Expression and potential regulation of miRNA­431 during lung development of Sprague­Dawley rats.

Deficiency of surfactant proteins (SPs) is the main cause of respiratory distress syndrome (RDS) and chronic lung diseases. Our previous study demonstrated that miR­431 was differentially expressed between infants with RDS and infants without RDS using microarray analysis. However, the potential role of miR­431 in the development of lung function is still unknown. In the present study, the morphological characteristics of lung tissues and the expression levels of miR­431 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 (SP­A, SP­B, SP­C and SP­D) were also validated by reverse transcription­quantitative polymerase chain reaction (RT­qPCR) and western blot analysis, respectively. The expression levels of miR­431 were gradually decreased over time periods of E19, E21 and P3, as determine using RT­qPCR and fluorescence in situ hybridization. Dual luciferase­reporter assays revealed that SMAD4 is a direct target of miR­431. 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 miR­431 decreased in the order of E19, E21 and P3 during rat lung development. The target gene of miR­431, SMAD4, was negatively regulated by miR­431, 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 miR­431 regulates pulmonary surfactant synthesis by targeting SMAD4.