Project description:We performed miRNA sequencing to comprehensively and unbiasedly examine the post-transcriptional landscape of lung tissue in female and male rats following bleomycin acute lung injury (ALI). Male animals displayed the greatest miRNA perturbation following lung injury with 112 miRNAs differentially expressed (75 upregulated, 37 downregulated). Female animals demonstrated markedly fewer differentially expressed miRNAs, with 66 total miRNAs (45 upregulated, 21 downregulated). Direct comparison between female and male animals’ post-injury revealed 54 differentially expressed miRNAs (8 upregulated, 46 downregulated). Baseline comparison between female and male miRNA profiles turned up 23 miRNAs (6 upregulated, 17 downregulated). Among the sex-biased miRNAs, miR-490-5p showed significant downregulation in females versus males following bleomycin-induced lung injury (log2FC = -2.40). This was particularly intriguing as miR-490-5p is predicted to target Bmpr2 (TargetScan score = 88), a critical component for vascular endothelial integrity and the BMP/TGF- signaling pathway. Correlation analysis of Bmpr2 expression and miR-490-5p expression revealed a negative correlation with females having a lower expression of miR-490-5p and a higher expression of Bmpr2 compared to males. Protein validation of BMPR2 revealed a baseline trend towards females having higher BMPR2 levels compared to males (p = 0.09), with females having significantly higher BMPR2 induction following lung injury compared to males (p = 0.009) . rno-miR-672-3p emerged as a noteworthy differentially expressed miRNA, a sex-specific regulator with opposing expression patterns between sexes. In males, miR-672-3p was significantly downregulated following bleomycin injury (log2FC = -2.46, p = 0.0003), while females showed no significant change (log2FC = 0.375, p = 0.202). Direct comparison revealed that miR-672-3p was significantly higher in bleomycin-treated females compared to bleomycin-treated males (log2FC = 2.21, p = 0.006). qPCR validation revealed a more nuanced pattern than the sequencing data suggested. Both sexes showed suppression of miR-672-3p expression following ALI compared to their respective sham controls. Male animals demonstrated the most dramatic reduction (p < 0.0001), with approximately 95% suppression relative to sham. Females also showed downregulation (approximately a 50% reduction, p = 0.0516), although the magnitude was substantially less pronounced. The direct comparison between injured males and females showed a trend toward significance (p = 0.0801), indicating high retention of miR-672-3p expression in injured females compared to males. Target prediction identified 242 potential miR-672-3p targets among male bleomycin versus sham and female versus male bleomycin DEGs, including high-confidence targeting of inflammatory mediators: Cxcr3, Cxcl11, C3ar1, Cd80, Cd4, and Serpina3a, all of which were upregulated in males in the parallel RNA-seq performed. The biological coherence of these targets, which encompass chemokine signaling, complement activation, and acute-phase responses, suggests a functional significance. To examine whether predicted miR-672-3p targets showed expression patterns consistent with mRNA expression, we analyzed correlations between miR-672-3p and target gene expression. In males, comparing injury to baseline, miR-672-3p showed negative correlations with inflammatory mediators including Cxcl11 (r = -0.88, p = 0.0008), Cxcr3 (r = -0.84, p =0.0022), Cd80 (r = -0.83, p = 0.0029), Cd4 (r = -0.78, p = 0.0083), Serpina3a (r = -0.73, p = 0.017), and C3ar1 (r = -0.77, p = 0.0086) (Figure 6D). These inverse relationships were also observed in the sex comparison with similar correlation strengths: C3ar1 (r = -0.81, p = 0.0045), Serpina3a (r = -0.80, p = 0.0054), Cd80 (r = -0.78, p = 0.0085), Cxcl11 (r = -0.78, p = 0.0148), Cxcr3 (r = -0.65, p = 0.042), and Cd4 (r = -0.77, p = 0.0089). A more granular analysis of top differentially expressed miRNAs uncovered multiple miRNAs that were upregulated in both bleomycin-treated females and males relative to their respective sham, notably miR-511-3p_R+1, though with males having higher miR-511-3p_R+1 expression relative to females following bleomycin-induced lung injury (Figure 5B. Figure S5A-D). Another miRNA mirroring a similar pattern of upregulation in both sexes post-ALI, but higher in males than females was miR-212-3p (Figure 5B). Together, these support the previous evidence of a more perturbed miRNA response in males than females. This coordinated dysregulation suggests that multiple miRNA networks beyond miR-672-3p alone contribute to sex-specific-post-transcriptional programs.
2026-04-01 | GSE324979 | GEO