Bta-miR-10b Secreted by Bovine Embryos Negatively Impacts Preimplantation Embryo Quality

Abstract

In a previous study, we found miR-10b to be more abundant in a conditioned culture medium of degenerate embryos compared to that of blastocysts. Here, we show that miR-10b mimics added to the culture medium can be taken up by embryos. This uptake results in an increase in embryonic cell apoptosis and aberrant expression of DNA methyltransferases (DNMTs). Using several algorithms, Homeobox A1 (HOXA1) was identified as one of the potential miR-10b target genes and dual-luciferase assay confirmed HOXA1 as a direct target of miR-10b. Microinjection of si-HOXA1 into embryos also resulted in an increase in embryonic cell apoptosis and downregulation of DNMTs. Cell progression analysis using Madin-Darby bovine kidney cells (MDBKs) showed that miR-10b overexpression and HOXA1 knockdown results in suppressed cell cycle progression and decreased cell viability. Overall, this work demonstrates that miR-10b negatively influences embryo quality and might do this through targeting HOXA1 and/or influencing DNA methylation.

Keywords: DNA methylation; HOXA1; apoptosis; bovine embryos; miR-10b; secreted miRNAs.

Figure 1

Effects of miR-10b mimics on embryo growth and apoptosis. (A) Embryos were treated with miR-10b mimics and the relative expression level of miR-10b was detected using RT-qPCR at 8 dpi. (B and C) Cleavage and blastocyst rate of embryos treated with miRNA mimics or control mimics were assessed. (D and E) Apoptosis rate of embryos was determined by TUNEL staining. The statistical analyses were performed using one-way ANOVA and data are presented as mean ± SD of three experiments (**P < 0.01; ns, no significance).

Figure 2

HOXA1 is a direct target of miR-10b. (A) 3′UTR analysis of HOXA1 containing putative regions that match the seed sequence of miR-10b, and the mutated nucleotides are underlined. (B) Overexpression of miR-10b inhibited Renilla luciferase activities. HEK-293T cells were cotransfected with 500 ng of reporter plasmid containing the MUT or WT-type UTRs and 5 nM miR-10b mimics. After 24 h, Renilla luciferase was normalized against firefly luciferase and then presented. (C and D) Embryos were treated with miR-10b mimics or control mimics and the relative levels of HOXA1 were detected with RT-qPCR and WB. (E and F) MiR-10b mimics were transfected into MDBKs. After 48 h or 24 h, cells were harvested for RT-qPCR or WB. The statistical analyses were performed using ANOVA and data are presented as mean ± SD of three experiments (**P < 0.01).

Figure 3

Effects of miR-10b mimics on DNMTs expression. Embryos were treated with miR-10b mimics and the relative expression levels of DNMTs were detected using RT-qPCR at 8 dpi. The statistical analyses were performed using two-way ANOVA, and data are presented as mean ± SD of three experiments (**P < 0.01; ns, no significance).

Figure 4

Effects of HOXA1 in embryo growth, apoptosis, and DNMTs. Embryos were injected with siHOXA1 and the cleavage rate (A) and blastocyst rate were assessed (B); cell apoptosis was determined by TUNEL staining (C and D); HOXA1 and DNMTs expressions were evaluated using RT-qPCR (E). The statistical analyses were performed using ANOVA and data are presented as mean ± SD of three experiments (*P < 0.05, **P < 0.01; ns, no significance).

Figure 5

Effects of miR-10b overexpression and HOXA1 knockdown on cell progression. (A and B) MDBKs were reverse transfected with miR-10b mimics or siHOXA1 for 48 h. RT-qPCR was then performed to assess the expression of miR-10b and HOXA1. (C and D) MDBKs were reverse transfected with miR-10b mimics or siHOXA1 for 48 h. (C) Cell viability was measured using the WST-1 assay, and (D) cell cycle assay by PI staining. Data are presented as mean ± SD of three experiments (**P < 0.01).