Fu Jiayao, Hao Lingyu, Tian Yawen, Liu Yang, Gu Yijing, Wu Junhua
Department of Prosthodontics, School and Hospital of Stomatology, Tongji University, Shanghai Engineering Research Center of Tooth Restoration and Regeneration, Shanghai, China.
J Cell Physiol. 2018 Mar;233(3):2292-2303. doi: 10.1002/jcp.26101. Epub 2017 Aug 25.
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.
迄今为止，有证据表明雌激素通过微小RNA部分调节细胞过程。自噬是一种分解代谢过程，受多种因素调节，与骨骼疾病相关。然而，雌激素是否通过微小RNA调节骨细胞自噬在很大程度上尚不清楚。在本研究中，我们观察到在去卵巢（OVX）小鼠的骨细胞区域中，转录后调节因子微小RNA-199a-3p上调。在骨细胞样MLO-Y4细胞中，miR-199a-3p的成熟形式和pri-miR-199a是对雌激素信号作出反应而产生的。蛋白质印迹法、自噬通量检测、mRFP-GFP-LC3荧光和电子显微镜证实，miR-199a-3p在MLO-Y4细胞中诱导自噬，尽管细胞凋亡未受影响。此外，我们记录了雌激素介导骨细胞自噬的能力。基于我们的体内数据，雌激素缺乏诱导骨细胞自噬。用17β-雌二醇处理饥饿的MLO-Y4细胞，通过激活雷帕霉素哺乳动物靶标（mTOR）相关信号级联反应，抑制了饥饿诱导的过度自噬，而给予雷帕霉素则逆转了17β-雌二醇的作用。同时，miR-199a-3p过表达逆转了17β-雌二醇介导的MLO-Y4细胞自噬调节。根据机制研究，miR-199a-3p通过直接结合胰岛素生长因子-1（IGF-1）和mTOR的3'-非翻译区来抑制mTOR途径。然而，miR-199a-3p过表达抑制了IGF-1磷酸化和mTOR相关途径。分别敲低mTOR和IGF-1可消除雌激素信号，并通过mTOR重新激活恢复LC3-II表达。因此，miR-199a-3p似乎参与了介导骨细胞自噬的雌激素调节网络，可能是通过靶向IGF-1和mTOR来实现的。
