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. 2021 Oct 19;12(11):964. doi: 10.1038/s41419-021-04250-1

Fig. 5. miR-4523-induced osteoblast cytoprotection against DEX requires activation of PGK1-Nrf2 cascade.

Fig. 5

Stable human osteoblasts with Nrf2 shRNA (shNrf2), a CRISPR/Cas9-Nrf2-knockout construct (“koNrf2”), or the CRISPR/Cas9 sgRNA control construct plus scramble control shRNA lentivirus (“shC+Cas9-C”) were established. These osteoblasts were further infected with pre-miR-4523-expressing lentivirus (“lv-pre-miR-4523”), with stable osteoblasts established following puromycin selection. Expression of listed genes was tested by western blotting (A) and qRT-PCR (B) assays, with relative ARE activity examined as well (B). Alternatively, these osteoblasts were transduced with lv-pre-miR-4523 or the control lentiviral miR construct (lv-miRC), and treated with or without DEX (1 μM) for applied time periods. Cell viability (MTT OD, C), apoptosis activation (TUNEL-positive nuclei ratio, D), and miR-4523 expression (E) were examined. Human osteoblasts with lv-miRC, lv-pre-miR-4523, or lv-pre-miR-4523 plus 3′-UTR-depleted PGK1 construct (“+UTR-null PGK1”) were treated with or without DEX (1 μM) for applied time periods. Cell viability (MTT OD, F), apoptosis activation (TUNEL-positive nuclei ratio, G), and miR-4523 expression (H) were examined. Stable human osteoblasts with the CRISPR/Cas9-PGK1-knockout construct (“koPGK1”) were further infected with or without lv-pre-miR-4523, control cells were with the CRISPR/Cas9 sgRNA control construct (“Cas9-C”). Expression of listed genes was tested by western blotting (I) and qRT-PCR (J) assays, with relative ARE activity tested as well (J). Cells were further treated with or without DEX (1 μM) for applied time periods; cell viability (MTT OD, K), apoptosis activation (TUNEL-positive nuclei ratio, L), and miR-4523 expression (M) were examined. Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 versus “Veh” treatment (C–E); #P < 0.05 versus “shC+Cas9-C” cells (B–E). *P < 0.05 (F–H). #P < 0.05 versus “Cas9-C” cells (J–M). “n.s.” stands for non-statistical difference (E, J–M). The experiments were repeated five times with similar results obtained.