Fig. 3. miR-29c attenuates FZD4 expression and induced porcine GC apoptosis.

a Potential miRNAs targeting FZD4 were predicted through four different algorithms. b Diagram showing the sequence of putative miR-29c binding site in FZD4 3′UTR and MFE (minimum free energy). Black line indicated miR-29c seed sequence. c Recombinant reporter vectors containing MRE wild-type (WT), mutated FZD4 3′UTR (top), and their luciferase activities in HEK293T cells with or without miR-29c were calculated (bottom). miR-29c levels (d) and FZD4 mRNA levels (e) in porcine GCs treated with miR-29c mimcis (miR-29c^OE) were assessed by qRT-PCR. f Western blot analysis (left) and quantification (right) for FZD4 and β-catenin protein levels in porcine GCs with miR-29c overexpression. GAPDH serves as control. miR-29c levels (g) and FZD4 mRNA levels (h) in porcine GCs treated with miR-29c inhibitor (miR-29-inhi) were assessed by qRT-PCR. i Western blot analysis (left) and quantification (right) for FZD4 and β-catenin protein levels in pGCs transfection with miR-29c knockdown. GAPDH serves as control. FACS analysis (left) and quantification (right) for the apoptosis rate of porcine GCs after miR-29c mimics treatment or with FZD4 overexpression (FZD4^OE) (j), and miR-29c inhibitor addition or with FZD4 knockdown (k). Data in c–k are represented as mean ± S.E.M. (n = 3, each). **p < 0.01, ns indicates no significance versus control or scrambled.