The role of miR-431-5p in regulating pulmonary surfactant expression in vitro
The role of miR-431-5p in regulating pulmonary surfactant expression in vitro
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DOI：
10.1186/s11658-019-0150-4
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发表时间：
2019-04-02
影响因子：
8.3
通讯作者：
Yang, Yang
中科院分区：
文献类型：
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作者：
Li, Shujun;Sun, Zhongyi;Yang, Yang
研究背景肺表面活性物质（pulmonary surfactant，PS）是覆盖肺泡表面的脂质和蛋白质的复杂混合物.肺表面活性物质缺乏是新生儿呼吸窘迫的主要原因之一。近年来的研究表明，miRNA在肺发育过程中发挥重要作用，但对miR-431调控肺表面活性物质的研究较少。本研究探讨miR-431- 5 p对肺表面活性物质表达的调控作用，并筛选其潜在靶基因Smad 4。方法利用生物信息学工具TargetScan预测miR-431的靶基因。通过转染miR-431- 5 p模拟物、miR-431 - 5 p抑制剂和相应的阴性对照实现miR-431- 5 p的表达。使用定量实时PCR测定miR-431- 5 p的水平。进行CCK 8测定以确认用miR-431- 5 p模拟物、抑制剂或NC转染后12小时的细胞生长。采用Western blot和实时荧光定量PCR方法分析Smad 4和A549细胞表面活性剂相关蛋白的表达。过表达miR-431促进A549细胞增殖，抑制A549细胞凋亡。结论miR-431 - 5 p在肺表面活性物质表达中起重要作用，其调控与TGF-/Smad 4通路密切相关。这些结果将有助于我们研究肺发育性疾病的病理生理机制。
BackgroundPulmonary surfactant is the complex mixture of lipid and protein that covers the alveolar surface. Pulmonary surfactant deficiency is one of the main causes of neonatal respiratory distress. Recent studies showed that miRNA plays an important role in lung development, but research into miR-431 regulation of pulmonary surfactant are sparse. In this study, we explored the regulatory role of miR-431-5p in the expression of pulmonary surfactant and identified its potential target gene, Smad4.MethodsThe bioinformatics tool TargetScan was used to predict the targets of miR-431. The expression of miR-431-5p was achieved via transfection of miR-431-5p mimics, an miR-431-5p inhibitor and corresponding negative control. The level of miR-431-5p was determined using quantitative real-time PCR. The CCK8 assay was conducted to confirm cell growth 12h after transfection with miR-431-5p mimics, inhibitor or NC. Smad4 and surfactant-associated proteins in A549 were analyzed using western blot and quantitative real-time PCR.ResultsSmad4 was validated as a target of miR-431 in A549 cells. Overexpression of miR-431 accelerated A549 proliferation and inhibited A549 apoptosis. The mRNA and protein levels for the surfactant proteins (SP-A, SP-B, SP-C and SP-D) were found to be differentially expressed in A549 cells over- or under-expressing miR-431-5p.ConclusionOur results show that miR-431-5p is critical for pulmonary surfactant expression and that its regulation is closely related to the TGF-/Smad4 pathway. These results will help us to study the pathophysiological mechanism of lung developmental diseases.