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

Jin-Qian Liang,Lin Bo,Hai-Ning Tan,Ming-Sheng Tan,Zhen-Tao Zhou,Jian-Hua Hu

doi:10.1038/s41419-021-04250-1

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.

Highlights

For patients with chronic inflammatory and auto-immune diseases, dexamethasone (DEX) and other glucocorticoids are commonly prescribed [1]
Stabilization (Fig. 1T) as well as increased mRNA expression of AREdependent genes (HO1, NAD(P)H quinone oxidoreductase 1 (NQO1), and glutamyl cysteine ligase catalytic subunit (GCLC)) (Fig. 1U) were detected as well in miR-4523-overexpressed hFOB1.19 cells. These results showed that miR-4523 silenced Phosphoglycerate kinase 1 (PGK1) and activated the nuclear-factor-E2-related factor 2 (Nrf2) signaling cascade in human osteoblasts
These results showed that miR-4523 overexpression attenuated DEX-induced oxidative injury in human osteoblasts

Summary

INTRODUCTION

For patients with chronic inflammatory and auto-immune diseases, dexamethasone (DEX) and other glucocorticoids are commonly prescribed [1]. In vitro studies have shown that DEX can induce direct and profound oxidative injury and cytotoxicity to cultured human osteoblasts and osteoblastic cells [2, 4,5,6,7]. Studies have shown that Nrf activation, using genetic strategies or pharmacological methods, can efficiently protect osteoblasts/osteoblastic cells from DEX-induced oxidative injury [6, 7, 9, 12,13,14,15]. Induced silencing or CRISPR/Cas9-induced knockout of PGK1 robustly activated Nrf signaling cascade and potently inhibited DEX-induced oxidative injury in human osteoblasts [9]. We hypothesized that targeting PGK1 by specific miRNAs should Transfection of miR mimic activate Nrf signaling and offer osteoblast cytoprotection against DEX. PGK1 3′-UTR activity Using the previously-described protocols [29], a firefly-luciferase reporter vector pGL4.13 (luc2/SV40) with the PGK1 3′-UTR

MATERIALS AND METHODS

DISCUSSION

Findings

10 REFERENCES