Fig. 2: Nrf2 cascade activation by miR-4523 is due to PGK1 silencing in human osteoblasts.

The sequences of the wild-type (WT) and mutant miR-4523 mimics were listed (A). RNA pull-down assay results confirmed the direct binding between biotinylated-WT-miR-4523 (but not the mutants) and PGK1 mRNA in primary human osteoblasts (B). The primary human osteoblasts were transfected with applied WT or mutant miR-4523 mimics (500 nM for 24 h, two rounds), relative PGK1 3′-UTR luciferase activity (C), expressions of listed mRNAs (D and G) and proteins (E) as well as the relative ARE activity (F) were tested. Stable human osteoblasts expressing the lentiviral construct encoding the antisense sequence of miR-4523 (lv-antagomiR-4523) or the control construct (lv-antaC) were established and cultured. Expression of miR-4523 was tested (H). The PGK1 3′-UTR luciferase reporter activity (I) as well as its mRNA and protein expression (J) were tested, with the relative ARE activity (K) examined as well. Primary human osteoblasts (L–N) or hFOB1.19 osteoblastic cells (O–Q), expressing the lentiviral construct encoding the pre-miR-4523 (“lv-pre-miR-4523”), were further transduced with or without 3′-UTR-depleted PGK1 construct (“+UTR-null PGK1”). Control cells were transduced with the control miR construct (lv-miRC). Expression of listed proteins was tested by western blotting assays (L and O). The ARE activities, HO1 mRNA levels (M and P), and miR-4523 expression (N and Q) were also tested. Data were presented as mean ± standard deviation (SD, n = 5). “miRC” stands for non-sense control miRNA mimic (B–G) *P < 0.05 versus “miRC” cells (B–G). *P < 0.05 versus “lv-antaC” cells (H–K). *P < 0.05 (M and P) “n.s.” stands for non-statistical difference (N and Q). The experiments were repeated five times with similar results obtained.