miR-107 inhibition upregulates CAB39 and activates AMPK-Nrf2 signaling to protect osteoblasts from dexamethasone-induced oxidative injury and cytotoxicity

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.


INTRODUCTION
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]. Furthermore, in the bones of DEX-taking patients significant osteoblast cell apoptosis and decreased number of viable osteoblasts were detected [6,7]. Our group has been dedicated to understanding the signaling mechanisms of DEX-induced osteoblast cytotoxicity, and to developing novel and efficient strategies to overcome them [2][3][4][5].
Here we discovered that inhibition of CAB39targeting miR-107, by antagomiR-107, induced significant AMPK cascade activation, causing AMPKα1-ACC phosphorylation and AMPK activity increase. Functional studies demonstrated that antagomiR-107 potently attenuated DEX-induced cell death and apoptosis in OB-6 cells and human osteoblasts. Such osteoblast cytoprotective actions by antagomiR-107 were however abolished with AMPK in-activation, through genetic strategies including AMPKα1 dominant negative mutation, silencing or KO. We concluded that miR-107 inhibition by antagomiR-107 activated AMPK signaling to protect osteoblasts from DEX-induced cytotoxicity.
In the present study we demonstrated that in OB-6 cells and primary human osteoblasts miR-107 inhibition induced Nrf2 signaling activation, causing Nrf2 protein stabilization, HO1-NQO1 expression and NQO1 activity increase. Importantly, blockage of AMPK signaling, by AMPKα1 mutation, silencing or KO, abolished antagomiR-107-induced Nrf2 activation in osteoblasts, suggesting that activation of AMPK is required for miR-107 inhibition-induced Nrf2 cascade activation. Further studies demonstrated that Nrf2 KO almost reversed antagomiR-107-induced osteoblast cytoprotection against DEX. Therefore, miR-107 inhibition provoked AMPK-dependent Nrf2 signaling, protecting osteoblasts from DEX-induced oxidative injury and cytotoxicity.

Chemicals and reagents
DEX, polybrene, neomycin and puromycin were provided by Sigma Aldrich Chemicals (St Louis, Mo). From Gibco Co. (Shanghai, China) fetal bovine serum (FBS) and other cell culture reagents were obtained. Antibodies were all provided by Cell Signaling Technology (Danvers, MA). TRIzol and all RNAassociated reagents were purchased from Thermo-Fisher Invitrogen (Suzhou, China). All viral constructs, microRNA mimic (wild-type and mutants), and other sequences were provided by Shanghai Genechem Co. (Shanghai, China), unless otherwise mentioned.

Cell culture
OB-6 human osteoblastic cells [6] and primary human osteoblasts [41,42] were differentiated and cultured as described previously. The protocols of the study were approved by IACUC and Ethics committee of Nanjing Medical University.

Forced expression or inhibition of miR-107
The lentiviral GV-369 vectors, encoding the pre-miR-107 sequence or the pre-miR-107 anti-sense sequence, were designed, synthesized and sequence-verified by Shanghai Genepharm Co. The construct and the lentivirus-packing plasmids (psPAX2 and pMD2.G) were co-transfected to HEK-293T cells, establishing pre-miR-107 expression lentivirus (LV-pre-miR-107) or the pre-miR-107 anti-sense lentivirus (antagomiR-107). The viruses were enriched, filtered, and added to cultured OB-6 cells or primary human osteoblasts (cultured in complete medium with polybrene) for 48h. When necessary puromycin was added in the complete medium for 6 days to select stable cells.

qPCR
Total cellular RNA was extracted by TRIzol reagents. Detailed protocols of quantitative Real-time PCR ("qPCR") were described previously [21,43]. Quantization of targeted mRNAs was through the 2 ΔΔCt method (relative to GAPDH). miR-107 expression was analyzed by the TaqMan microRNA assay (Applied Biosystems, Shanghai, China), from 10ng of total RNA of each sample. Primers for the Nrf2 pathway genes and GAPDH were provided by Dr. Jiang at Nanjing Medical University [44,45]. All other primers were listed in Table 1.

Cell functional assays
After the applied DEX treatment, cell viability assaying through Cell Counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) assay kit, cell death detection via the medium lactate dehydrogenase (LDH) release procedure, and cell apoptosis studies by TUNEL staining and caspase-3 activity assays were described in detail in our previous studies [3][4][5].

miR mimic transfection
OB-6 osteoblastic cells were seeded into the six-well plates, transfected with 500 nM of the applied miR-107 mimic (wild-type and mutants) through Lipofectamine 2000 for 48h.

AMPKα1 KO
The CRISPR/Cas9 AMPKα1-KO construct (from Dr. Pan at Shanghai Jiao Tong University [32]) was transfected to OB-6 cells via Lipofectamine 2000, with stable cells selected by puromycin. AMPKα1 KO in stable cells was confirmed by Western blotting.

AMPK activity assay
From each treatment 150 μg of total cellular lysates were incubated with anti-AMPKα1 antibody (Santa Cruz Biotech, Shanghai, China). The AMPK activity was tested in the kinase assay buffer by adding AMP-[γ-32 P] ATP mixture and AMPK substrate SAMS (HMRSAMSGLHLVKRR) peptide. Phosphocellulose paper was added to stop the reactions. The AMPK radioactivity was examined by a scintillation counter.

Superoxide detection
Using a previously-described protocol [35,36] OB-6 cells or human osteoblasts were seeded into the 96-well tissue-culturing plates, treated with DEX, and tested by a superoxide colorimetric assay kit (BioVision, Shanghai, China), with the superoxide's absorbance tested at the 450 nm [44].

Lipid peroxidation
Using a previously-described protocol [44] OB-6 cells or human osteoblasts were seeded into six-well plates, treated with DEX, and assayed by a lipid peroxidation kit (Abcam, Shanghai, China) through the thiobarbituric acid reactive (TBAR) method [44,50].

NQO1 activity
The assay of NQO1 activity, using menadione as the substrate, was performed via a previously-described protocol [51]. NQO1 activity in stimulated osteoblasts was normalized to that of untreated control cells.

Nrf2 knockout
From Dr. Xu at Central South University [52] the lenti-CRISPR-GFP-Nrf2 knockout (KO) construct was obtained and transfected to OB-6 cells by Lipofectamine 2000. GFP-positive OB-6 cells were sorted by FACS, and monoclonal single stable cells achieved. Nrf2 KO was screened by qPCR and Western blotting assays.

Mitochondrial depolarization
JC-1, a fluorescence dye, will aggregate in the mitochondria in stressed cells with mitochondrial depolarization, forming green monomers [53]. OB-6 cells or human osteoblasts were initially seeded into the 24-well plates, treated with DEX, and stained with JC-1 (5.0 μg/mL, Sigma). Cells were then tested via a fluorescence spectrofluorometer (Hitachi, Japan) at wavelength of 545 nm (green).

Statistical analysis
The investigators were blinded to the group allocation. Experiments were repeated three times. Data were expressed as mean ± standard deviation (SD). Statistics AGING were analyzed by one-way ANOVA through the Scheffe's f-test. When compare significance between two groups, the two-tailed unpaired T test (Excel 2007) was applied. p values < 0.05 were considered statistically significant.