Fig. 2: MiR-132 expression is salt concentration dependent and directly targets Cox-2 in macula densa cells.

a Schematic representation of miR-132 binding site in 3′UTR of Cox-2. b Luciferase assay confirms functional, 3′UTR-dependent repression of Cox-2 expression by miR-132. pCOX is Cox2 3′UTR reporter construct, pMIR is empty control reporter construct, pCOXmut is Cox2 3′UTR reporter construct with a single point mutation in the miR-132 binding site, n = 2–3 (independent experiments). c–f Representative western blot for COX-2 in NIH3T3 (c) and mpkccd (e) cells, with β-actin as housekeeping protein (separately loaded), and corresponding quantification (d, f) shows increased COX-2 expression following miR-132 silencing. n = 3 (independent experiments), numbers adjacent to blots indicate kD. Relative expression is normalized to β-actin and sc set a 1. g–j Using a macula densa cell line (MMDD-1 cells) we confirmed that miR-132 inhibition (g) and miR-132 overexpression (h) for 24 h resulted in increased (i) and decreased (j) Cox-2 gene expression, respectively, as determined by RT-qPCR. MiR-132 is expressed relative to U6, Cox-2 is expressed relative to Gapdh, n = 5–6 (independent wells). k MiR-132 inhibition increases PGE2 formation after 24 h as compared to scramblemir control treated cells, as determined in the supernatant using ELISA, n = 5–6. l A low or high salt stimulus of these MMDD-1 cells resulted in initial decreased or increased miR-132 levels followed by increased and decreased, respectively, as determined by RT-qPCR, indicating an active regulatory role for miR-132 upon changes in salt concentration. MiR-132 is expressed relative to U6, n = 3 (independent experiments). m Cox-2 levels, as determined by RT-qPCR and expressed relative to Gapdh, increase and decrease upon low and high salt treatment, respectively, n = 5–6. sc scramblemir, a132 antagomir-132, *P < 0.05, **P < 0.01, ***P < 0.001, data are represented as mean ± SD, except for g that is represented as median ± interquartile range (IQ1–IQ3).