Sex-Specific Differences in MicroRNA Expression During Human Fetal Lung Development

Abstract

Background: Sex-specific differences in fetal lung maturation have been well described; however, little is known about the sex-specific differences in microRNA (miRNA) expression during human fetal lung development. Interestingly, many adult chronic lung diseases also demonstrate sex-specific differences in prevalence. The developmental origins of health and disease hypothesis suggests that these sex-specific differences in fetal lung development may influence disease susceptibility later in life. In this study, we performed miRNA sequencing on human fetal lung tissue samples to investigate differential expression of miRNAs between males and females in the pseudoglandular stage of lung development. We hypothesized that differences in miRNA expression are present between sexes in early human lung development and may contribute to the sex-specific differences seen in pulmonary diseases later in life. Methods: RNA was isolated from human fetal lung tissue samples for miRNA sequencing. The count of each miRNA was modeled by sex using negative binomial regression models in DESeq2, adjusting for post-conception age, age², smoke exposure, batch, and RUV factors. We tested for differential expression of miRNAs by sex, and for the presence of sex-by-age interactions to determine if miRNA expression levels by age were distinct between males and females. Results: miRNA expression profiles were generated on 298 samples (166 males and 132 females). Of the 809 miRNAs expressed in human fetal lung tissue during the pseudoglandular stage of lung development, we identified 93 autosomal miRNAs that were significantly differentially expressed by sex and 129 miRNAs with a sex-specific pattern of miRNA expression across the course of the pseudoglandular period. Conclusion: Our study demonstrates differential expression of numerous autosomal miRNAs between the male and female developing human lung. Additionally, the expression of some miRNAs are modified by age across the pseudoglandular stage in a sex-specific way. Some of these differences in miRNA expression may impact susceptibility to pulmonary disease later in life. Our results suggest that sex-specific miRNA expression during human lung development may be a potential mechanism to explain sex-specific differences in lung development and may impact subsequent disease susceptibility.

Keywords: gene expression; human; lung development; microRNA; pulmonary disease; sex-specific.

FIGURE 1

Human fetal lung tissue samples used for this analysis demonstrates a similar distribution of estimated gestational ages observed in each sex.

FIGURE 2

(A) Top 10 miRNAs by p-value are depicted with their effect size expressed as log₂ (fold change) with 95% confidence intervals. Estimated fold changes (miRNA counts in males compared to female samples) and q-values are also presented. (B,C) show example scatter plots of residualized miRNA levels (y-axis) plotted by sex (x-axis). Residuals are displayed to illustrate sex-differences after adjusting for covariates including, age, age (Torday et al., 1981), smoke exposure, technical batch, and the four RUV-inferred covariates to emphasize effects of sex.

FIGURE 3

Examples of sex-specific trajectories. Two miRNAs with statistically significant age-by-sex interactions are plotted with residualized expression (y-axis) across age (x-axis) during the pseudoglandular period differs by sex (indicated by color). Residuals are displayed to illustrate sex-by-age differences after adjusting for smoke exposure, technical batch, and the four RUV-inferred covariates to emphasize patterns by age and sex.