Fig. 1: miR-4523 silences PGK1 and activates Nrf2 signaling cascade in human osteoblasts.

miR-4523 putatively targets PGK1 mRNA 3′-UTR (at position 898–905) (A). RNA immunoprecipitation (RNA-IP) demonstrated that the Ago2 protein immunoprecipitated with endogenous PGK1 mRNA and miR-4523 (but not miR-4533, a negative control) in human osteoblasts (B). RNA pull-down assay results confirmed a direct binding between biotinylated-miR-4523 and PGK1 mRNA in primary human osteoblasts (C). RNA fluorescence in situ hybridization (FISH) confirmed miR-4523 (in green) and PGK1 mRNA (in red) colocalization in primary human osteoblasts (D). Primary human osteoblasts were transduced with a lentiviral construct encoding the pre-miR-4523 (“lv-pre-miR-4523”) or control miR construct (lv-miRC), and stable cells established after puromycin selection; Expression of mature miR-4523 (E), listed mRNAs (G and M) and proteins (in total cell lysates and nuclear lysates, H, J, K, N) were tested by qRT-PCR and western blotting assays. The relative PGK1 3′-UTR luciferase reporter activity (F) and the relative ARE activity (L) were tested as well; Keap1–Nrf2 association was tested by co-immunoprecipitation (Co-IP) assays (I). Ago2 RNA-IP (O) and RNA-FISH (P) confirmed the direct binding between miR-4523 and PGK1 mRNA in hFOB1.19 osteoblastic cells. Stable hFOB1.19 cells expressing lv-pre-miR-4523 or lv-miRC were established; Expression of mature miR-4523 (Q), listed mRNAs (S and U) and proteins (in total cell lysates, T) were tested by qRT-PCR and western blotting assays; The relative PGK1 3′-UTR luciferase reporter activity was tested as well (R). Data were presented as mean ± standard deviation (SD, n = 5). *P < 0.05 versus “lv-miRC” group. The experiments were repeated five times with similar results obtained. Scale bar = 100 μm (D and P).