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

Chun K Kim,Mengjie Zhang,Toni R Pak,Sarah Flury,Mikayla L Newby,Megan L Linscott

doi:10.3390/ncrna7030053

Abstract

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.

Highlights

MicroRNAs are small non-coding RNAs that post-transcriptionally regulate up to 60% of all cellular proteins [1]
We used a previously validated miRNA degradation assay to determine whether E2 altered the degradation rate of mature miR-9-5p and miR-9-3p using neuronal cell lines, as
We used a previously validated miRNA degradation assay to determine whether E2 altered the degradation rate of mature miR-9-5p and miR-9-3p using neuronal cell lines, daesscdreisbcerdibpedrepvrioevuisoluys[l1y6[]1.6F].irFsitr,swt, ewteestetestdedthteheefeffefcetcstsoof fEE22uussiinnggaa rraatt nneeuurroonnaall cceelllllliinnee ddeerirviveeddfrforommththeeppaararavveenntrtirciuculalarrnnuuclceleuuss(P(PVVNN) )oof fththeehhyyppooththalaalmamuus.s.OOuurrddaatatashshoowwededa saigsniigfinciafinctamntaminaeinffeecffteocft ToifmTeimone moniRm-9iR-5-p9-h5palfh-alilffe-liinfenineunroeunraolncaelllcse[lFls(1,[F1(21), 1=2)7=.573.5;3p; p==00..001188]] (F(Figiguurere11AA––CC).)

Summary

Introduction

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate up to 60% of all cellular proteins [1]. Our previous data demonstrated that the ability of E2 to regulate these miRNAs in aged rats was dependent on the length of time between ovarian hormone depletion (via ovariectomy) and the subsequent timing of replacement E2 treatment [6]. Those data further supported the notion that differential regulation of miR-9-5p and miR-9-3p could, in part, explain the disparate functional outcomes of E2 treatment in women closer to the menopausal transition compared to those given E2 many years past menopause

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