LncRNA GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cells by targeting miR-429/DUSP1

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Abstract

Acute lung injury (ALI) is a life-threatening syndrome characterized by excessive inflammation and apoptosis of alveolar epithelial cells. This study firstly investigated the role and mechanism of long non-coding RNA (lncRNA) growth arrest-specific 5 (GAS5) in regulating lipopolysaccharide (LPS)-induced inflammatory response and apoptosis of murine alveolar epithelial cell line MLE-12. The expression of GAS5, miR-429, and dual-specificity phosphatase 1 (DUSP1) were examined using quantitative Real-Time PCR (qRT-PCR) and western blot. The inflammatory responses were evaluated by detecting the levels of pro-inflammatory cytokines using ELISA. Cell apoptosis was assessed by TUNEL assay. The interactions among GAS5, miR-429, and DUSP1 were examined using luciferase reporter assay. The results showed that GAS5 and DUSP1 expression were decreased, whereas miR-429 was increased in lung tissues from LPS-induced ALI mice and LPS-treated MLE-12 cells. Furthermore, GAS5 overexpression decreased cell inflammatory responses and apoptosis in LPS-treated MLE-12 cells, which was reversed by miR-429 mimic and DUSP1 knockdown. Mechanistically, GAS5 acted as a competitive endogenous RNA by sponging miR-429 to facilitate DUSP1 expression. Our findings suggest that GAS5 suppresses inflammatory responses and apoptosis of alveolar epithelial cell MLE-12 by targeting miR-429/DUSP1 axis.

Introduction

Acute lung injury (ALI), along with its severe form acute respiratory distress syndrome (ARDS), is a life-threatening syndrome with high rates of morbidity and mortality (Butt et al., 2016). ALI is characterized by increased alveolar-capillary membrane permeability, diffuse alveolar damage, edema, excessive pulmonary inflammation and apoptosis of alveolar epithelial cells (Butt et al., 2016; Favarin et al., 2013). Lipopolysaccharide (LPS), a major constituent of the outer membrane of Gram-negative bacteria, is one of the primary causative factors of ALI caused by bacterial infections (Yan et al., 2019). Therefore, LPS administration has been widely used as a common model of ALI, which was characterized by enhanced alveolar epithelial cell inflammation and apoptosis (Jiang et al., 2018; Ju et al., 2018; Lei et al., 2018a; Li et al., 2018e).

Long non-coding RNAs (lncRNAs) are a class of non-protein coding transcripts with the length longer than 200 nucleotides. LncRNAs are implicated in various biological functions and play an important role in the pathogenesis of many inflammatory diseases including ALI. For example, knockdown of lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcription 1) led to inhibition of inflammatory responses in LPS-induced ALI rat model (Dai et al., 2018). Growth arrest-specific 5 (GAS5) is a lncRNA that generally plays a tumor suppressor role and acts as a putative therapeutic target in human malignancies (Ghaforui-Fard and Taheri, 2019). Sun et al. (2016) screened the differentially expressed lncRNAs in the lung tissues from paraquat-induced ALI mice, and found that GAS5 (ENSMUST00000065709) was significantly down-regulated in lung tissues of ALI mice, suggesting that GAS5 may play a protective role in ALI. However, the exact role and mechanisms of GAS5 in ALI remain unclear.

Recently, lncRNAs have been well known to exert roles by functioning as a competitive endogenous RNA (ceRNA) to segregate microRNAs (miRNAs) away from the target mRNAs (Gu et al., 2018; Wang et al., 2017; Xiong et al., 2018). Our bioinformatics analysis (Starbase) revealed that GAS5 harbors putative binding sites of miR-429, suggesting that GAS5 may play a role by binding to miR-429. miR-429 has been reported to be highly expressed in the lung tissues from LPS-induced ALI rats and can facilitate the LPS-induced inflammatory responses by targeting dual-specificity phosphatase 1 (DUSP1) (Xiao et al., 2015). Thus, we speculated that GAS5 might play a role in the LPS-induced ALI by acting as ceRNA for miR-429 to modulate DUSP1 expression.

In the present study, we detected the expression of GAS5, miR-429, and DUSP1 in lung tissues from LPS-induced ALI mice and LPS-treated murine alveolar epithelial cell line MLE-12. Furthermore, we investigated their interaction and effects on LPS-induced inflammation and apoptosis of MLE-12 cells.

Section snippets

ALI mice model

Male C57BL/6 mice (8–10 weeks old, weight 25–30 g) were raised in cages under a specific pathogen-free (SPF) environment (20–25 °C, 50–55% humidity, with adequate food and water) with a 12-h light-dark cycle. After acclimation for around one week, mice were randomly divided into two groups (n = 10/group): The normal group and ALI group. To establish the murine model of ALI, LPS (5 mg/kg, O111: B4 from Escherichia coli; Sigma-Aldrich, St. Louis, MO, USA) in 50 μl sterile saline was intratracheally

GAS5 and DUSP1 expression were decreased, whereas miR-429 expression was increased in ALI mice

Compared with the normal group, the expression of GAS5 in the lung tissues from mice in the ALI group was significantly decreased (Fig. 1A). In contrast, miR-429 expression in the ALI group was notably higher than that in the normal group (Fig. 1B). Furthermore, DUSP1 expression was significantly decreased in the ALI group compared with the normal group, both at mRNA (Fig. 1C) and protein levels (Fig. 1D).

LPS treatment decreased GAS5 and DUSP1 expression while it increased miR-429 expression in MLE-12 cells

Compared with the control group, GAS5 expression in the LPS-treated MLE-12 cells was

Discussion

ALI is a life-threatening syndrome with high rates of morbidity and mortality. LPS is an important pathogenic factor in ALI and has been widely used to induce ALI characterized by excessive uncontrolled inflammation and apoptosis of alveolar epithelial cells both in vivo and in vitro (Jiang et al., 2018; Ju et al., 2018; Lei et al., 2018a; Li et al., 2018e). Considerable evidence has emerged to suggest that pro-inflammatory cytokines, notably TNF-α, IL-1β and IL-6, play critical roles in the

Conclusions

In conclusion, our findings first revealed that GAS5 acted as a ceRNA by sponging miR-429 to facilitate DUSP1 expression, and thereby suppressed inflammatory responses and apoptosis of alveolar epithelial cell MLE-12 (Fig. 7). These findings suggest that GAS5 might serve as a potential therapeutic target for the treatment of ALI.

Declaration of Competing Interest

The authors declare no c.onflicts of interest.

Acknowledgements

None declared.

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