To date, evidence indicates that estrogen partially modulates cellular processes through microRNAs. Autophagy is a catabolic process that is regulated by multiple factors and is associated with skeletal diseases. However, whether estrogen regulates osteocyte autophagy via microRNAs is largely unknown. In this study, we observed the up-regulation of microRNA-199a-3p, a post-transcriptional regulatory factor, in osteocytic areas in ovariectomized (OVX) mice. The mature forms of miR-199a-3p and pri-miR-199a were produced in response to estrogen signaling in osteocyte-like MLO-Y4 cells. Western blotting, autophagic flux detection, mRFP-GFP-LC3 fluorescence, and electron microscopy confirmed that miR-199a-3p induced autophagy in MLO-Y4 cells, although cellular apoptosis was not affected. Additionally, we documented the ability of estrogen to mediate osteocyte autophagy. Based on our in vivo data, estrogen deficiency induced autophagy in osteocytes. Treatment of starved MLO-Y4 cells with 17β-estradiol suppressed the excess autophagy induced by starvation via activation of mammalian target of rapamycin (mTOR)-related signaling cascades, while administration of rapamycin reversed the effects of 17β-estradiol. Meanwhile, miR-199a-3p overexpression reversed 17β-estradiol-mediated regulation of autophagy in MLO-Y4 cells. According to mechanistic studies, miR-199a-3p inhibited the mTOR pathway by directly binding to the 3'-untranslated regions of insulin growth factor-1 (IGF-1) and mTOR. However, overexpression of miR-199a-3p inhibited IGF-1 phosphorylation and mTOR-related pathways. Knockdown of mTOR and IGF-1 abolished estrogen signaling and restored LC3-II expression through mTOR re-activation, respectively. Thus, miR-199a-3p appears to be involved in the estrogen regulatory networks that mediate bone cell autophagy, potentially by targeting IGF-1 and mTOR.
Keywords: autophagy; bone; estrogen signaling; miR-199a-3p; microRNA; osteocyte-like MLO-Y4 cells; ovariectomized mice.
© 2017 Wiley Periodicals, Inc.