Expression profile of androgen-modulated microRNAs in the fetal murine lung

Abstract

Background: Androgens are known to delay lung development. As a consequence, the incidence and morbidity of respiratory distress syndrome of the neonate are higher for male than for female premature infants. We previously reported that many genes were expressed with a sex difference in the mouse developing lung and that several genes were under the control of androgens in the male fetal lung. microRNAs are small non-coding RNAs known to negatively regulate the expression of specific genes. In this study, we examined whether murine miRNAs are under the control of androgens in the male developing lung.

Methods: Expression profiling of microRNAs was performed by microarrays using RNA extracted from male fetal lungs isolated on gestational day (GD) 17.0 and GD 18.0 after daily injection of pregnant mice from GD 10.0 with the antiandrogen flutamide or vehicle only. To identify putative miRNA target genes, the data obtained here were combined with gene profiling data reported previously using the same RNA preparations. qPCR was used to confirm microarray data with fetal lungs from other litters than those used in microarrays.

Results: Flutamide induced downregulation and upregulation of several miRNAs on GD 17.0 and GD 18.0. Of the 43 mature miRNAs modulated by flutamide on GD 17.0, 60 % were downregulated, whereas this proportion was only of 34 % for the 35 mature miRNAs modulated on GD 18.0. For 29 and 26 flutamide-responsive miRNAs, we found a corresponding target inversely regulated by androgens on GD 17.0 and 18.0, respectively. The androgen-regulated target genes were involved in several biological processes (lipid metabolism, cell proliferation, and lung development) and molecular functions, mainly transcription factor binding.

Conclusions: Regulation of male lung development involves several miRNAs that are under androgen modulation in vivo.

Keywords: Androgens; Lung development; Preterm birth; Respiratory distress syndrome; Sex differences; Surfactant; microRNA.

Fig. 1

Overview of the microarray data obtained from fetal mouse lungs exposed or not to flutamide. a Number of androgen-regulated miRNAs on GD 17.0 and GD 18.0. b Expression profile of differentially expressed miRNAs is presented for each replica and each gestation time. Red and blue indicate the intensity level above and below the median, respectively, for each miRNA and each replicate. For each age, results are given for the two replicates (1, 2) for male exposed to vehicle (mv), and male exposed to flutamide (mf). c Number of androgen-modulated mature miRNA according to their reactivity to flutamide at GD 17.0 and GD 18.0

Fig. 2

Validation of microarray data by qPCR for several miRNAs. a Selection of reference genes by geNorm among the five tested candidates. The average of expression stability M is presented for each tested reference gene candidate (left panel). The two most stable genes yielded the lower pairwise variation value (0.179) (right panel). Quantification of several miRNAs by qPCR on GD 17.0 (b) and GD 18.0 (c). d Comparison of expression ratios (flutamide/vehicle) obtained by microarray and qPCR for seven miRNA genes

Fig. 3

Number of androgen-regulated miRNAs and their potential androgen-regulated targets according to biological processes on GD 17.0 (a) and GD 18.0 (b)

Fig. 4

Number of androgen-regulated miRNAs and their potential androgen-regulated targets according to molecular functions on GD 17.0 (a) and GD 18.0 (b)