Fig. 4: AQP1 and ANKH are direct targets of miR-328-3p.

a IHC staining was conducted to evaluate the expression levels of AQP1 and ANKH in normal and osteoarthritic cartilage samples (scale bar = 200 μm). The quantification of cells positive for AQP1 and ANKH staining is shown in the right panel. b The expression levels of AQP1 and ANKH in normal and osteoarthritic cartilage were evaluated by qRT-PCR (n = 39). c Left: A Venn diagram showing that AQP1 and ANKH are potential targets of miR-328-3p based on three different target prediction algorithms (miRmap, TargetScan, and miRanda). Right: The putative miR-328-3p binding sites in the 3′UTRs of AQP1 and ANKH and the designed WT/MUT sequences (top); luciferase reporter assay in HEK293T cells transfected with pGL3-AQP1-3′UTR (WT or MUT), pGL3-ANKH-3′UTR (WT or MUT), and the miR-328-3p mimic or its control (bottom). d, e qRT-PCR (d) and western blot analysis (e) were performed to evaluate the mRNA and protein levels of AQP1 and ANKH in chondrocytes after transfection with the miR-328-3p mimic, the miR-328-3p inhibitor, or their respective controls. f Associations between miR-328-3p and AQP1 or ANKH expression in osteoarthritic cartilage were assessed by Spearman correlation analysis. OA osteoarthritis, WT wild-type, MUT mutant, ns no significant difference. The data are presented as the means ± SDs. Statistical differences were determined using unpaired two-tailed Student’s t test (a–d) or Spearman correlation analysis (f). ^**P < 0.01; ^***P < 0.001.