A primary STAT3 activation by circulating pro-PAH factors accounts for miR-204 down-regulation in PAH-PASMCs. (A) siSTAT3 increases miR-204 expression in PAH-PASMCs. miR-204 level measured by qRT-PCR in PAH treated with control siRNA (siRNA ctrl) or siSTAT3 as indicated (n = 3). (B) STAT3 activation and miR-204 expression are inversely correlated in PASMCs. Analysis of the correlation between STAT3 activation (measured by the pY705-STAT3/STAT3 ratio monitored by Western blot) and miR-204 expression (measured by qRT-PCR; n = 2 experiments/patient in three PAH and five control patients). (C) Pro-PAH factors decrease miR-204 and TRPM3 expression similarly by a STAT3-dependent mechanism in control PASMCs. miR-204 (top) and TRPM3 (middle) expression were measured by qRT-PCR performed on control cells treated with the pro-PAH factors PDGF, endothelin-1 (ET-1), or angiotensin II (AII) as indicated (n = 3 experiments/patient/in three PAH and five controls). (bottom) Analysis of the similarities between miR-204 and TRPM3 pattern of expression measured by qRT-PCR in control, PAH, and PAH treated with siSTAT3 as indicated. (D) p-STAT3–binding sites are detected downstream of the TRPM3 gene. ChIP-PCR experiments studying STAT3-binding sites upstream (Up1) and downstream (Dw1 and Dw2) on TRPM3 genes. The OR8J1 gene was used as a negative control, whereas the VEGF gene was used as a positive control. Graphs represent means ± SEM (*, P < 0.05; **, P < 0.01; ***, P < 0.001).