Abstract
MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR‐20a‐5p had such regulatory functions on alveolar type II (AT‐II) cells. To accomplish this, miR‐20a‐5p–overexpressed and miR‐20a‐5p–inhibited adenoviral vectors were constructed and transfected into cultured AT‐II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR‐20a‐5p levels were verified by real‐time PCR. The CCK‐8 assay was used to compare the proliferation ability of AT‐II cells that had over‐ or underexpressed miR‐20a‐5p. The expression of surfactant‐associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real‐time PCR and Western blotting. In AT‐II cells, transfection resulted in over‐ or under‐regulation of miR‐20a‐5p. While overexpression of miR‐20a‐5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR‐20a‐5p regulation of PTEN. As a result, when miR‐20a‐5p was rendered overexpressed, PTEN was down‐regulated. By contrast, when miR‐20a‐5p was underexpressed, PTEN was up‐regulated. Neither overexpression nor underexpression of miR‐20a‐5p altered the cell proliferation. miR‐20a‐5p plays no role in proliferation of foetal AT‐II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.
Highlights
The alveolar type II (AT‐II) epithelial cells at the alveolar horn have highly specialized functions for the synthesis, secretion and reutili‐ zation of pulmonary surfactant.[1]
The result is consistent with our previous work in rats that showed that the expression of miR‐20a gradually decreased in the late stage of foetal lung development.[12]. Given these results, which were further validated by real‐time qPCR, and in view of the role of PS in respiratory distress syndrome (RDS), the association between miR‐20a and RDS and the role of miR‐20a in lung development, we further explored the role of miR‐20a‐5p in pulmonary surfactant gene expression
Dual‐luciferase reporter assay system re‐ vealed that the relative luciferase activity of wild‐type pmirGLO‐PTEN‐3′UTR (WT)‐3′UTR vector and miR‐20a‐5p mimic cotransfected group was decreased by 31.39% comparing with miRNA controls, the differences were statistically significant, while the luciferase activity of mutant pmir‐ GLO‐PTEN‐3′UTR (MUT)‐3′UTR vector was not affected obviously (Figure 6A,B), which is consistent with the results reported in the related literature.[13,14]
Summary
The alveolar type II (AT‐II) epithelial cells at the alveolar horn have highly specialized functions for the synthesis, secretion and reutili‐ zation of pulmonary surfactant.[1]. Whether and in what way miRNAs regulate pulmonary surfactant synthesis re‐ mains unknown In this present study, we used miRNA microarrays to first detect the differential expression of miRNAs in peripheral blood of premature infants with versus without RDS. | 7665 for the first time we have shown that miR‐20a‐5p plays an important role in regulation of synthesis of pulmonary surfactant. Based upon these findings, we postulate that miR‐20a‐5p might be important in controlling essential developmental and physiological events in the lung, including the synthesis and metabolism of pulmonary surfac‐ tant in AT‐II, and may be involved in the occurrence and devel‐ opment of RDS
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