MicroRNA-451 plays a role in murine embryo implantation through targeting Ankrd46, as implicated by a microarray-based analysis  Zhengyu Li, M.D., Jia Jia, M.B.B.S., Jinhai Gou, M.B.B.S., Xia Zhao, M.D., Tao Yi, Ph.D.  Fertility and Sterility  Volume 103, Issue 3, Pages 834-844.e4 (March 2015) DOI: 10.1016/j.fertnstert.2014.11.024 Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 1 (A) Comparison of the expression of miR-1839-5p, miR-451, and miR-676 between implantation sites and interimplantation sites, quantified by qRT-PCR; (B) Expression profile of miR-1839-5p, miR-451, and miR-676 in models of pseudopregnancy, delayed implantation, and artificial decidualization, quantified by qRT-PCR. Data were recorded as mean ± SD, and represent 3 independent experiments, each in triplicate. **P<.01, t test. Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 2 Prediction and confirmation of target genes of miRNAs. (A) HEK293T cells were transfected with miRNA mimics, inhibitors, and the corresponding control (NC), and the up-regulation and down-regulation of miR-451 and miR-676 expression were quantified by qRT-PCR. Cotransfection with miRNA mimics, inhibitors, or NC and the luciferase reporter vectors containing wide-type putative binding sites in 3′UTR region (wt-Ankrd46, wt-Ankrd13a, and wt-Kif3b) for luciferase assay; (B) A schematic representation of the structure of the luciferase reporter vector. The 3′UTR fragment of putative target gene (Ankrd46, Ankrd13a, and Kif3b) containing the binding sites of miRNA (miR-451 and miR-676) was cloned into the downstream of firefly luciferase reporter gene in psiCHECK-2 vector (Promega); (C) HEK293T cells were cotransfected with miR-451 mimic, inhibitor, or NC and wt- or mu-Ankrd46 for luciferase assay. A mutant reporter vector (mu-Ankrd46) was generated by mutation of the putative binding site in the 3′UTR region to remove all complementarities to miR-451. The mutant sequence of binding sites is underlined and italicized; (D) Expression of Ankrd46 mRNA and protein in HEK293T cells transfected with miR-451 mimic, inhibitor, or NC by qRT-PCR or western blotting. The value of the Blank group in qRT-PCR and luciferase assay was arbitrarily set at 100%, and all data in other groups were expressed as ratios to the Blank group. Data were recorded as mean ± SD, and represent 3 independent experiments, each in triplicate. Comparisons between 2 groups were performed by unpaired t test; comparisons among multiple groups were performed by 1-way ANOVA. *P<.05; **P<.01. Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Figure 3 In vivo effect of miR-451 loss-of-function by miRNA sponge vector. Pregnant mice were injected with LV-miR-451 sponge, miR-451 inhibitor, or the corresponding control (NC). (A) Comparison of the number of implantation sites between injected cornu (left cornu) and control (right cornu). Data were recorded as mean ± SD, and represent 5 mice in 1 group. To verify the efficacy of miR-451 loss-of-function, the expression of miR-451 and Ankrd46 mRNA in injected cornu and control was quantified by qRT-PCR. The value in the Blank group was arbitrarily set at 100%, and all data in other groups were expressed as ratios to the Blank group. Data were recorded as mean ± SD, and represent 3 independent experiments, each in triplicate. (B) A schematic representation of the Lentiviral miR-451 sponge vector (LV-miR-451 sponge). Four pairs of binding sites complementary to the miR-451 seeding sequence were inserted into the downstream of the H1 promoter and the green fluorescent protein (GFP) opening reading frame (ORF) of the Lentiviral vector, with a bulge to prevent RNA interference-type cleavage and degradation of the sponge RNA. (C) Expression of Ankrd46 protein in injected cornu and control was examined by western blotting. The bands are representative of 3 independent experiments. Comparisons between 2 groups were performed by unpaired t test; comparisons among multiple groups were performed by 1-way ANOVA. *P<.05; **P<.01. Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 1 (A) Comparison of the distributions of expression values for the 6 samples (3 in the D1 group and 3 in the D5 group) after normalization, in a box-plot view. (B) Assessment of the variation between chips in a scatter-plot view, a tool for comparison of the expression profile between 2 groups. In the scatter plot, the expression values of each group are along the 2 axes, allowing us to see any trends between the 2 groups. D = day (eg, D5-2 = day 5, sample 2). Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 2 The heat map of the normalized intensity of the 11 selected miRNAs in the microarray analysis (3 samples in the D1 group, and 3 samples in the D5 group). D = day (eg, D5-2 = day 5, sample 2). Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 3 Expression profile during peri-implantation period of 6 validated miRNAs in (A) luminal epithelium, and (B) whole endometrium, quantified by qRT-PCR. Data were recorded as mean ± SD, and represent 3 independent experiments, each in triplicate. Comparisons among multiple groups were performed by 1-way ANOVA. *P<.05; **P<.01. Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions

Supplemental Figure 4 (A) Expressions of miR-451 in NIH3T3 cells transfected with miRNA mimics, inhibitors, and the corresponding control (NC) were quantified by qRT-PCR; (B) Expressions of Ankrd46 mRNA in NIH3T3 cells transfected with miR-451 mimic, inhibitor, or NC were quantified by qRT-PCR; (C) NIH3T3 cells were cotransfected with miR-451 mimic, inhibitor, or NC and wt- or mu-Ankrd46 for luciferase assay. A mutant reporter vector (mu-Ankrd46) was generated by mutation of the putative binding site in the 3′UTR region to remove all complementarities to miR-451. The value of the Blank group in qRT-PCR and luciferase assay was arbitrarily set at 100%, and all data in other groups were expressed as ratios to the Blank group. Data were recorded as mean ± SD, and represent 3 independent experiments, each in triplicate. Comparisons between 2 groups were performed by unpaired t test; comparisons among multiple groups were performed by 1-way ANOVA. *P<.05; **P<.01. Fertility and Sterility 2015 103, 834-844.e4DOI: (10.1016/j.fertnstert.2014.11.024) Copyright © 2015 American Society for Reproductive Medicine Terms and Conditions