Fig. 3: NORFA acts as a ceRNA and sponges miR-126 in porcine granulosa cells.

a Schematic showing the locations of NORFA (red) and its nearby coding genes on porcine chromosome 1. b, c The expression levels of NORFA nearby coding genes in porcine granulosa cells were detected by qRT-PCR after transfection with pcDNA3.1-NORFA (b) or NORFA-siRNA (c). d, e The expression levels of miR-126 in porcine granulosa cells were measured after pcDNA3.1-NORFA (d) or NORFA-siRNA (e) treatment. Level of miR-126 was detected by stem-loop qRT-PCR. f Subcellular localization of NORFA in porcine granulosa cells. Expression levels of NORFA and marker gene (GAPDH and RPLP0 for cytoplasm and U6 for nuclear) in isolated nuclear and cytoplasm fraction from porcine granulosa cells were detected by qRT-PCR. g Subcellular localization of NORFA in porcine granulosa cells was detected by FISH (Scale bars, 50 µm). Nucleus was dyed with DAPI (blue) and NORFA was dyed with NORFA-specific probe (green). h Schematic showing an miRNA response element (MRE) of miR-126 in exon 2 (E2) of porcine NORFA. i Schematic showing the interactions of miR-126 with wild-type NORFA (red) and the mutant version (green). j Porcine granulosa cells were co-transfected with dual-luciferase reporter vector containing the wild-type NORFA or the mutant version and miR-126 mimics, and luciferase activity was detected. k, l qRT-PCR analysis showing the expression levels of NORFA in porcine granulosa cells when miR-126 was overexpressed (k) or silenced (l). m Porcine granulosa cells lysates were incubated with biotin-labeled wild-type or mutated NORFA. After pull-down, miRNAs were quantified by qRT-PCR. miR-34c and miR-143 were used as negative controls. Experiments were conducted in triplicate. Data in b–e and j–m are shown as mean ± S.E.M. with three independent experiments. P values were calculated by a two-tailed Student’s t test. **P < 0.01.