The focus of the present study was the biological function of NEAT1 and underlying molecular mechanism in lipopolysaccharide (LPS)-induced human middle ear epithelial cells (HMEECs). The expression of NEAT1, miR-301b-3p and toll-like receptor 4 (TLR4) protein were determined by reverse transcription-quantitative PCR and western blot assays, respectively. Dual-luciferase reporter assay was performed to investigate the combination of miR-301b-3p and NEAT1 or . In addition, cell viability, apoptosis and the levels of pro-inflammatory factors (IL-1β, TNF-α and IL-6) were measured by Cell Counting Kit-8 assay, flow cytometry and ELISA, respectively. Cell viability was significantly decreased, whereas apoptosis and inflammation were increased in LPS-stimulated HMEECs. Functional analyses demonstrated that NEAT1 was upregulated following LPS treatment, whereas knockdown of NEAT1 significantly increased cell viability and alleviated apoptosis and inflammation. Mechanistically, NEAT1 directly bound to and negatively regulated miR-301b-3p expression, whereas miR-301b-3p inhibitors abolished the inhibitory effect of NEAT1 knockdown on cell apoptosis and inflammation. As a target of miR-301b-3p, TLR4 was regulated by NEAT1 and miR-301b-3p. TLR4 overexpression alleviated NEAT1 silencing-induced inflammatory suppression. Rescue experiments demonstrated that NEAT1 promoted TLR4 expression by inhibiting miR-301b-3p. Collectively, the results of the present study suggested that NEAT1 may attenuate LPS-induced inflammation and apoptosis in HMEECs by modulating the miR-301b-3p/TLR4 axis, and may provide a new therapeutic target for the clinical treatment of AOM.
急性中耳炎（AOM）是儿童常见的传染病，伴有中耳炎症和感染的体征及症状。既往研究表明，长链非编码（lnc）RNA核富集丰富转录本1（NEAT1）参与多种炎症反应，并发挥重要的调控作用。本研究的重点是NEAT1在脂多糖（LPS）诱导的人中耳上皮细胞（HMEECs）中的生物学功能及潜在分子机制。分别通过逆转录定量PCR和蛋白质印迹法检测NEAT1、miR-301b-3p和Toll样受体4（TLR4）蛋白的表达。进行双荧光素酶报告基因检测以研究miR-301b-3p与NEAT1或……的结合情况。此外，分别通过细胞计数试剂盒-8检测、流式细胞术和酶联免疫吸附测定法测量细胞活力、细胞凋亡及促炎因子（IL-1β、TNF-α和IL-6）水平。在LPS刺激的HMEECs中，细胞活力显著降低，而细胞凋亡和炎症反应增加。功能分析表明，LPS处理后NEAT1上调，而敲低NEAT1可显著提高细胞活力并减轻细胞凋亡和炎症反应。机制上，NEAT1直接结合并负向调节miR-^301b-3p的表达，而miR-301b-3p抑制剂消除了敲低NEAT1对细胞凋亡和炎症反应的抑制作用。作为miR-301b-3p的靶标，TLR4受NEAT1和miR-301b-3p的调控。TLR4过表达减轻了NEAT1沉默诱导的炎症抑制。挽救实验表明，NEAT1通过抑制miR-301b-^3p促进TLR4表达。总体而言，本研究结果提示，NEAT1可能通过调节miR-301b-3p/TLR4轴减轻LPS诱导的HMEECs炎症和凋亡，可能为AOM的临床治疗提供新的治疗靶点。
