Fig 1: The SNV g.-398C > T affects miR-23a promoter activity by recruiting the transcription factor SMAD4. (A) A point variant C > T was detected at -398 nt in the miR-23a promoter. (B) Identification of the core promoter region of miR-23a. The image contains a schematic representation of the four deletion constructions (left) and their luciferase activity (right). The first base of pre-miR-23a was taken as +1. (C) Schematic showing the constructions of pGL3-C and pGL3-T. (D) Reporter activity assays. pGL3-C and pGL3-T were transfected into pGCs; luciferase activity was detected. (E) miR-23a levels were determined in pGCs with different genotypes for the SNV g.-398C > T. GCs were isolated from the ovaries of sows with genotype CC (n = 3) and genotype TT (n = 3). (F) Prediction of binding sites for transcription factors in the miR-23a promoter with allele C and allele T by JASPAR. (G) Effects of transcription factors SMAD4 and NR2C1 on the activity of the miR-23a promoter with allele C and allele T. (H) ChIP assay. The image contains a schematic diagram of the porcine miR-23a (top) and ChIP images (bottom). F/R are the primers used to amplify a DNA fragment containing the SMAD4 binding element (SBE) motif. Data are expressed as the mean ± S.E.M., and significance was tested using a t-test (B,E) and a one-way ANOVA (D,G) (ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001).