MicroRNA-7-5p regulates human alveolar epithelial sodium channels by targeting the mTORC2/SGK-1 signaling pathway
MicroRNA-7-5p regulates human alveolar epithelial sodium channels by targeting the mTORC2/SGK-1 signaling pathway
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DOI：
10.1080/01902148.2016.1197347
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发表时间：
2016-06-01
影响因子：
1.7
通讯作者：
Wang, Daoxin
中科院分区：
文献类型：
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作者：
Qin, Ke;Zhong, Xi;Wang, Daoxin
目的：本研究旨在探讨miRNA-7-5p对人肺泡上皮钠通道的影响，阐明miRNA-7-5p调节ARDS中ENaC表达的通路。材料与方法：采用双荧光素酶靶基因验证实验来确认mTOR和SGK-1是否为miRNA-7-5p的靶基因。然后，我们分别在人肺泡上皮细胞系 A549 中过度表达和抑制 miRNA-7-5p。使用浓度为 100 ng/mL 的 LPS 刺激细胞。通过定量RT聚合酶链反应（qRT-PCR）和蛋白质印迹法检测miRNA-7-5p、mTOR、SGK-1、p-Akt-Ser473、α-、β-和γ-ENaC的表达。结果：本研究首次证实mTOR和SGK-1是miRNA-7-5p的靶基因。然后，我们发现 miRNA-7-5p 模拟组中 mTOR 和 SGK-1 的 mRNA 表达水平分别比空白对照下调 0.54 和 0.3 倍（P < 0.01）。 miRNA-7-5p 过表达抑制 mTORC2/SGK-1 信号通路活性并减少 ENaC 表达。脂多糖（LPS）刺激的 A549 细胞中 miRNA-7-5p 的表达显着上调，mTOR 和 SGK-1 的 mRNA 表达水平下调。转染miRNA-7-5p抑制剂后，我们发现与仅LPS刺激组相比，mTORC2/SGK-1通路活性恢复，并且ENaC表达也明显增加。结论：我们的结果表明 miRNA-7-5p 可以通过靶向 mTORC2/SGK-1 信号通路来调节人肺泡 ENaC 的表达。抑制miRNA-7-5p可以增强ENaC的表达，这可能为ARDS的治疗提供新的靶点。
Purpose: The aim of this study was to investigate the effect of miRNA-7-5p on human alveolar epithelial sodium channels and clarify the pathway in which miRNA-7-5p regulates the expression of ENaC in ARDS. Materials and Methods: Dual luciferase target gene validation experiments were used to confirm whether mTOR and SGK-1 are the target genes of miRNA-7-5p. Then, we overexpressed and inhibited miRNA-7-5p in the human alveolar epithelial cell line A549, respectively. LPS at a concentration of 100 ng/mL was used to stimulate the cells. The expressions of miRNA-7-5p, mTOR, SGK-1, p-Akt-Ser473, alpha-, beta-, and gamma-ENaC were detected by quantitative RT polymerase chain reaction (qRT-PCR) and western blotting. Results: In this study, we first confirmed that mTOR and SGK-1 are the target genes of miRNA-7-5p. Then, we found that mRNA expression levels of both mTOR and SGK-1 were downregulated to 0.54- and 0.3-fold, respectively, in the miRNA-7-5p mimic groups than the blank controls (P < 0.01). MiRNA-7-5p overexpression inhibited mTORC2/SGK-1 signaling pathway activity and reduced ENaC expression. The expression of miRNA-7-5p was significantly upregulated in A549 cells stimulated with lipopolysaccharide (LPS) and downregulated mRNA expression levels of both mTOR and SGK-1. After transfection with miRNA-7-5p inhibitors, we found that the mTORC2/SGK-1 pathway activity was restored compared to the group with LPS stimulation only, and the ENaC expression was also obviously increased. Conclusion: Our results demonstrate that miRNA-7-5p can regulate the expression of human alveolar ENaC by targeting the mTORC2/SGK-1 signaling pathway. The inhibition of miRNA-7-5p can enhance the expression of ENaC, which may provide a new target for the treatment of ARDS.