Pulmonary Arterial Hypertension (PAH) is a progressive devastating disease characterized by excessive proliferation of the Pulmonary Arterial Smooth Muscle Cells (PASMCs). Studies suggest that PAH and cancers share an apoptosis-resistant state featuring excessive cell proliferation. MicroRNA-206 (miR-206) is known to regulate proliferation and is implicated in various types of cancers. However, the role of miR-206 in PAH has not been studied. In this study, it is hypothesized that miR-206 could play a role in the proliferation of PASMCs. In the present study, the expression patterns of miR-206 were investigated in normal and hypertensive mouse PASMCs. The effects of miR-206 in modulating cell proliferation, apoptosis and smooth muscle cell markers in human pulmonary artery smooth muscle cells (hPASMCs) were investigated in vitro. miR-206 expression in mouse PASMCs was correlated with an increase in right ventricular systolic pressure. Reduction of miR-206 levels in hPASMCs causes increased proliferation and reduced apoptosis and these effects were reversed by the overexpression of miR-206. miR-206 over expression also increased the levels of smooth muscle cell differentiation markers α-smooth muscle actin and calponin implicating its importance in the differentiation of SMCs. miR-206 overexpression down regulated Notch-3 expression, which is key a factor in PAH development. These results suggest that miR-206 is a potential regulator of proliferation, apoptosis and differentiation of PASMCs, and that it could be used as a novel treatment strategy in PAH.
Acute lung injury (ALI) is a devastating disease characterized by pulmonary edema. Removal of edema from the air spaces 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.
ObjectiveMicroRNAs (miRNAs) have emerged as a novel class of gene regulators which play a critical role in complex diseases like acute lung injury (ALI). Our miRNA profiling studies in animal models of ALI revealed that miR‐146a, mir‐16 and miR‐155 were dysregulated. Mir16 targets Serotonin transporter (SERT) which is involved in serotonin uptake. Elevated levels of serotonin in ALI are known to regulate epithelial sodium channel (ENac) expression. The aim of this study is to investigate the functional effects of mir16 on SERT and ENaC in human alveolar epithelial cells (A549) in vitro.MethodsA549 cells were commercially obtained and cultured according to the manufacturer instructions. Cells were transfected with plasmid containing miR16 expressing or control vector by Lipofectamine. q PCR and immunoblot analysis were performed to assess the effects of miR‐16 on SERT, ENaC and other downstream signalling molecules.ResultsA549 cells transfected with miR16 decreased the expression levels of SERT when compared to controls. Dose dependant regulation of SERT was observed in untransfected A549 cells treated with serotonin. Increased expression of ENaC β was observed in miR16 transfected cells compared to controls.ConclusionmiR16 over expression altered SERT and ENaC expression in human alveolar epithelial cells.Research supported by:RO1HL105932(NIH) and 09SDG2260957(AHA)
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