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

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.

Figure 1

The levels of proinflammatory cytokines and MUC proteins in LPS- or IL-17A-stimulated HMEECs. (a–c) ELISA was used to measure concentrations of inflammatory cytokines (TNF-α, IL-β, and IL-6) in HMEECs under LPS or IL-17A treatment. (d, e) The protein levels of MUC5AC and MUC8 were measured by western blot in HMEECs under LPS or IL-17A treatment. ^∗∗p < 0.01.

Figure 2

The NF-κB pathway is activated by LPS or IL-17A treatment in HMEECs. (a, b) The effect of LPS on protein levels of cytoplasm p65 and nucleus p65 was determined by western blot analysis. (c, d) The effect of LPS on protein levels p-IκBα and IκBα in HMEECs. (e, f) The effect of IL-17A on protein levels of cytoplasm p65 and nucleus p65 was determined by western blot analysis. (g, h) The effect of IL-17A on protein levels p-IκBα and IκBα in HMEECs. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 3

Knockdown of MALAT1 inactivates the NF-κB pathway. (a) The level of MALAT1 was explored by RT-qPCR assay in control, LPS, or IL-17A group. (b) The knockdown efficacy of sh-MALAT1 in HMEECs was explored by RT-qPCR analysis. (c–h) The effect of MALAT1 on protein levels of cytoplasm p65, nucleus p65, p-IκBα, and IκBα in HMEECs under LPS or IL-17A treatment. (i, j) Immunofluorescence assay was conducted to assess the nuclear translocation of p-NF-κB in HMEECs under LPS or IL-17A treatment. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 4

Role of MALAT1 in proinflammatory cytokine production and MUC protein levels. (a–f) The effect of MALAT1 on concentrations of inflammatory cytokines (TNF-α, IL-β, and IL-6) was evaluated by ELISA assay in HMEECs under LPS or IL-17A treatment. (g, h) The effect of MALAT1 on protein levels of MUC5AC and MUC8 was measured by western blot in HMEECs under LPS or IL-17A treatment. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 5

MALAT1 promotes LPS- or IL-17A-induced inflammatory response through the NF-κB pathway. (a) The overexpression efficacy of pcDNA3.1/MALAT1 in HMEECs was detected by RT-qPCR analysis. (b–g) The effects of MALAT1 and QNZ on concentrations of inflammatory cytokines (TNF-α, IL-β, and IL-6) were evaluated by ELISA assay in HMEECs under LPS or IL-17A treatment. (h, i) The effects of MALAT1 and QNZ on protein levels of MUC5AC and MUC8 were measured by western blot in LPS- or IL-17A-induced HMEECs. ^∗p < 0.05, ^∗∗p < 0.01.