Figure 6

Regulation of PKD1/PKD2 expression by lineage-specific miRNAs during nephron progenitor differentiation. (A) Taqman qPCR analysis showing the expression tendency of miR-372-3p during KD (n = 3 independent cultures for each time-point). (B) Correlation plot revealing reverse-correlations between miR-372-3p and PKD1/PKD2, respectively. (C) Luciferase reporter assay results confirming that miR-372-3p could inhibit the 3′UTR of both PKD1 and PKD2 (n = 4 independent cultures for each group). (D,E) Western blot results (upper panel) showing expression of Polycystin 1 and Polycystin 2 in KD 6 cells (D) with transfection of miR-372-3p mimics or non-targeting controls, and expression of Polycystin 1 and Polycystin 2 in KD 14 cells (E) upon transfection of miR-372-3p inhibitors or non-targeting controls. Normalized protein expressions of Polycystin 1 and Polycystin 2 are shown in lower panel (D,E). The expression of NC group of each experiment is set as 1. n = 3 independent cultures for each group. (F) KD 12 cells (left column) and KD 18 cells (right column) were examined by immunofluorescent staining (IFC) analysis. The cells transfected with miR-372-3p mimics (upper row) and scramble controls (lower row) were compared (Scale bars represent 200 μm). Table showing the percentage of HOXD11⁺ cells at KD 12, SIX2⁺ cells and HOXD11⁺ cells at KD 18, with the transfection of miR-372-3p mimics and non-targeting controls (data are presented as the means ± SD. n = 3 independent cultures for each group). In (A), data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 for statistical comparisons between day 0 and other time-points (Paired two-tailed t-test). In (C–E), data are presented as the means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 for statistical comparisons between control groups and experimental groups (Paired two-tailed t-test). OE: overexpression; KnD: Knockdown; KD: nephron progenitor differentiation, NC: non-targeting control.