Fig. 5

Preliminary identification of target genes of miR-124-3p in the endometrium and embryo. a Gene ontology (GO) annotation was performed for the target genes of miR-124-3p, and the terms according to the protein function are plotted. b KEGG pathway analysis of target genes of miR-124-3p. c Complementarity between target genes and seed sequence of miR-124-3p. d, e Western blot analyses show increased levels of LIF, MUC1, and BCL2 proteins in Ishikawa cells transfected with miR-124-3p inhibitor. Data represent the mean ± SD of three independent experiments. Different superscripts indicate significance at p < 0.05 (one-way ANOVA with Duncan’s test). f, g Immunofluorescence staining was performed to compare the expression levels of Lif, Muc1, and Bcl2 in the uteri from the control and miR-124-3p-AAV groups on day 5 of pregnancy. Scale bar: 500 μm. Histogram shows the ratio of integrated density level to the area. Data represent the mean ± SD in replicates. *p < 0.05; **p < 0.01 (Student’s t-test). h, i Dnmt1 localization in control and miR-124-3p-AAV-treated blastocytes following 2-cells formation for 60 h. Dnmt1 signals (red) localize to both TE and ICM cells. Severe inhibition of Dnmt1 was characterized by a lack of signal in the miR-124-3p-AAV group. Scale bar: 20 µm. **p < 0.01 (Student’s t-test). j, k Western blotting analysis of Dnmt1expression in blastocytes after control and miR-124-3p-AAV transduction. Actin was used as an internal control. Data represent the mean ± SD in replicates. p < 0.05 (one-way ANOVA with Duncan’s test)