LncRNA MALAT1 Affects Mycoplasma pneumoniae Pneumonia via NF-κB Regulation

Abstract

Our aim was to determine whether the long non-coding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) is involved in Mycoplasma pneumoniae pneumonia (MPP), and its possible mechanism of action. MALAT1 expression in the bronchoalveolar lavage fluid of 50 hospitalized children with MPP was compared to its expression in 30 children with intrabronchial foreign bodies. MALAT1 expression was higher in children with MPP, accompanied by increased inflammatory mediators interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α), compared to the controls. In human airway epithelial cells infected with wild-type Mycoplasma pneumoniae (strain M129), MALAT1, IL-8, and TNF-α expression significantly increased, and increased expression of IL-8 and TNF-α could be suppressed by MALAT1 knockdown. Luciferase reporter gene assay and western blot showed that knockdown of MALAT1 reduced nuclear factor-κB (NF-κB) activation. In vivo, RNAi packaged with adenovirus (Adv) was nasally transfected into BALB/c mice to silence MALAT1, and an MP-infected mouse pneumonia model was prepared. The results demonstrated that the degree of pulmonary inflammatory injury, vascular permeability, secretion of inflammatory factors, and expression of phosphorylated p65 (pp65) in MP-infected mice were partly reversed after MALAT1 knockdown compared to those in the controls. In conclusion, MALAT1 is involved in the regulation of airway and pulmonary inflammation caused by MP infection via NF-κB regulation.

Keywords: Mycoplasma pneumoniae pneumonia; inflammation; lncRNA; metastasis associated lung adenocarcinoma transcript 1; nuclear factor-κB.

FIGURE 1

The expression of MALAT1 and inflammatory cytokines in BALF. (A) MALAT1 was increased in BALF of 50 children with MPP compared with 30 children with FB. The expression of MALAT1 was measured by qRT-PCR and normalized to GAPDH. Results are shown as median with interquartile range (**P < 0.01 by Mann-Whitney test). (B,C) Levels of cytokines (IL-8 and TNF-α) in BALF of children with MPP were much higher than that in BALF of children with FB. Data are shown as mean ± standard deviation (B) or median with interquartile range (C) (***P < 0.001). (D,E) The expression of MALAT1 in BALF of children with MPP was positively correlated with the levels of IL-8 and TNF-α (***P < 0.001, ****P < 0.0001).

FIGURE 2

Knockdown of MALAT1 inhibited the secretion of inflammatory cytokines by airway epithelial cells induced by Mycoplasma pneumoniae infection. SiRNA transfection was used to knockdown MALAT1. The expression of MALAT1 was detected by qRT-PCR and normalized to GAPDH. 24 h after MP infection, the expression of MALAT1 in A549 and BEAS-2B cells increased (A,D) along with increased levels of IL-8 and TNF-α in cell supernatant (B,C,E,F). The above changes were reversed after MALAT1 was knocked down. Data are shown as mean ± standard deviation (SD) from three experiments (n = 6, ***P < 0.001, **P < 0.01, *P < 0.05).

FIGURE 3

Knockdown of MALAT1 inhibited NF-κB activation of airway epithelial cells induced by MP infection. The expression of phosphorylated p65 protein in A549 (A) and BEAS-2B (B) cells was detected by western blot. Compared with the normal group, the expression of phosphorylated p65 decreased after MP infection in the knockdown group. The activity of NF-κB was measured by the dual- luciferase reporter gene assay. Compared with the control group, the NF-κB activity of the MALAT1 silent group decreased after MP infection (C,D). Data are shown as mean ± standard deviation (SD) from three experiments (n = 3, ***P < 0.001, **P < 0.01, *P < 0.05).

FIGURE 4

MALAT1 knockdown reduced histopathological damage in BALB/c mice caused by MP infection. Lung tissue samples were collected from each group of mice, then the expression of MALAT1 was detected by qRT-PCR. (A) The expression of MALAT1 in the lung tissues of mice was up-regulated in shNC + MP group, while it was decreased in shMALAT1 + PBS group, compared to the shNC + PBS group. In addition, the expression of MALAT1 was inhibited in shMALAT1 + MP group compared with the shNC + MP group. Data are shown as mean ± standard deviation (SD) from three experiments (n = 8, ***P < 0.001). (B) Histopathological changes were evaluated by HE staining of lung-tissue samples. The shNC + MP group showed significant histological changes, including cell infiltration, alveolar wall thickening, significantly increased secretion, and even structural collapse, while the above changes were reversed in shMALAT1 + MP group. (a) Representative scans (original magnification × 5) of HE stained FFPE sections of left lungs; (b) digital zoom of the lung mid region; (c) representative images (original magnification × 200) for histopathology changes (n = 6).

FIGURE 5

MALAT1 knockdown reduced lung injury in BALB/c mice caused by MP infection. (A,B) The shNC + MP group mice exhibited increases in protein concentration in lung homogenate and ratio of protein concentration in lung homogenate and serum compared to the shNC+PBS group. The two indexes were decreased upon MALAT1 knocked down in MP infected group. (C,D) Compared with shNC + MP group, SOD level in lung homogenate of mice in the shMALAT1 + MP group was significantly increased, while MDA level was decreased. Data are shown as mean ± standard deviation (SD) from three experiments (n = 6, ***P < 0.001,*P < 0.05).

FIGURE 6

MALAT1 knockdown reduced the expression of inflammatory cytokines in mice with MP infection. The mRNA expressions of IL-8 and TNF-α in lung tissues were detected by qRT-PCR (A,B), and the protein level of IL-8 and TNF-α in mice lung homogenate (C,D) and BALF (E,F) were detected by ELISA. Data are shown as mean ± standard deviation (SD) from three experiments (n = 6, ***P < 0.001, **P < 0.01, *P < 0.05).

FIGURE 7

NF-κB is involved in the role of MALAT1 in regulating the inflammatory response induced by MP infection. The expression of phosphorylated p65 in lung tissues of each group was detected by western blot. Densitometry results of the western-blot are shown in the bar graph. The expression of phosphorylated p65 in the shNC + MP group was significantly increased, comparing with the shNC + PBS group, however, the above change was reversed after MALAT1 knocking down. Data are shown as mean ± standard deviation (SD) from three experiments (n = 6, ***P < 0.001).