Targeting MALAT1 and miRNA-181a-5p for the intervention of acute lung injury/acute respiratory distress syndrome

Abstract

Background: ALI/ARDS is a severe lung injury leading to refractory respiratory failure, accounting for high morbidity and mortality. However, therapeutic approaches are rather limited. Targeting long non-coding RNA MALAT1 and microRNA miR-181a-5p might be potential option for ALI/ARDS intervention.

Objective: We aimed to investigate the role of MALAT and miR-181a-5p in the pathogenesis of ALI/ARDS, and test the therapeutic effects of targeting MALAT and miR-181a-5p for ALI/ARDS intervention in vitro.

Methods: MALAT1 and miR-181a-5p levels were measured in plasma from ALI/ARDS patients. In vitro human pulmonary microvascular endothelial cell (HPMEC) injury was induced by LPS treatment, and molecular targets of MALAT1 and miR-181a-5p were explored by molecular biology approaches, mainly focusing on cell apoptosis and vascular inflammation. Interaction between MALAT1 and miR-181a-5p was also detected. Finally, the effects of targeting MALAT1 and miR-181a-5p for ALI/ARDS intervention were validated in a rat ALI/ARDS model.

Results: MALAT1 upregulation and miR-181a-5p downregulation were observed in ALI/ARDS patients. Transfection of mimic miR-181a-5p into HPMECs revealed decreased Fas and apoptosis, along with reduced inflammatory factors. Fas was proved to be a direct target of miR-181a-5p. Similar effects were also present upon MALAT1 knockdown. As for the interaction between MALAT1 and miR-181a-5p, MALAT1 knockdown increased miR-181a-5p expression. Knocking down of MALAT1 and miR-181a-5p could both improve the outcome in ALI/ARDS rats.

Conclusion: MALAT1 antagonism or miR-181a-5p could both be potential therapeutic strategies for ALI/ARDS. Mechanistically, miR-181a-5p directly inhibits Fas and apoptosis, along with reduced inflammation. MALAT1 negatively regulates miR-181a-5p.

Keywords: Acute lung injury; Factor associated suicide; Lipopolysaccharide; Metastasis-associated lung adenocarcinoma transcript-1; Pro-inflammatory factor; miRNA-181a-5p.

Fig. 1

Increased expression of MALAT1 and decreased miRNA-181a-5p in ALI/ARDS patients. The RNA from plasma samples of patients was extracted and measured by qRT-PCR. (A-B) Increased MALAT1 expression and decreased miR-181a-5p expression in ALI/ARDS patients. (C) The negative correlation between MALAT1 and miRNA-181a-5p in ALI/ARDS patients determine by Pearson’s correlation (R =  − 0.508, P = 0.0031). (D-F) Increased expression of apoptotic receptor Fas and proinflammatory factors TNF-α, IL-1β and IL-6 measured by qRT-PCR in ALI/ARDS patients. *, ** p < 0.05, 0.01 vs control

Fig. 2

miRNA-181a-5p inhibits LPS-induced HPMEC apoptosis through directly targeting Fas. Mimic miR-181a-5p or mimic miR-NC was transfected to HPMECs, which were then treated with LPS. a Increased miR-181a-5p expression by mimic miR-181a-5p. b Decreased LPS-induced Fas mRNA level by mimic miR-181a-5p. c Decreased LPS-induced Fas protein level by mimic miR-181a-5p. d Decreased LPS-induced HPMEC apoptosis by mimic-miR-181a-5p. e pGL3-FAS-WT or pGL3-FAS-MUT plasmids were co-transfected with mimic miR-181a-5p or miR-NC. f Luciferase assay revealed that mimic miR-181a-5p decreased the luciferase activity in pGL3-FAS-WT but not in pGL3-FAS-MUT. *, **p < 0.05, 0.01.

Fig. 3

miRNA-181a-5p alleviates LPS-induced inflammation in HPMECs. Mimic miR-181a-5p or mimic miR-NC was transfected to HPMECs, which were then treated with LPS. a Decreased LPS-induced TNF-α mRNA level by mimic miR-181a-5p. b, c Decreased LPS-induced TNF-α protein level by mimic miR-181a-5p. d Decreased LPS-induced IL-1β mRNA level by mimic miR-181a-5p. e Decreased LPS-induced IL-6 mRNA level by mimic miR-181a-5p. *, **p < 0.05, 0.01.

Fig. 4

Knockdown of MALAT1 inhibits LPS-induced apoptosis in HPMECs. SiR-MALAT1 or siR-NC was transfected to HPMECs, which were then treated with LPS. a Decreased MALAT1 expression by siR-MALAT1. b Decreased LPS-induced Fas mRNA level by siR-MALAT1. c, d Decreased LPS-induced Fas protein level by mimic siR-MALAT1. e Decreased LPS-induced HPMEC apoptosis by siR-MALAT1. *, **p < 0.05, 0.01

Fig. 5

Knockdown of MALAT1 inhibits LPS-induced inflammation in HPMECs. SiR-MALAT1 or siR-NC was transfected to HPMECs, which were then treated with LPS. a Decreased LPS-induced TNF-α mRNA level by siR-MALAT1. b, c Decreased LPS-induced TNF-α protein level by siR-MALAT1. d Decreased LPS-induced IL-1β mRNA level by siR-MALAT1. e Decreased LPS-induced IL-6 mRNA level by siR-MALAT1. *, **p < 0.05, 0.01

Fig. 6

Interaction between MALAT1 and miR-181a-5p. a SiR-MALAT1 or siR-NC was transfected to HPMECs, which were then treated with LPS. SiR-MALAT1 decreased miR-181a-5p expression. b Mimic miR-181a-5p or mimic miR-NC was transfected to HPMECs, which were then treated with LPS. Mimic miR-181a-5p failed to alter MALAT1 expression. c pGL3-MALAT1-WT or pGL3-MALAT1-MUT plasmids were co-transfected with mimic miR-181a-5p or miR-NC. Luciferase assay revealed that mimic miR-181a-5p decreased the luciferase activity in pGL3-MALAT1-WT but not in pGL3-MALAT1-MUT. *, **p < 0.05, 0.01

Fig. 7

miR-181a-5p and anti-MALAT1 improved outcome in ALI/ARDS rats. LPS was injected i.p. to induced ALI/ARDS in rats 48 h after transfection of mimic miR-181a-5p or mimic NC and siR-MALAT1 or siR-NC. a, b H&E staining showing increased septal thickness, intra-alveolar transudates, and increased inflammatory cell infiltration in LPS group. c, d Increased Fas and TNF-α protein expression in lung tissue extracts determined by Western blotting. e H&E staining showed that both miR-181a-5p and siR-MALAT1 improved LPS-induced lung injury. f IQA scores were reduced by both miR-181a-5p and siR-MALAT1. *, **p < 0.05, 0.01.

Fig. 8

miR-181a-5p and anti-MALAT1 decreased Fas expression in ALI/ARDS rats. LPS was injected i.p. to induced ALI/ARDS in rats 48 h after transfection of mimic miR-181a-5p or mimic NC and siR-MALAT1 or siR-NC. Lung immunohistochemical staining of Fas and its quantification revealed decreased Fas expression by both miR-181a-5p and siR-MALAT1.

Fig. 9

Immunoreactive density was analyzed with ImageJ software *, **p < 0.05, 0.01