Department of Neonates, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu 210008, P.R. China.
Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Int J Mol Med. 2020 May;45(5):1571-1582. doi: 10.3892/ijmm.2020.4511. Epub 2020 Feb 24.
The synthesis and secretion of surfactant proteins (SPs) is an important sign of lung maturation. Furthermore, the morbidity of lung developmental diseases, including respiratory distress syndrome and bronchopulmonary dysplasia which are mainly caused by immature lung development and lack of SPs, is increasing. As is well known, multiple microRNAs (miRs/miRNAs) are able to influence lung development via numerous different signaling pathways. However, few studies examine the association between the miRNAs and lung developmental diseases. A previous study has demonstrated that miR‑431 was significantly (F=33.49; P<0.001) downregulated in the lung tissues of Sprague‑Dawley rats at 3 time points, embryonic day 19, embryonic day 21 and postnatal day 3. The present study reported that the regulation of miR‑431 may influence the expression of SPs. Thus, the further potential mechanisms of miR‑431 in negatively regulating lung development were examined in the present study. Stable A549 cell lines overexpressing or knocking down SMAD family member 4 (SMAD4) transfected with miR‑431 overexpressed or knocked down, and their control groups were established. Subsequently, the expression of bone morphogenetic protein 4 (BMP4), SMAD4 and SPs (SP‑A, SP‑B and SP‑C) at the RNA and protein levels were validated respectively by reverse transcription quantitative PCR and western blotting. miR‑431 exhibited a decreased expression, while BMP4 and SPs exhibited increased expression at the mRNA and protein levels in the SMAD4 knockdown group. Meanwhile, the expression of SPs were reduced in the SMAD4‑knockdown group via overexpressing miR‑431 and increased in the SMAD4‑overexpression group via inhibiting miR‑431. The present results indicate that SMAD4 negatively regulates the expression of SPs, and that miR‑431 negatively regulates the expression of SPs through inhibiting the BMP4/activin/transforming growth factor‑β signaling pathway by targeting SMAD4.
表面活性蛋白（SPs）的合成和分泌是肺成熟的一个重要标志。此外，肺发育疾病的发病率正在上升，这些疾病主要由肺发育不成熟和缺乏 SPs 引起，包括呼吸窘迫综合征和支气管肺发育不良。众所周知，多种 microRNAs（miRs/miRNAs）可以通过多种不同的信号通路影响肺发育。然而，很少有研究探讨 miRNAs 与肺发育疾病之间的关系。先前的一项研究表明，miR-431 在 Sprague-Dawley 大鼠的肺组织中，在 3 个时间点（胚胎第 19 天、胚胎第 21 天和出生后第 3 天）显著下调（F=33.49；P<0.001）。本研究报道，miR-431 的调节可能影响 SPs 的表达。因此，本研究进一步研究了 miR-431 负调控肺发育的潜在机制。建立了稳定过表达或敲低 SMAD 家族成员 4（SMAD4）的 A549 细胞系，转染过表达或敲低 miR-431，并建立其对照组。随后，通过逆转录定量 PCR 和 Western blot 分别验证了骨形态发生蛋白 4（BMP4）、SMAD4 和 SPs（SP-A、SP-B 和 SP-C）在 RNA 和蛋白水平的表达。miR-431 的表达降低，而 BMP4 和 SPs 的表达在 SMAD4 敲低组中在 mRNA 和蛋白水平上均增加。同时，通过过表达 miR-431 降低了 SMAD4 敲低组中 SPs 的表达，通过抑制 miR-431 增加了 SMAD4 过表达组中 SPs 的表达。本研究结果表明，SMAD4 负调控 SPs 的表达，miR-431 通过靶向 SMAD4 抑制 BMP4/激活素/转化生长因子-β信号通路负调控 SPs 的表达。
