Fig. 3

Clock-controlled miR-449c-5p inhibited ATP2B4 mRNA and protein expression by directly targeting ATP2B4 in uterine tubular gland cells. A The target position of the miR-449c-5p seed sequence on the ATP2B4–3′UTR sequence (red characters) was predicted using TargetScan software. B, C Relative expression of miR-449c-5p and ATP2B4 at the time point of ZT0 (ZT24), ZT4, ZT8, ZT12, ZT16, and ZT20 in the chicken uterus, respectively. D Chicken DF-1 cells were co-transfected with ATP2B4–3′UTR wild or mutant dual-luciferase vector and miR-449c-5p mimic or mimic-NC. Relative luciferase activity was assayed 48 h later. E Immunofluorescence analysis was performed to identify uterine tubular gland cells. F, G qRT-PCR was used to determine miR-449c-5p expression levels after transfection of miR-449c-5p overexpression and miR-449c-5p inhibition plasmid, respectively. H, I mRNA expression of ATP2B4 in chicken uterine tubular gland cells was detected by qRT-PCR after overexpression and inhibition of miR-449c-5p, respectively. J, K Protein expression of ATP2B4 in chicken uterine tubular gland cells was detected by Western blot analysis after a gain or loss of miR-449c-5p. UTR: untranslated region; miR: microRNA; DAPI: 4′, 6-diamidino-2-phenylindole; NC: negative control. Replications = 3. The samples were derived from the same experiment and the gels/blots were processed in parallel. The data are presented as mean ± standard error (SE); *P < 0.05 and **P < 0.01