17β-Estradiol Regulates miR-9-5p and miR-9-3p Stability and Function in the Aged Female Rat Brain

Clinical studies demonstrated that the ovarian hormone 17β-estradiol (E₂) is neuroprotective within a narrow window of time following menopause, suggesting that there is a biological switch in E₂ action that is temporally dependent. However, the molecular mechanisms mediating this temporal switch have not been determined. Our previous studies focused on microRNAs (miRNA) as one potential molecular mediator and showed that E₂ differentially regulated a subset of mature miRNAs which was dependent on age and the length of time following E₂ deprivation. Notably, E₂ significantly increased both strands of the miR-9 duplex (miR-9-5p and miR-9-3p) in the hypothalamus, raising the possibility that E₂ could regulate miRNA stability/degradation. We tested this hypothesis using a biochemical approach to measure miRNA decay in a hypothalamic neuronal cell line and in hypothalamic brain tissue from a rat model of surgical menopause. Notably, we found that E₂ treatment stabilized both miRNAs in neuronal cells and in the rat hypothalamus. We also used polysome profiling as a proxy for miR-9-5p and miR-9-3p function and found that E₂ was able to shift polysome loading of the miRNAs, which repressed the translation of a predicted miR-9-3p target. Moreover, miR-9-5p and miR-9-3p transcripts appeared to occupy different fractions of the polysome profile, indicating differential subcellular. localization. Together, these studies reveal a novel role for E₂ in modulating mature miRNA behavior, independent of its effects at regulating the primary and/or precursor form of miRNAs.