MicroRNA regulation of nonmuscle myosin light chain kinase expression in human lung endothelium

Abstract

Increased lung vascular permeability, the consequence of endothelial cell (EC) barrier dysfunction, is a cardinal feature of inflammatory conditions such as acute lung injury and sepsis and leads to lethal physiological dysfunction characterized by alveolar flooding, hypoxemia, and pulmonary edema. We previously demonstrated that the nonmuscle myosin light chain kinase isoform (nmMLCK) plays a key role in agonist-induced pulmonary EC barrier regulation. The present study evaluated posttranscriptional regulation of MYLK expression, the gene encoding nmMLCK, via 3' untranslated region (UTR) binding by microRNAs (miRNAs) with in silico analysis identifying hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290 as miRNA candidates. We identified increased MYLK gene transcription induced by TNF-α (24 h; 4.7 ± 0.45 fold increase [FI]), LPS (4 h; 2.85 ± 0.15 [FI]), and 18% cyclic stretch (24 h; 4.6 ± 0.24 FI) that was attenuated by transfection of human lung ECs with mimics of hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, or hsa-miR-1290 (20-80% reductions by each miRNA). TNF-α, LPS, and 18% cyclic stretch each increased the activity of a MYLK 3'UTR luciferase reporter (2.5-7.0 FI) with induction reduced by mimics of each miRNA (30-60% reduction). MiRNA inhibitors (antagomirs) for each MYLK miRNA significantly increased 3'UTR luciferase activity (1.2-2.3 FI) and rescued the decreased MLCK-3'UTR reporter activity produced by miRNA mimics (70-110% increases for each miRNA; P < 0.05). These data demonstrate that increased human lung EC expression of MYLK by bioactive agonists (excessive mechanical stress, LPS, TNF-α) is regulated in part by specific miRNAs (hsa-miR-374a, hsa-miR-374b, hsa-miR-520c-3p, and hsa-miR-1290), representing a novel therapeutic strategy for reducing inflammatory lung injury.

Figure 1.

Effects of inflammatory agonists on nonmuscle myosin light chain kinase isoform (nmMLCK) expression and MYLK 3′untranslated region reporter activity. (A) Total RNA was isolated from human pulmonary artery endothelial cells (ECs) and treated with control vehicle or LPS (100 ng/ml) or TNF-α (10 ng/ml) or exposed to 18% cyclic stretch (CS) for 0 to 24 hours, and nmMLCK mRNA level was detected via real-time PCR. Data are presented as fold change in mRNA level over vehicle-treated control and expressed as means ± SE from three independent experiments. (B) Human pulmonary artery ECs were cotransfected with MYLK 3′UTR reporter along with phRL-TK, a Renilla luciferase normalization control vector, and treated with LPS or TNF-α or exposed to 18% CS (24 h), and luciferase activity was measured using the Dual Luciferase Assay System (Promega) according the manufacturer’s protocol. The bar graph represents relative luciferase unit (RLU). Data are presented as RLUs over vehicle-treated control and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control.

Figure 2.

Time-dependent effects of inflammatory agonist on miR-374a, miR-374b, miR-520c-3p, and miR-1290 expression in human lung ECs. Total RNA was isolated from human pulmonary artery ECs and treated with control vehicle or 18% CS (A), TNF-α (10 ng/ml) (B), or LPS (100 ng/ml) (C) for 0 to 24 hours, and the levels of miR-374a, miR-374b, miR-520c-3p, and miR-1290 were determined via real-time PCR. Data are presented as fold change in micro RNA (miRNA) level over vehicle-treated control and expressed as means ± SE from three independent experiments. *P < 0.05 versus unstimulated control. ^#P < 0.01 versus unstimulated control.

Figure 3.

Effect of miRNA mimics on LPS-induced human lung EC permeability. ECs grown in chambers on gold microelectrodes were transfected with miR-374a mimic (A), miR-374b mimic (B), miR-1290 mimic (C), miR-520c-3p (D), or a combination of miR-374a, miR-374b, miR-1290, and miR-520c-3p mimics (4 miRs) (E) or were treated with nonspecific negative control mimic (nc) as described in Materials and Methods and used for transendothelial electrical resistance (TER) measurements. At time = 0, cells were stimulated with LPS (100 ng/ml) or vehicle control. Shown are pooled data of five independent experiments. (F) Pooled TER data (n = 5). Maximal value of permeability in endothelial cells treated with nc reagent (Control) achieved within 10 hours was taken as 100% ± SE. *Significantly different from cells treated with nc reagent with LPS (P < 0.01).

Figure 4.

Effects of miRNA mimics on inflammatory agonist-induced nmMLCK expression in human lung ECs. Total RNA was isolated from ECs that were transfected with negative control mimic (nc) or with the indicated miRNA mimics and untreated (A) or exposed to 18% CS (B; 24 h), treated with LPS (C; 4 h), or treated with TNF-α (D; 24 h). nmMLCK mRNA level was detected via real-time PCR. Transfection with miR-568 was used as the second negative control. Data are presented as fold change in mRNA level over vehicle-treated control and expressed as means ± SE from four independent experiments. *Significantly different from control cells without stimulation (P < 0.05). ^#Significantly different from control cells without stimulation (P < 0.01). **Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.05). ^##Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.01).

Figure 5.

Effects of miRNA mimics on inflammatory agonist-induced MYLK 3′ untranslated region (UTR) reporter activity in human lung ECs. ECs were cotransfected with MYLK 3′UTR reporter along with negative control mimic or with the indicated miRNA mimics, and phRL-TK, a Renilla luciferase normalization control vector, and untreated (A) exposed to 18% CS (B), treated with LPS (C), or treated with TNF-α (D) (24 h) and luciferase activity was measured according the manufacturer’s protocol. Transfection with miR-568 was used as the second negative control. Data are presented as RLU over vehicle-treated control and expressed as means ± SE from four independent experiments. **Significantly different from control cells without stimulation (P < 0.05). *Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.05). ^#Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.01).

Figure 6.

Effects of miRNA antagomirs on inflammatory agonist–induced nmMLCK expression and MYLK 3′UTR reporter activity. Total RNA was isolated from ECs that were transfected with negative control antagomir (nc) or with the indicated miRNA anti- and nmMLCK. mRNA level was detected via real-time PCR (A). ECs were cotransfected with the MYLK 3′UTR reporter along with phRL-TK, controls (nc = negative antagomir [anti], negative mimic control, or a combination of negative anti and negative mimic) or with the indicated miRNA anti, miRNA anti, and mimics combined. Cells were untreated (B) exposed to 18% CS (C), treated with LPS (D), or treated with TNF-α (E) (24 h), and luciferase activity was measured according the manufacturer’s protocol. Transfection with miR-568 was used as the second negative control. Data are presented as RLU over vehicle-treated control and expressed as means ± SE from four independent experiments. *Significantly different from control cells without stimulation (P < 0.05). ^#Significantly different from control cells without stimulation (P < 0.01). **Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.05). ^##Significantly different from control cells stimulated with 18% CS, LPS, or TNF-α (P < 0.01).