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Published on: May 4, 2018
DCAF1-targeting microRNA-3175 activates Nrf2 signaling and inhibits dexamethasone-induced oxidative injury in human
Jing Chen1, Jin-Qian Liang2, Yun-Fang Zhen3
1Department of Endocrinology, Hunan Provincial People's Hospital, The First Affiliated Hospital of Hunan Normal University, Changsha, China.
Abstract:
Activation of nuclear-factor-E2-related factor 2 (Nrf2) signaling can protect human osteoblasts from dexamethasone-induced oxidative injury. DDB1 and CUL4 associated factor 1 (DCAF1) is a novel ubiquitin E3 ligase for Nrf2 protein degradation. We identified a novel DCAF1-targeting miRNA, miR-3175. RNA pull-down, Argonaute 2 RNA-immunoprecipitation, and RNA fluorescent in situ hybridization results confirmed a direct binding between miR-3175 and DCAF1 mRNA in primary human osteoblasts. DCAF1 3'-untranslated region luciferase activity and its expression were significantly decreased after miR-3175 overexpression but were augmented with miR-3175 inhibition in human osteoblasts and hFOB1.19 osteoblastic cells. miR-3175 overexpression activated Nrf2 signaling, causing Nrf2 protein stabilization, antioxidant response (ARE) activity increase, and transcription activation of Nrf2-dependent genes in human osteoblasts and hFOB1.19 cells. Furthermore, dexamethasone-induced oxidative injury and apoptosis were largely attenuated by miR-3175 overexpression in human osteoblasts and hFOB1.19 cells. Importantly, shRNA-induced silencing or CRISPR/Cas9-mediated Nrf2 knockout abolished miR-3175 overexpression-induced osteoblast cytoprotection against dexamethasone. Conversely, DFAC1 knockout, by the CRISPR/Cas9 method, activated the Nrf2 cascade and inhibited dexamethasone-induced cytotoxicity in hFOB1.19 cells. Importantly, miR-3175 expression was decreased in necrotic femoral head tissues of dexamethasone-taking patients, where DCAF1 mRNA was upregulated. Together, silencing DCAF1 by miR-3175 activated Nrf2 signaling to inhibit dexamethasone-induced oxidative injury and apoptosis in human osteoblasts.
Insights
MicroRNA-3175 protects human osteoblasts from dexamethasone damage by targeting DCAF1, stabilizing Nrf2, and reducing oxidative stress and apoptosis. This finding offers a potential therapeutic strategy for steroid-induced bone injury.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Nuclear-factor-E2-related factor 2 (Nrf2) signaling protects osteoblasts from oxidative stress.
- DDB1 and CUL4 associated factor 1 (DCAF1) is a novel E3 ligase that degrades Nrf2.
- Dexamethasone can induce oxidative injury in human osteoblasts.
Purpose of the Study:
- To investigate the role of a novel microRNA, miR-3175, in regulating Nrf2 signaling and protecting osteoblasts.
- To explore the therapeutic potential of modulating miR-3175 and DCAF1 in dexamethasone-induced osteoblast injury.
Main Methods:
- RNA pull-down assays, Argonaute 2 RNA-immunoprecipitation, and in situ hybridization to confirm miR-3175 and DCAF1 mRNA interaction.
- Luciferase reporter assays and gene expression analysis to assess the effect of miR-3175 on DCAF1.
- Overexpression and knockout studies (shRNA, CRISPR/Cas9) in human osteoblasts and hFOB1.19 cells to evaluate Nrf2 activation and cytoprotection.
- Analysis of patient tissues to correlate miR-3175 and DCAF1 levels with dexamethasone-induced necrosis.
Main Results:
- miR-3175 directly binds to DCAF1 mRNA in human osteoblasts, leading to decreased DCAF1 expression.
- Overexpression of miR-3175 stabilizes Nrf2, enhances antioxidant response element (ARE) activity, and upregulates Nrf2-dependent genes.
- miR-3175 overexpression significantly attenuates dexamethasone-induced oxidative injury and apoptosis in osteoblasts.
- Nrf2 knockout abolishes miR-3175's protective effects, while DCAF1 knockout mimics these effects by activating Nrf2.
- Decreased miR-3175 and increased DCAF1 mRNA were observed in necrotic femoral head tissues from patients treated with dexamethasone.
Conclusions:
- Silencing DCAF1 via miR-3175 activates Nrf2 signaling, providing cytoprotection against dexamethasone-induced oxidative injury and apoptosis in human osteoblasts.
- miR-3175 represents a potential therapeutic target for mitigating steroid-induced osteoblast toxicity.
