MicroRNA-127 modulates fetal lung development

Abstract

MicroRNAs (miRNAs) are small endogenous RNAs and are widely regarded as one of the most important regulators of gene expression in both plants and animals. To define the roles of miRNAs in fetal lung development, we profiled the miRNA expression pattern during lung development with a miRNA microarray. We identified 21 miRNAs that showed significant changes in expression during lung development. These miRNAs were grouped into four distinct clusters based on their expression pattern. Cluster 1 contained miRNAs whose expression increased as development progressed, while clusters 2 and 3 showed the opposite trend of expression. miRNAs in cluster 4 including miRNA-127 (miR-127) had the highest expression at the late stage of fetal lung development. Quantitative real-time PCR validated the microarray results of six selected miRNAs. In situ hybridization demonstrated that miR-127 expression gradually shifted from mesenchymal cells to epithelial cells as development progressed. Overexpression of miR-127 in fetal lung organ culture significantly decreased the terminal bud count, increased terminal and internal bud sizes, and caused unevenness in bud sizes, indicating improper development. These findings suggest that miR-127 may have an important role in fetal lung development.

Fig. 1.

Cluster analysis of micro RNAs (miRNAs) significantly changed during rat lung development. miRNAs from lungs on gestational days 16, 19 and 21 (E16, E19, and E21) or postnatal days 0, 6, and 14 (P0, P6, and P14) or adult lungs (AD) were cohybridized with the common reference. Normalized data were subjected to Significant Analysis of Microarrays (SAM) test to identify miRNAs whose expression was significantly changed during this process (q < 0.05). The identified miRNAs were grouped into 4 clusters by K-means clustering and viewed by TreeView. Each column represents 1 stage, and each row represents 1 miRNA. Each value of expression is the average of 6 replicates and is then log₂ transformed. Red represents positive values, green negative values, and black zero.

Fig. 2.

miRNA expression patterns during rat lung development. The relative expression of miRNAs in each cluster of Fig. 1 is plotted against each stage of development. The relative expression level is the ratio of the sample signal to the common reference signal. The data shown are the means of 6 replicates.

Fig. 3.

Validation of miRNA microarray results by quantitative real-time PCR (qRT-PCR). Total RNA was extracted from fetal lungs at E16, E19 and E21 and lungs at P0, P6, P14, and AD. miRNA levels were measured by real-time PCR. Relative expression levels of miR-29a (A), miR-195 (B), miR-18 (C), miR-20a (D), miR-127 (E), miR-351 (F), miR-136 (G), and miR-337 (H) are expressed as % of maximum expression. Error bars represent SE; n = 3 independent preparations, each assay performed in duplicate. Microarray data shown are averages from 6 replicates.

Fig. 4.

In situ hybridization for miRNAs. In situ hybridization was carried out in dewaxed and rehydrated fetal rat lung tissue sections at E16, E19 and E21 and lungs at P0 and AD. The sections were hybridized with 5′ DIG-labeled LNA probes against miR-20 (A), miR-127 (B), and miR-351 (C). A probe with scrambled sequence unrelated to known miRNAs was used as a negative control (Neg), and a probe for U6 was used as a positive control (Pos). Positive signals were visualized as dark blue/purple color. Arrowheads denote signals from epithelial cells; arrows denote signals from mesenchymal cells; insets represent enlarged images. Scalebars, 40 μm.

Fig. 5.

miR-127 overexpression in fetal lung culture. A: E14 fetal lungs were cultured in an insert. miR-127 overexpression adenovirus or virus control (VC) was added to the culture on day 0 (D0), and culture continued for 2 days (D1 and D2). The blank control (BC) was treated with medium alone. Images and green fluorescent protein (GFP) fluorescence were taken at D0–D2. Each treatment was carried out in at least 10 lungs from 3 different mothers in 3 separate experiments. B: qRT-PCR to quantify overexpression of miR-127. Total RNAs from D1 and D2 of the fetal lung culture were isolated. miR-127 levels were measured by qRT-PCR. Error bars represent SE; n = 3 independent preparations, each assay performed in duplicate. *P < 0.05 vs. VC, **P ≤ 0.02 vs. VC.

Fig. 6.

Effect of miR-127 overexpression on fetal lung development. E14 fetal lungs cultured in inserts were treated with miR-127 overexpression adenovirus or VC or BC for 2 days. Images were taken at the end of culture. A: enlarged image from Fig. 5A. B: terminal bud width was measured with MetaVue software. The width of each bud was measured at its longest diameter and expressed in relative units. C: variability in terminal bud width. Terminal bud width values were arranged in ascending order in each treatment and plotted against the number of buds to demonstrate variability in terminal bud size. Data were obtained from >250 terminal buds from at least 10 fetuses obtained from 3 mothers. D: average internal bud width was calculated with MetaVue software and the same measuring parameter for terminal buds. The value is expressed in relative units. E: no. of terminal buds formed at the end of D2 in miR-127-overexpressed lungs was compared with VC and BC after normalizing with the terminal buds on D0. Number of terminal buds was counted in a blinded manner by at least 2 different individuals, and the relative number at the end of D2 was expressed as a ratio to number of terminal buds on D0. At least 25 terminal or random internal buds from each lung were used for the respective analyses. Data are from at least 10 lungs obtained from 3 different mothers (n = 3). Error bars represent SE. *P < 0.05 vs. VC, **P ≤ 0.02 vs. VC.