Figure 5.

miR-30c-2-3p overexpression decreases MR expression in renal KC3AC1 cells and compromises MR-mediated target gene expression. Renal KC3AC1 cells were transfected with 10 nM Mimics or 10 nM negative Control Mimics (CTR mimic). (A) Specific overexpression of miR-30c-2-3p in renal KC3AC1 cells was confirmed by RT-qPCR. (B) Quantitative RT-qPCR of endogenous Nr3c2 (MR) expression, analyzed 18 h after transfection. Data are means ± SEM from three independent experiments performed in 4–6 replicates (n = 12–18). NS = not significant, **** p < 0.0001 compared to CTR Mimics, arbitrarily set at 100%. (C) Western blot analysis of MR expression, 48 h following transfection of CTR Mimics or 30c-2-3p Mimics (left panel) and quantification of the corresponding signals (right panel) in which MR expression with CTR Mimics was arbitrarily set at 100%. Data are means ± SEMs (n = 6). (D,E) Renal KC3AC1 cells were transfected with 10 nM Inhibitors or negative Control Inhibitors (CTR inhibitor). (D) Specific overexpression of miR-30c-2-3p in renal KC3AC1 cells was confirmed by RT-qPCR. (E) Quantitative RT-qPCR of endogenous Nr3c2 (MR) expression, analyzed 18 h after transfection. Data are means ± SEMs (n = 6). NS = not significant, *** p < 0.001, **** p < 0.0001 compared to CTR inhibitor, arbitrarily set at 100%. (F,G) Overexpression of miR-30c-2-3p prevented aldosterone-induced expression of Tsc22d3 (Gilz) (F) or of Sgk1 (G) in renal KC3AC1 cells. KC3AC1 cells were deprived for 24 h in minimal medium then cells were transfected with 10 nM CTR Mimics or 10 nM 30c-2-3p Mimics. Eighteen hours later, cells were stimulated with 10 nM Aldosterone for 1 h then Tsc22d3 (Gilz) or Sgk1 expression was measured by RT-qPCR. Data are means ± SEM from three independent experiments performed in six replicates (n = 18). NS = not significant, ** p < 0.01, **** p < 0.0001 compared to condition without aldosterone stimulation. ^§§§§ p < 0.0001 between the 2 conditions indicated by line.