Up-regulation of Lin28 and Dicer are required for the activation of mouse blastocysts

Abstract

MicroRNA Let-7 family is down-regulated during the pre-implantation embryo development and upon estradiol-activation of the dormant blastocysts. Induction of Let-7a in mouse blastocysts attenuates trophoblast attachment and outgrowth in vitro and compromises pregnancy in vivo partly via modulation of the expression of integrin beta-3 and Vav3. However, the mechanism that regulates Let-7a expression in mouse blastocysts remains unknown. It is known that Let-7 expression is regulated by Dicer and Lin28 (Lin28A and Lin28B) in other cell types. Dicer is a RNAase III enzyme that cleaves precursor microRNAs into their mature form. The RNA-binding protein Lin28 is a negative regulator of Let-7. It recruits the TUTases to assemble the Ago-mediated microRNA degradation complex. Although Dicer and Lin28 modulate Let-7a expression, they are putative targets of Let-7a. In this study, we hypothesized that Dicer and Lin28 were mediators of Let-7a action upon blastocyst activation. During pre-implantation development towards blastocysts, the expression of primary and mature Let-7a in embryos decreased, while that of Lin28 and Dicer increased. Force-expression of Let-7a inhibited Dicer and Lin28 protein expression in the blastocysts. In the dormant mouse blastocyst, Let-7a expression was high. Estradiol adminstration down-regulated Let-7a expression, but upregulated that of the dicer protein in the these blastocysts. Knockdown of dicer at one-hour post-estradiol activation led to a shift of the microRNAome towards that of the dormant blastocysts, and repressed implantation in vivo and in vitro as indicated by a decrease in epidermal growth factor binding ability. Similarly, Lin28 was up-regulated along the mural trophectoderm of the dormant blastocysts after 3-hour post estradiol activation. The temporal change in Lin28 expression among the estradiol activated dormant blastocysts was yet lagged from that of dicer. Knocking down of Lin28 at one-hour post estradiol activation increased the mature Let-7a expression and concurrently lowered the implantation rate in vivo. The data thus supported that during activation of dormant blastocyst, the down-regulation of Let-7a enhanced Dicer expression for microRNA processing, and Lin28 expression, which degraded the primary and mature Let-7a to potentiate implantation. [The research work is partly supported by a GRF grant to WSBY].