Étude de l'expression des microARNs et des enzymes de synthèse des corticostéroïdes dans le développement pulmonaire

Le syndrome de détresse respiratoire du nouveau-né (SDR) est l’une des pathologies les plus fréquentes dont souffrent les bébés prématurés. Le SDR est causé par un déficit dans la synthèse du surfactant pulmonaire en raison de l’immaturité du poumon lors d’une naissance prématurée. Plusieurs éléments régulent le développement pulmonaire notamment les stéroïdes sexuels et les corticostéroïdes. Le sexe est aussi un élément régulateur du développement pulmonaire. En effet, les garçons sont plus atteints que les filles par le SDR. Ce dimorphisme sexuel est attribué aux androgènes. Le traitement anténatal aux glucocorticoïdes est prescrit aux femmes qui sont à risque d’accoucher prématurément. En effet, les corticostéroïdes favorisent la maturation pulmonaire anténatale. Également, il a été démontré que les microARNs sont primordiaux pour le développement pulmonaire. Ceci nous a conduit à étudier l’impact des androgènes sur le profil d’expression des microARNs lors de la transition du stade canaliculaire au stade sacculaire (jour gestationnel (JG)17.0 au JG18.0), période qui coïncide avec la montée de la synthèse et de la sécrétion du surfactant chez la souris. Tout d’abord, nous avons étudié la stabilité des gènes de normalisation (snoRNAs) afin de quantifier les microARNs par qPCR. Cette analyse a été effectuée avec 3 logiciels différents et sur plusieurs stades du développement notamment de la période pseudoglandulaire jusqu’au stade alvéolaire chez les deux sexes. On a identifié les meilleures combinaisons de gènes de normalisation les plus stables pour chaque stade du développement étudié ainsi que pour la période couvrant tous les stades étudiés. Ensuite nous avons analysé à GD17.0 et GD18.0 le profil d’expression des microARNs chez des fœtus mâles dont les mères ont été traitées au flutamide (anti-androgènes pure). Les résultats ont montré que 43 microARNs matures sont modulés par les androgènes à GD17.0 et 35 microARNs à GD18.0. Pour certains microARNs, nous avons identifié des cibles potentielles qui sont inversement modulées par les androgènes par rapport aux microARNs. Ces cibles sont impliquées dans plusieurs processus biologiques tels que le métabolisme des lipides et la prolifération cellulaire ainsi que dans des fonctions moléculaires tels que la liaison des facteurs de transcription. Des expériences de validation ont été effectuées par qPCR. Nos résultats ont montré que les androgènes régulent des processus qui peuvent être impliqués dans la maturation pulmonaire via la régulation des microARNs. En plus de l’intérêt porté aux androgènes dans la maturation pulmonaire, nous avons analysé l’expression d’enzymes de synthèse des corticostéroïdes dans le poumon fœtal humain. L’expression de l’enzyme 21-hydroxylase a été étudiée par qPCR et par immunobuvardage. Également la localisation de l’ARNm de cette enzyme clé de la synthèse des glucocorticoïdes, a été effectuée par hybridation in situ. L’ARNm de CYP21A2 a été détecté par qPCR dans les 34 échantillons analysés et dont les âges variaient entre 17 et 40 semaines de grossesse. Aucune corrélation, avec l’âge gestationnel ou le sexe, n’a été observée. Des niveaux significatifs de la protéine 21-hydroxylase ont été détectés dans nos échantillons. Nous avons investigué l’expression d’autres enzymes impliquées dans la voie de synthèse des glucocorticoïdes notamment CYP11B1, CYP11B2 et CYP17A1. Les ARNm des gènes CYP11B1, CYP11B2 n’ont pas été détectés dans nos échantillons, contrairement à CYP17A1 dont l’ARNm a été détecté dans tous nos tissus fœtaux analysés. La protéine de la 17α-hydroxylase a été détectée à de faibles niveaux. Nos résultats d’hybridation in situ ont montré que l’expression de CYP21A2 est localisée presqu’exclusivement dans l’épithélium pulmonaire distal. Nos résultats suggèrent que les produits de la 21-hydroxylase agiront via une action intracrine sur l’épithélium distal en activant le récepteur des glucocorticoïdes (GR). L’activation du récepteur des minéralocorticoïdes (MR) ne semble pas dépendre de produits de la 21-hydroxylase en raison des quantités importantes d’aldostérone circulante.Respiratory distress syndrome of the newborn (RDS) is one of the most common diseases affecting preterm babies. RDS is caused by a deficiency in the synthesis and secretion of pulmonary surfactant as a result of lung immaturity caused by a premature birth. Several elements and factors regulate lung development including sex steroids and corticosteroids and the sex of the infant. In fact, boys are more affected than girls by RDS. This sexual dimorphism is attributed to the presence of androgens in male lungs. In contrast, corticosteroids are given to mother at higher risk to deliver prematurely to promote antenatal lung maturation of the fetuses. As other factors, it has been shown that microRNAs are essential to lung development. This led us to study the impact of androgen on the expression profile of microRNAs in the transition period between canalicular and saccular stages (gestational day (GD)17.0 and GD18.0). This period overlap the surge of surfactant synthesis in the mouse. First, we studied the stability of normalization genes (snoRNAs) to quantify microRNAs by qPCR. This analysis was performed by 3 methods of calculation at several stages of lung development from the pseudoglandular to the alveolar stages and this for both sexes. We identified the best combinations of the most stable normalization genes for each individual developmental stage studied as well as for the period covering all the studied stages. Then, we analyzed the expression profile of microRNAs on GD17.0 and GD18.0 in male fetuses whose mothers were treated with flutamide (pure anti-androgen). The results showed that 43 mature microRNAs are modulated by endogenous androgens on GD17.0 whereas 35 microRNAs on GD18.0. We have identified some microRNAs and potential targets that are inversely modulated by androgens compared with microRNAs. These targets are involved in several biological processes such as lipid metabolism and cell proliferation as well as in molecular functions such as transcription factor binding. Validation experiments were performed by qPCR. Our results showed that androgens regulate processes that may be involved in lung maturation via the regulation of microRNAs. In addition to the interest in the impact of androgens on lung maturation, we analyzed the expression of corticosteroid synthesis enzymes in the human fetal lung. Expression of the CYP21A2 and the presence of its corresponding 21-hydroxylase enzyme have been studied by qPCR and immunoblot. Also mRNA localization of this key enzyme in the synthesis of glucocorticoids has been also assessed by in situ hybridization. CYP21A2 mRNA was detected by qPCR in all the 34 analyzed samples, whose ages ranged between 17 and 40 weeks of pregnancy. No correlation with gestational age or sex was observed. Significant levels of 21-hydroxylase protein were detected in our samples. We investigated the expression of other enzymes involved in the pathway of glucocorticoid synthesis including CYP11B1, CYP11B2 and CYP17A1. CYP11B1, CYP11B2 mRNA were not detected in our samples, unlike CYP17A1 whose mRNA was detected in all our analyzed fetal tissues. CYP17A1 protein was detected at low levels. In situ hybridization data showed that CYP21A2 expression is localized almost exclusively in the distal epithelium of human fetal lung. Our results suggest that 21-hydroxylase products act via an intracrine action on the distal epithelium by activating the glucocorticoid receptor (GR). Activation of the mineralocorticoid receptor (MR) at this site does not seem to depend on the 21-hydroxylase products due to the large amounts of circulating aldosterone

Identification of most stable endogenous control genes for microRNA quantification in the developing mouse lung.

MicroRNAs (miRNAs) are endogenous small non coding RNAs acting as negative regulators. miRNA are involved in lung development and pulmonary diseases. Measurement of their levels by qPCR is directly influenced by the stability of normalization gene(s), which can be affected by the experimental conditions. The developing lung is a changing tissue and one normalization gene showing stability on one developmental day may be modulated over time. Moreover, some developmental events are affected by sex, which also has to be considered. In this study, we compared stability of five putative control genes in the lung between sexes from the pseudoglandular to the alveolar stages and in adult lungs. Expression of sno135, sno142, sno202, sno234, and sno251 was studied by qPCR in male and female lung samples collected at seven time points from GD 15.5 to PN 30. Cq values of sno251 showed the highest variation across the different developmental stages, while sno234 was the most stable gene. Gene expression stability was studied by geNorm, NormFinder and BestKeeper. Our data showed that ranking of genes based on expression stability changed according to developmental time and sex. sno135/sno234 and sno142/sno234 were proposed as best combinations of normalization genes when both sexes and all the studied developmental stages are considered. Normalization of let7-a RNA levels with different pairs of control genes proposed by geNorm and NormFinder gave similar data, while the use of less stable genes introduced a statistically significant difference on PN 0. In conclusion, variations in stability of normalization gene expression are observed over time and according to sex during lung development. Best pairs of normalization genes are presented for specific developmental stages, and for the period extending from the pseudoglandular to the alveolar stages. The use of normalization genes selected for their expression stability is essential in lung development studies

Additional file 1: Table S1. of Expression profile of androgen-modulated microRNAs in the fetal murine lung

List of primers used for reverse transcription and qPCR for selected miRNA genes. (DOCX 20 kb

Putative endogenous control genes and primers used for reverse transcription and qPCR.

<p>^<i>a</i>Nucleotide sequences in bold are specific to each gene.</p><p>^<i>b</i>RT: Reverse transcription.</p><p>^<i>c</i>The reverse primer for qPCR is the same for all the genes and corresponds to a segment of the reverse transcription primers: 5′-GTGCAGGGTCCGAGGT-3′<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111855#pone.0111855-VarkonyiGasic1" target="_blank">[16]</a>.</p><p>^<i>d</i>F: Forward.</p><p>Putative endogenous control genes and primers used for reverse transcription and qPCR.</p

Expression levels of putative housekeeping genes in the mouse lung at different developmental stages and in the adult lung.

<p>Cq (mean ± SEM) obtained by qPCR are presented for the five putative control genes for the indicated developmental stages and for adult lungs. Pools of male and female lungs were used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111855#pone-0111855-t001" target="_blank">Table 1</a> for details).</p

Relative expression levels of <i>Let-7a</i> in canalicular-stage lungs normalized with different pairs of housekeeping genes.

<p>Let7-a RNA levels obtained by qPCR were normalized using the best pair of control genes calculated with: A) geNorm (<i>sno135</i>/<i>sno234</i>); B) NormFinder (<i>sno135</i>/<i>sno142</i>). C) qPCR data were normalized using a pair of less stable genes as estimated by geNorm and NormFinder (<i>sno202</i>/<i>sno251</i>). Pools of male and female lungs were used (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111855#pone-0111855-t001" target="_blank">Table 1</a> for details).</p

Lung developmental stages and number of fetuses used.

<p>GD, gestational day.</p><p>PN, postnatal day.</p><p>Lung developmental stages and number of fetuses used.</p

Stability values of housekeeping gene expression in lungs at different developmental stages for each sex.

<p>^<i>a</i><i>M</i> stability values are calculated by geNorm. Gene stability increases while <i>M</i> value decreases.</p><p>^<i>b</i>Stability values are calculated by NormFinder, Gene stability increases while stability value decreases.</p><p>^<i>c</i>Standard deviation (SD) is calculated by BestKeeper. Gene stability increases while SD value decreases.</p><p>^<i>d</i>All studied developmental stages.</p><p>Stability values of housekeeping gene expression in lungs at different developmental stages for each sex.</p

Housekeeping gene stability values in lungs at different developmental stages with both sexes combined.

<p>^<i>a</i><i>M</i> stability values calculated with the geNorm software.</p><p>^<i>b</i>Stability values calculated with the NormFinder software.</p><p>^<i>c</i>Standard deviation (SD) is calculated by BestKeeper. Gene stability increases while SD value decreases.</p><p>^<i>d</i>All studied developmental stages.</p><p>Housekeeping gene stability values in lungs at different developmental stages with both sexes combined.</p

Determination of optimal number of reference genes for normalization.

<p>The pairwise variation (Vn/Vn+1) was analyzed between two sequential normalization factors by geNorm. The optimal number of reference genes varies according to the developmental stage: two genes for the pseudoglandular and the canalicular stages as well as for the adult, three genes for the alveolar stage, and four genes for the saccular stage and the developing lung (from GD 15.5 to PN 30) as indicated by an asterisk.</p