Department of Pediatrics, The First Affiliated Hospital, Nanjing Medical University, Nanjing, Jiangsu 210029, P.R. China.
Department of Neonates, Children's Hospital of Nanjing Medical University, Nanjing, Jiangsu 210008, P.R. China.
Mol Med Rep. 2019 Jun;19(6):4980-4988. doi: 10.3892/mmr.2019.10154. Epub 2019 Apr 11.
Deficiency of surfactant proteins (SPs) is the main cause of respiratory distress syndrome (RDS) and chronic lung diseases. Our previous study demonstrated that miR‑431 was differentially expressed between infants with RDS and infants without RDS using microarray analysis. However, the potential role of miR‑431 in the development of lung function is still unknown. In the present study, the morphological characteristics of lung tissues and the expression levels of miR‑431 were examined at three time points of rat lung development [gestational days 19 and 21 (E19, and E21) and postnatal day (P3)]. The protein and mRNA levels of SMAD4 and SPs (SP‑A, SP‑B, SP‑C and SP‑D) were also validated by reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis, respectively. The expression levels of miR‑431 were gradually decreased over time periods of E19, E21 and P3, as determine using RT‑qPCR and fluorescence in situ hybridization. Dual luciferase‑reporter assays revealed that SMAD4 is a direct target of miR‑431. The mRNA and protein expression levels of SMAD4 and SPs increased gradually in rat lung tissues from E19 to P3. The order of magnitude was as follows: E19, E21 and P3. The present study demonstrated that the expression level of miR‑431 decreased in the order of E19, E21 and P3 during rat lung development. The target gene of miR‑431, SMAD4, was negatively regulated by miR‑431, and its expression levels in the rat lung tissue increased from E19 to the P3. Surfactant synthesis was further increased over the E19 to P3 time period. Further studies are required to determine how miR‑431 regulates pulmonary surfactant synthesis by targeting SMAD4.
表面活性蛋白（SPs）缺乏是呼吸窘迫综合征（RDS）和慢性肺部疾病的主要原因。我们之前的研究通过微阵列分析表明，miR-431 在 RDS 婴儿和非 RDS 婴儿之间存在差异表达。然而，miR-431 在肺功能发育中的潜在作用尚不清楚。在本研究中，我们在大鼠肺发育的三个时间点（妊娠第 19 天和第 21 天（E19 和 E21）和出生后第 3 天（P3））检查了肺组织的形态特征和 miR-431 的表达水平。SMAD4 和 SPs（SP-A、SP-B、SP-C 和 SP-D）的蛋白和 mRNA 水平也分别通过逆转录-定量聚合酶链反应（RT-qPCR）和蛋白质印迹分析进行了验证。使用 RT-qPCR 和荧光原位杂交技术，确定 miR-431 的表达水平随 E19、E21 和 P3 时间的推移逐渐降低。双荧光素酶报告基因实验表明，SMAD4 是 miR-431 的直接靶标。从 E19 到 P3，大鼠肺组织中 SMAD4 和 SPs 的 mRNA 和蛋白表达水平逐渐增加。数量级顺序为：E19、E21 和 P3。本研究表明，在大鼠肺发育过程中，miR-431 的表达水平呈 E19、E21 和 P3 的顺序降低。miR-431 的靶基因 SMAD4 受 miR-431 的负调控，其在大鼠肺组织中的表达水平从 E19 增加到 P3。表面活性剂合成在 E19 到 P3 期间进一步增加。需要进一步研究确定 miR-431 通过靶向 SMAD4 如何调节肺表面活性剂的合成。
