Abstract
To human osteoblasts dexamethasone (DEX) treatment induces significant oxidative injury and cytotoxicity. Inhibition of CAB39 (calcium binding protein 39)-targeting microRNA can induce CAB39 upregulation, activating AMP-activated protein kinase (AMPK) signaling and offering osteoblast cytoprotection. Here we identified a novel CAB39-targeting miRNA: the microRNA-107 (miR-107). RNA-Pull down assay results demonstrated that the biotinylated-miR-107 directly binds to CAB39 mRNA in OB-6 human osteoblastic cells. Forced overexpression of miR-107, by infection of pre-miR-107 lentivirus or transfection of wild-type miR-107 mimic, largely inhibited CAB39 expression in OB-6 cells and primary human osteoblasts. Contrarily, miR-107 inhibition, by antagomiR-107, increased its expression, resulting in AMPK cascade activation. AntagomiR-107 largely attenuated DEX-induced cell death and apoptosis in OB-6 cells and human osteoblasts. Importantly, osteoblast cytoprotection by antagomiR-107 was abolished with AMPK in-activation by AMPKα1 dominant negative mutation, silencing or knockout. Further studies demonstrated that antagomiR-107 activated AMPK downstream Nrf2 cascade to inhibit DEX-induced oxidative injury. Conversely, Nrf2 knockout almost abolished antagomiR-107-induced osteoblast cytoprotection against DEX. Collectively, miR-107 inhibition induced CAB39 upregulation and activated AMPK-Nrf2 signaling to protect osteoblasts from DEX-induced oxidative injury and cytotoxicity.
Highlights
Sustained and/or excessive long-term usage of Dexamethasone (DEX) could induce osteoporosis or even osteonecrosis [1]
By performing the RNA-Pull down assay in OB-6 human osteoblastic cells, we show that the biotinylated-miR-107 directly associated with CAB39 mRNA (Figure 1B)
In the present study we show that miR-107 inhibition activated AMPK signaling in OB-6 cells, as phosphorylation (“p”) of AMPKα1 (Thr-172) and its major downstream target protein acetyl-CoA carboxylase (ACC, Ser-79) was significantly enhanced in antagomiR-107expressing OB-6 cells (Figure 2E)
Summary
Sustained and/or excessive long-term usage of Dexamethasone (DEX) could induce osteoporosis or even osteonecrosis [1]. To cultured human osteoblasts or osteoblastic cells DEX treatment will induce profound cytotoxicity and cell apoptosis [2,3,4,5]. Activated AMPK can promote cell survival under stress conditions [9]. Under oxidative stress AMPK activation can suppress reactive oxygen species (ROS) production and oxidative injury [5, 12,13,14,15]. AMPK will activate its potential downstream Nrf signaling to alleviate oxidative www.aging-us.com injury [16, 17]. AMPK activation blocks mammalian target of rapamycin complex 1 (mTORC1) signaling, favoring cell survival under energy crisis conditions [18, 19]. AMPK is a pro-survival signaling in stressed human cells
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