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Fig. 3 | Journal of Ovarian Research

Fig. 3

From: Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: cellular mechanism involved in polycystic ovaries

Fig. 3

mir-379-5p regulates rat granulosa cell proliferation through PDK1 suppression in vitro. A Binding of mir-379-5p mimic to PDK1 3’UTR mRNA, but not to its mutant (binding site deleted) significantly reduced luciferase activity; B & C mir-379-5p mimic reduced PDK1 protein content and cell proliferation (MCM2 protein content) but, miR-379-5p inhibitor, like DHT, increased these responses in vitro. Granulosa cells were isolated from preantral follicles (Diethylstilbestrol-primed 21-day old rats; subcutaneous injection for 3 consecutive days) and either transfected with (A) the reporter vector containing a complementary mir-379-5p sequence in the 3ʹ-UTR of the PDK1 (or its mutant as control) or (B & C) with mir-379-5p mimic (mirVana Mimics, MC10316, Thermo-Fisher) and inhibitor (mirVana inhibitor, MH10316) for 24 h before culture without or with DHT (1 µM; 24 h and 36 h). Protein extraction and Western blotting were performed as described previously [15]. Signal intensities generated on the film were measured densitometrically using Image J and normalized over that of GAPDH or β-Actin. Results are expressed as means ± SEM (n = 3 replicates, each from 2 rats). Data were analyzed by t-test: **P < 0.001 (A); one-way ANOVA & tukey post hoc (B); three-way ANOVA and tukey post hoc (C). *P < 0.05; **P < 0.01, ***P < 0.001

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