MicroRNA 16 modulates epithelial sodium channel in human alveolar epithelial cells

Abstract

Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces of lung is a critical function of the epithelial sodium channel (ENaC) in ALI. The molecular mechanisms behind resolution of pulmonary edema are incompletely understood. MicroRNA's (miRNA) are crucial gene regulators and are dysregulated in various diseases including ALI. Recent studies suggest that microRNA-16 (miR-16) targets serotonin transporter (SERT) involved in the serotonin (5-HT) transmitter system. Alterations in serotonin levels have been reported in various pulmonary diseases. However, the role of miR-16 on its target SERT, and ENaC, a key ion channel involved in the resolution of pulmonary edema, have not been studied. In the present study, the expression patterns of miR-16, SERT, ENaC and serotonin were investigated in mice exposed to room air and hyperoxia. The effects of miR-16 overexpression on ENaC, SERT, TGF-β and Nedd4 in human alveolar epithelial cells were analyzed. miR-16 and ENaC were downregulated in mice exposed to hyperoxia. miR-16 downregulation in mouse lung was correlated with an increase in SERT expression and pulmonary edema. Overexpression of miR-16 in human alveolar epithelial cells (A549) suppressed SERT and increased ENaCβ levels when compared to control-vector transfected cells. In addition, miR-16 over expression suppressed TGFβ release, a critical inhibitor of ENaC. Interestingly Nedd4, a negative regulator of ENaC remained unaltered in miR-16 over expressed A549 cells when compared to controls. Taken together, our data suggests that miR-16 upregulates ENaC, a major sodium channel involved in resolution of pulmonary edema in ALI.

Figure 1

Elevated serotonin and SERT expression in mice exposed to hyperoxia (100% O₂). A) Lung serotonin levels in mice exposed to room air and hyperoxic conditions. Lung homogenates were obtained from mice and subjected to capture ELISA to assess serotonin concentrations. B) Lung edema was measured using wet to dry ratio of mouse lungs exposed to hyperoxia and room air. C) Quantitative RT-PCR analysis to measure SERT mRNA expression in lung homogenates of mice exposed to hyperoxia and their room air controls. SERT mRNA was measured using reverse transcription and qPCR with SERT primers. 18S rRNA was used as internal control D) Relative SERT protein expression in mice lung homogenates was measured using Western blot analysis. **P<0.001 compared with room air controls.

Figure 2

Expression levels of miR-16 and ENaC in hyperoxia induced ALI mouse models. A) miR-16 levels were analyzed by qRT-PCR in lung homogenates of mice exposed to hyperoxia and room air. Total RNA from the lung was isolated, reverse transcribed and analyzed for miR-16 using miR-16 specific assay primers and SYBR green method. miR-16 expression was normalized to 18S. B) ENaC-α and ENaC-β protein levels in mouse lung homogenates exposed to normoxia and hyperoxia were measured by western blot analysis. Lung homogenates (n=4) were subjected to immunoblotting with the indicated antibodies. **P<0.001 compared with room air controls.

Figure 3

Over expression of miR-16 downregulated SERT expression in A549 cells in vitro. A) Serotonin transporter expression varied dose dependently with the addition of different concentrations of serotonin. A549 cells were treated with increasing concentrations of serotonin and were subjected to qRT-PCR to assess SERT mRNA expression levels. SERT mRNA expression was normalized to GAPDH. B) qRT-PCR analysis of SERT in miR-16 overexpressed A549 cells post-treated with serotonin. A549 cells were transfected with miR-16; 4 h post-transfection, cells were treated with either 1 or 2 mM serotonin, and 24 h later, RNA was isolated and subjected to qRT-PCR analysis. All experiments were repeated at least 3 independent times. C) SERT protein expression was analyzed in miR-16 overexpressed A549 cells post-treated with serotonin. A549 cells were transfected with miR-16 or control plasmid and post-treated with serotonin for 24 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. *P<0.01 and **P<0.001 compared with control.

Figure 4

miR-16 overexpression in A549 cells modulates ENaC and TGF-β expression via Nedd4 independent pathway. A) Immunoblot analysis of different subunits of ENaC in miR-16 and control plasmid transfected A549 cells. A549 cells transfected with miR-16 or control plasmid were post-treated with or without serotonin and protein lysates were analyzed for ENaC-α, -β and -γ by immunoblot. B) TGF-β concentration in cell supernatants was analyzed by capture ELISA. Human alveolar epithelial cells were transfected with miR-16 or control plasmid and 24 h post-transfection, the supernatants were subjected to ELISA. C) Mice were exposed to hyperoxia and room air, and lung homogenates were analyzed for Nedd4 levels by immunoblot. D) Nedd4 immunoblot in A549 cells transfected with miR-16 or control plasmid in the presence or absence of serotonin treatment post-transfection. *P<0.01 compared with control.