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. 2017 May 22;7:2238. doi: 10.1038/s41598-017-02071-x

Figure 1.

Figure 1

miR-125a-3p down-regulates Fyn expression in oocytes by a direct binding to Fyn 3′UTR. (A,B) Inverse correlation between miR-125a-3p and Fyn protein during maturation: RNA (30 oocytes at each experimental group) and protein (75 oocytes at each experimental group) were extracted from GV, GVBD and in-vivo matured MII oocytes. Whole cell lysates were subjected to (A) qPCR analysis for the detection of miR-125a-3p and U6-snRNA, which served as an internal control; or to (B) western blot analysis for detection of Fyn protein expression, with actin serving as an internal control (upper – graph; lower – a representative blot). Results were normalized to the value of GV oocytes. Data were analyzed by one-way ANOVA followed by Tukey post hoc test. Bars are Mean ± SD of four (A) or three (B) independent experiments. a,bp < 0.05; aversus GV oocytes; bversus MII oocytes. (C–E) GV oocytes were microinjected with miR-125a-3p mimic (miR-125a-3p) or scramble-miR (Scramble) as control, and incubated for four hours at 37 °C in the presence of 1 µM milrinone. RNA (30 oocytes at each experimental group) and proteins (75 oocytes at each experimental group) were extracted. Whole cell lysates were subjected to qPCR analysis for detection of (C) miR-125a-3p and U6-snRNA, which served as an internal control, or of (D) Fyn mRNA; or subjected to western blot analysis for detection of (E) Fyn protein expression, with actin serving as an internal control (upper – graph; lower – a representative blot: Scramble-miR – Scr; miR-125a-3p - miR). Results were normalized to the value of scramble-miR. Data were analyzed by one-sample two-tailed student t-test. Bars are Mean ± SD of three independent experiments, *p < 0.05. (F) GV oocytes were microinjected with miR-125a-3p mimic (miR-125a-3p) or scramble-miR (Scramble) combined with a Renilla-luciferase RNA conjugated to either WT Fyn-3′UTR (WT) or to mutated Fyn-3′UTR (MUT). Oocytes were incubated for 4 hours at 37 °C in the presence of milrinone, prior to lysis. The results of each experimental group were normalized to the value of scramble-miR. Data were analyzed by one-way ANOVA followed by Tukey post hoc test. Bars are Mean ± SD of three independent experiments, *p < 0.05. (G) A scheme showing the binding-site of miR-125a-3p within WT Fyn 3′UTR of mouse origin, and the site-directed mutagenesis employed to prevent miR-125a-3p from binding to Fyn 3′UTR.