Phosphoglycerate kinase 1 silencing by a novel microRNA microRNA-4523 protects human osteoblasts from dexamethasone through activation of Nrf2 signaling cascade.

ABSTRACT

Nuclear-factor-E2-related factor 2 (Nrf2) cascade activation can ameliorate dexamethasone (DEX)-induced oxidative injury and death in human osteoblasts. Phosphoglycerate kinase 1 (PGK1) depletion is shown to efficiently activate Nrf2 signaling by inducing methylglyoxal modification of Kelch-like ECH-associated protein 1 (Keap1). We here identified a novel PGK1-targeting microRNA microRNA-4523 (miR-4523). RNA fluorescent in situ hybridization, RNA pull-down, and Argonaute-2 RNA immunoprecipitation results confirmed a direct binding between miR-4523 and PGK1 mRNA in primary human osteoblasts and hFOB1.19 osteoblastic cells. Forced overexpression of miR-4523, using a lentiviral construct, robustly decreased PGK1 3'-UTR (untranslated region) luciferase activity and downregulated its expression in human osteoblasts and hFOB1.19 cells. Furthermore, miR-4523 overexpression activated the Nrf2 signaling cascade, causing Keap1-Nrf2 disassociation, Nrf2 protein stabilization, and its nuclear translocation as well as transcription activation of Nrf2-dependent genes (NQO1, GCLC, and HO1) in human osteoblasts. By expressing a UTR-null PGK1 construct, miR-4523 overexpression-induced Nrf2 cascade activation was however largely inhibited. Importantly, DEX-induced reactive oxygen species production, oxidative injury, and cell apoptosis were significantly attenuated by miR-4523 overexpression in human osteoblasts and hFOB1.19 cells. Such actions by miR-4523 were abolished by Nrf2 shRNA or knockout, but mimicked by PGK1 knockout (using CRISPR/Cas9 method). In PGK1 knockout human osteoblasts, miR-4523 overexpression failed to further increase Nrf2 cascade activation and offer osteoblast cytoprotection against DEX. Significantly, miR-4523 is downregulated in human necrotic femoral head tissues of DEX-taking patients. Together, PGK1 silencing by miR-4523 protected human osteoblasts from DEX through activation of the Nrf2 signaling cascade.