MicroRNA-200a activates Nrf2 signaling to protect osteoblasts from dexamethasone

Treatment with dexamethasone in human osteoblasts leads to oxidative stress and cell injures. NF-E2-related factor 2 (Nrf2) is a key anti-oxidant signaling. We want to induce Nrf2 activation via microRNA-mediated silencing its suppressor Keap1. Our results show that microRNA-200a (“miR-200a”) expression depleted Keap1, causing Nrf2 protein stabilization in OB-6 osteoblastic cells. Reversely, the miR-200a anti-sense led to Keap1 upregulation and Nrf2 degradation. miR-200a expression activated Nrf2 signaling, which inhibited dexamethasone-induced reactive oxygen species production and OB-6 cell death/apoptosis. Keap1 shRNA also activated Nrf2 and protected OB-6 cells from dexamethasone. Importantly, miR-200a was in-effective in Keap1-silenced (by shRNA) OB-6 cells. In the primary human osteoblasts, Keap1 silence by targeted-shRNA or miR-200a protected cells from dexamethasone. Significantly, miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. Together, miR-200a expression activates Nrf2 signaling and protects human osteoblasts from dexamethasone.

If Keap1 is the primary target of miR-200a, then miR-200a should be ineffective in Keap1-silenced cells. To test this hypothesis, miR-200a vector was introduced to Keap1 shRNA ("Sequ1")-expressing OB-6 cells ( Figure 4F). MiR-200a (-3p) level was again increased after expressing the construct in Keap1-silenced cells ( Figure 4F). Remarkably, miR-200a expression was unable to further protect OB-6 cells from Dex, when Keap1 was already silenced by targeted-shRNA ("Sequ1", Figure 4G and 4H). Thus, miR-200a expression was very much invalid against Dex when Keap1 was already silenced ( Figure 4G and 4H). These results suggest that Keap1 should be the primary target of miR-200a in mediating its pro-survival function in OB-6 cells.

miR-200a downregulation and Keap1 mRNA upregulation in human necrotic femoral head tissues
At last, we tested expression of miR-200a in human necrotic femoral head tissues. A total of 10 different necrotic femoral head tissues ("Necrosis") from Dex-taking patients were tested. As compared to the surrounding normal femoral head tissues ("Normal"), expression of miR-200a (-3p) was significantly decreased in the "Necrosis" tissues ( Figure 6A). Reversely, Keap1 mRNA was increased in the "Necrosis" tissues ( Figure 6B). The miR-200a downregulation in human necrotic femoral head tissues indicates a potential function of this miRNA in the pathogenesis of femoral head necrosis in the Dex-taking patients.

DISCUSSIONS
Recent studies have suggested that Dex-induced ROS production and subsequent oxidative stress are the major reason of osteoblast injuries [7,9,11,13,34]. On the other hand, inhibition of ROS could efficiently protect osteoblasts from Dex [7,9,11,13,34]. For
Very recent studies have identified potential miRNAs that could possibly activate Nrf2 via depleting Keap1. For example, Yang et al., showed that miR-200a activates Nrf2 by targeting Keap1 in hepatic stellate cells [38]. Shi et al., showed that miR-141 activates Nrf2dependent antioxidant pathway also via depleting Keap1 [39]. Similarly, a very recent study by Gong et al., showed that miRNA-141 attenuated UV-induced oxidative stress by activating Keap1-Nrf2 signaling [40]. Therefore, miRmediated silence of Keap1 is effective in activating Nrf2 signaling.
In the current study, we demonstrate that miR-200a activated Nrf2 signaling in OB-6 cells and primary human osteoblasts. First, forced-expression of miR-200a induced Keap1 degradation, causing Nrf2 stabilization in human osteoblasts/osteoblastic cells. Reversely, miR-200a depletion by the antamiR-200a led to Keap1 upregulation and Nrf2 degradation. Second, stabilized Nrf2 induced transcription of several Nrf2-regulated genes (HO1, NOQ1 and GCLC) in miR-200a-expressing cells. Significantly, expression of miR-200a, by inducing Nrf2 activation, largely inhibited Dex-induced oxidative stress and subsequent osteoblasts/osteoblastic cell death and apoptosis. Third, Keap1 shRNA, mimicking miR-200a, activated Nrf2 and protected OB-6 cells from Dex. More importantly, miR-200a was unable to further protect OB-6 cells from Dex when Keap1 was silenced by shRNA. We therefore conclude that Keap1 is the primary target of miR-200a in osteoblasts in mediating its anti-Dex functions. Significantly, we found that miR-200a level was decreased in necrotic femoral head tissues, which was correlated with Keap1 mRNA upregulation. It will be interesting to further test the potential function of miR-200a on Dex-damaged human osteoblasts in vivo.

Chemicals and reagents
Dex and puromycin were purchased from Sigma Aldrich (Nantong, China). Cell culture reagents were from Gibco (Nantong, China). Antibodies of this study were purchased from Cell Signaling Technology (Nanjing, China).

Culture of human osteoblastic cells
The OB-6 human osteoblastic cells [4], obtained from the Cell Bank of Shanghai Institute of Biological Science (Shanghai, China), were described as described previously [10].

Culture of primary human osteoblasts
The primary human osteoblasts were achieved from the redundant trabecular bone fragments of healthy donors. The trabecular bone fragments were minced into small pieces and washed. The bone pieces were then digested with 2 mg/mL collagenase type II (300 U/mg; Sigma) for 2 hours, which were placed in culture flasks with the described medium [41]. Medium was changed twice a week until cells reached confluence. Writteninformed consent was obtained from each participant. The protocols were approved by Ethics Review Board of Nanjing Medical University.

Human femoral head tissues
A total of 10 Dex-taking patients undergoing femoral head surgery were included. The necrotic femoral head tissues along with paired surrounding normal femoral head tissues were obtained. Tissues were subjected to tissues lysis buffer (Biyuntian, Wuxi, China) incubation. Samples were stored in liquid nitrogen for further analysis. Written-informed consent was obtained from each participant. The protocols were approved by Ethics Review Board of Nanjing Medical University.

Western blot assay
The detailed protocol of Western blotting assay was descibred previously, and blot results were quantified via ImageJ software [7][8][9].

Statistical analysis
All values were expressed as means ± standard deviation (SD). The statistical significance of differences among groups were determined by one-way analysis of variance (ANOVA) followed by the Tukey's post hoc multiple comparison tests. p < 0.05 was considered significant.

CONCLUSIONS
MiR-200a expression activates Nrf2 signaling and protects human osteoblasts/osteoblastic cells from Dex. Keap1 is the primary target of miR-200a in mediating its actions.

Author contributions
All authors carried out the experiments, participated in the design of the study and performed the statistical analysis, participated in its design and coordination and helped to draft the manuscript.

ACKNOWLEDGMENTS
This work is supported by the National Natural Science Foundation (81672170).

CONFLICTS OF INTEREST
The listed authors have no conflicts of interest.