Suppression of lncRNA MALAT1 Reduces LPS- or IL-17A-Induced Inflammatory Response in Human Middle Ear Epithelial Cells via the NF-κB Signaling Pathway.

Xiangru Yang,Lijun Xia,Qinna Zhang,Hui Lu,Chenxin Wang

doi:10.1155/2021/8844119

Abstract

Otitis media (OM) is a common inflammatory disease of the middle ear cavity and mainly occurs in children. As a critical regulator of inflammation response, the nuclear factor kappa B (NF-κB) pathway has been found to play an essential role in the pathogenesis of various human diseases. The aim of this study was to explore the potential mechanism under the inflammatory response of human middle ear epithelial cells (HMEECs). We established in vitro models of OM by treating HMEECs with lipopolysaccharide (LPS) or interleukin 17A (IL-17A). Enzyme-linked immunosorbent assay and western blot analysis were used to measure the inflammatory response of HMEECs under LPS or IL-17A stimulation. The results revealed that the concentrations of proinflammatory cytokines (p < 0.001) and protein levels of mucin (MUC) (for MUC5AC, p = 0.002, p = 0.004; for MUC8, p = 0.004, p < 0.001) were significantly elevated by LPS or IL-17A stimulation in HMEECs. Moreover, we found that LPS or IL-17A treatment promoted the phosphorylation of IκBα (for p-IκBα, p = 0.018, p = 0.002; for IκBα, p = 0.238, p = 0.057) and the translocation of p65 from cytoplasm to nucleus in HMEECs (for nucleus p65, p = 0.01; for cytoplasm p65, p < 0.001). In addition, RT-qPCR analysis revealed that long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was verified to be upregulated in LPS- or IL-17A-stimulated HMEECs (p < 0.001). Western blot analysis and immunofluorescence staining assay revealed that that MALAT1 knockdown significantly suppressed the activation of the NF-κB pathway by reducing phosphorylated IκBα levels and inhibiting the nuclear translocation of p65 (p < 0.001) in LPS- or IL-17A-stimulated HMEECs (for p-IκBα, p < 0.001; for IκBα, p = 0.242, p = 0.647). Silence of MALAT1 decreased the proinflammatory cytokine production and MUC protein levels (p < 0.001). Furthermore, rescue assays revealed that the increase of proinflammatory cytokine production (for TNF-α, p = 0.002, p = 0.015; for IL-1β, p < 0.001, p = 0.006; for IL-6, p = 0.002, p < 0.001) and MUC protein levels (for MUC5AC, p = 0.001, p < 0.001; for MUC8, p < 0.001, p = 0.001) induced by MALAT1 overexpression was neutralized by 4-N-[2-(4-phenoxyphenyl) ethyl] quinazoline-4, 6-diamine (QNZ) treatment in LPS- or IL-17A-stimulated HMEECs. In conclusion, MALAT1 promotes inflammatory response in LPS- or IL-17A- stimulated HMEECs via the NF-κB signaling pathway, which may provide a potential novel insight for the treatment of OM.

Highlights

Otitis media (OM) is a common inflammatory disease occurring in the middle ear cavity among children [1]
We examined MUC5AC and MUC8 protein levels and found that the MUC5AC and MUC8 protein levels were elevated in human middle ear epithelial cells (HMEECs) exposed to LPS or interleukin 17A (IL-17A) (Figures 1(d) and 1(e)) (Figure 1(d), p = 0:002, p = 0:004; Figure 1(e), p = 0:004, p < 0:001)
IL-17A stimulated the NFκB signaling in HMEECs (Figures 2(e)–2(h)) (Figure 2(f), for nucleus p65, p = 0:01; for cytoplasm p65, p < 0:001; Figure 2(h), for p-IκBα, p = 0:002; for p-IκBα, p = 0:057)

Summary

Introduction

Otitis media (OM) is a common inflammatory disease occurring in the middle ear cavity among children [1]. OM evolves into a more chronic stage accompanied with persistent inflammation and effusion in the middle ear [2]. OM has become a great threat to children’s health, which may lead to conductive hearing loss, otalgia, and even induce behavioral problems [3, 4]. The spectrum of otitis media includes acute and chronic forms, each of which can be either suppurative of nonsuppurative [5]. Inflammation in the middle ear, which can be provoked by different primary factors such as bacterial and viral infection, local allergic reactions, and reflux, is the crucial event in the pathogenesis of OM. Unresolved acute inflammatory responses or defective immunoregulation of middle inflammation can promote chronic

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