Knockdown of lncRNA MALAT1 Alleviates LPS-Induced Acute Lung Injury via Inhibiting Apoptosis Through the miR-194-5p/FOXP2 Axis

Abstract

Purpose: We aimed to identify and verify the key genes and lncRNAs associated with acute lung injury (ALI) and explore the pathogenesis of ALI. Research showed that lower expression of the lncRNA metastasis-associated lung carcinoma transcript 1 (MALAT1) alleviates lung injury induced by lipopolysaccharide (LPS). Nevertheless, the mechanisms of MALAT1 on cellular apoptosis remain unclear in LPS-stimulated ALI. We investigated the mechanism of MALAT1 in modulating the apoptosis of LPS-induced human pulmonary alveolar epithelial cells (HPAEpiC).

Methods: Differentially expressed lncRNAs between the ALI samples and normal controls were identified using gene expression profiles. ALI-related genes were determined by the overlap of differentially expressed genes (DEGs), genes correlated with lung, genes correlated with key lncRNAs, and genes sharing significantly high proportions of microRNA targets with MALAT1. Quantitative real-time PCR (qPCR) was applied to detect the expression of MALAT1, microRNA (miR)-194-5p, and forkhead box P2 (FOXP2) mRNA in 1 μg/ml LPS-treated HPAEpiC. MALAT1 knockdown vectors, miR-194-5p inhibitors, and ov-FOXP2 were constructed and used to transfect HPAEpiC. The influence of MALAT1 knockdown on LPS-induced HPAEpiC proliferation and apoptosis via the miR-194-5p/FOXP2 axis was determined using Cell counting kit-8 (CCK-8) assay, flow cytometry, and Western blotting analysis, respectively. The interactions between MALAT1, miR-194-5p, and FOXP2 were verified using dual-luciferase reporter gene assay.

Results: We identified a key lncRNA (MALAT1) and three key genes (EYA1, WNT5A, and FOXP2) that are closely correlated with the pathogenesis of ALI. LPS stimulation promoted MALAT1 expression and apoptosis and also inhibited HPAEpiC viability. MALAT1 knockdown significantly improved viability and suppressed the apoptosis of LPS-stimulated HPAEpiC. Moreover, MALAT1 directly targeted miR-194-5p, a downregulated miRNA in LPS-stimulated HPAEpiC, when FOXP2 was overexpressed. MALAT1 knockdown led to the overexpression of miR-194-5p and restrained FOXP2 expression. Furthermore, inhibition of miR-194-5p exerted a rescue effect on MALAT1 knockdown of FOXP2, whereas the overexpression of FOXP2 reversed the effect of MALAT1 knockdown on viability and apoptosis of LPS-stimulated HPAEpiC.

Conclusion: Our results demonstrated that MALAT1 knockdown alleviated HPAEpiC apoptosis by competitively binding to miR-194-5p and then elevating the inhibitory effect on its target FOXP2. These data provide a novel insight into the role of MALAT1 in the progression of ALI and potential diagnostic and therapeutic strategies for ALI patients.

Keywords: FOXP2; MALAT1; acute lung injury; apoptosis; miR-194-5p.

FIGURE 1

Hypothetical model of the function of MALAT1 in ALI. MALAT1 acts as a sponge of miR-194-5p. Knockdown of MALAT1 alleviates apoptosis of LPS-stimulated HPAEpiC by restoring FOXP2 expression. ALI, acute lung injury; LPS, lipopolysaccharide.

FIGURE 2

Volcano plot of lncRNA (A) and mRNA (B) expressions in the microarray dataset GSE18341. Genes with P value < 0.01 and | log FC| > 1.5 were considered to be differentially expressed. Blue represents downregulated and red represents upregulated. (C) The heatmap depicts the correlation of 2015 genes correlated with Malat1 and most correlations were positive. (D) Venn diagram showing the four ALI-related genes determined through the intersection of differentially expressed genes (DEGs), genes correlated with lung, genes correlated with DELs, and genes sharing a significantly high proportion of miRNA targets with Malat1. (E) Boxplot illustrating Foxp2 and Malat1 expression levels in control and ALI samples. (F) Scatter plot indicating the positive correlation between Foxp2 and Malat1.

FIGURE 3

LPS promotes MALAT1 expression and apoptosis, and suppresses the viability of HPAEpiC. (A) Viability of HPAEpiC treated with 1 μg/ml LPS analyzed at 24, 48, and 72 h. (B) Flow cytometry was used to detect apoptosis of HPAEpiC treated with LPS at 24 h. (C) MALAT1 expression in 1 μg/ml LPS-treated HPAEpiC after 24 h was determined using RT-qPCR. ^∗P < 0.05 vs. respective control. OD, optical density; FL1H, fluorescence of PI; FL2H, fluorescence of Annexin V-FITC.

FIGURE 4

MALAT1 knockdown reverses the LPS-induced effects on viability and apoptosis of HPAEpiC. HPAEpiC transfected with si-MALAT1 or si-NC at 48 h were treated with 1 μg/ml LPS for 24 h. (A) RT-qPCR analysis of MALAT1 expression in HPAEpiC after transfection with si-NC or si-MALAT1 at 24 h. (B) RT-qPCR analysis of MALAT1 expression in 1 μg/ml LPS-treated HPAEpiC after transfection with si-NC or si-MALAT1 at 24 h. (C) Viability was evaluated in HPAEpiC treated with 1 μg/ml LPS and transfected with si-NC or si-MALAT1 at 24, 48, and 72 h. (D) Flow cytometry was used to detect the apoptosis of HPAEpiC treated with 1 μg/ml LPS and transfected with si-NC or si-MALAT1. ^∗P < 0.05 vs. respective control; ^#P < 0.05 vs. LPS; ^&P < 0.05 vs. LPS+si-NC.

FIGURE 5

MALAT1 targets miR-194-5p to regulate FOXP2 expression in LPS-treated HPAEpiC. (A) The predicted binding sites between MALAT1 and miR-194-5p, with the dual-luciferase reporter gene assay used to verify the relationship between MALAT1 and miR-194-5p. (B) The binding sites between miR-194-5p and FOXP2 predicted using dual-luciferase reporter gene assay were used to verify the relationship between miR-194-5p and FOXP2. (C) RT-qPCR was used to detect the expression of miR-194-5p in HPAEpiC treated with LPS at 24 h. (D) mRNA and (E) protein levels of FOXP2 in 1 μg/ml LPS-treated HPAEpiC were evaluated using RT-qPCR and Western blotting analysis, respectively, at 48 h. (F) RT-qPCR was used to detect the expression of miR-194-5p in LPS-treated HPAEpiC transfected with si-MALAT1 at 24 h. FOXP2 (G) mRNA and (H) protein levels in 1 μg/ml LPS-treated HPAEpiC transfected with si-MALAT1 were evaluated using RT-qPCR and Western blotting analysis, respectively, at 24 h. *P < 0.05 vs. respective control.

FIGURE 6

miR-194-5p inhibitor reverses the effect of MALAT1 knockdown on FOXP2 expression in LPS-induced HPAEpiC. (A) mRNA and (B) protein levels of FOXP2 in 1 μg/ml LPS-treated cells co-transfected with miR-194-5p inhibitor and si-MALAT1 were evaluated using RT-qPCR and Western blotting analysis, respectively, at 24 h. ^∗P < 0.05 vs. respective control.

FIGURE 7

FOXP2 overexpression reverses the effect of MALAT1 knockdown on viability and apoptosis of LPS-treated HPAEpiC. (A) FOXP2 expression in 1 μg/ml LPS-treated HPAEpiC co-transfected with ov-FOXP2 and si-MALAT1 was evaluated using Western blotting at 24 h. (B) Viability of the HPAEpiC treated with 1 μg/ml LPS and co-transfected with ov-FOXP2 and si-MALAT1 was determined at 24, 48, and 72 h. (C) Apoptosis of HPAEpiC treated with 1 μg/ml LPS and co-transfected with ov-FOXP2 and si-MALAT1 was determined using flow cytometry at 24 h. *P < 0.05 vs. respective control.