194 EXPRESSION OF microRNA-15a, 16, AND 21 AND THEIR POSSIBLE ROLES IN MOUSE EMBRYOS DEVELOPING IN VITRO

Abstract

MicroRNAs (miRNAs) are small (~22 nucleotides) non-coding RNA molecules that can regulate gene expression by base-pairing with fully or partially sequence-complementary target mRNAs. Hundreds of miRNAs have been identified in various multicellular organisms and many miRNAs are evolutionarily conserved. While miRNAs play an important role in animal development, little is known about their biological function during early mammalian development. In order to obtain insight into the role of miRNAs in early embryogenesis, we first determined the expression levels of three apoptosis-related miRNAs, miR-15a, -16, and -21 in mouse preimplantation embryos using TaqMan^® MicroRNA Assays. Five embryos of each developmental stage were snap-frozen and amplified by stem-loop RT primer and TaqMan Universal PCR Master Mix (Applied Biosystems Inc., Foster City, CA, USA). The miRNA concentrations (10^–X) in embryo samples were calculated by standard curve from synthetic lin-4 miRNA and the absolute copy number per embryo was obtained based on the formula of 6.02 × 10(^8–X). All three miRNAs had low expression levels from the zygote to the 8-cell stage and were up-regulated thereafter. In general, among the three miRNAs, miR-15a exhibited the lowest expression in preimplantation embryos, while miR-16 exhibited the highest. Because of the low levels of miRNA-15a, we determined developmental ability and apoptosis of embryos following microinjection of miRNA-15a. The microinjection of miR-15a into zygotes did not affect embryo development up to the blastocyst stage (miR-15a, 90 ± 4.5% v. buffer 94.6 ± 5.8%); however, it did induce a significant degree of apoptosis (P < 0.05; Tukey's multiple range test). Furthermore, the expression levels of miR-15a and -16 were increased in microinjected blastocysts compared to the control group (copy number per blastocyst, miR-15a, 6991 ± 1223 v. 3098 ± 592; miR-16, 196216 ± 958 v. 133514 ± 6059). Real-time RT-PCR data showed that the gene expression levels of the housekeeping gene GAPDH, the anti-apoptotic gene Bcl-xL, and the miRNA pathway-related genes GW182 and Dicer remained unchanged in miR-15a-injected blastocysts compared to the control group. In contrast, the expression of the stem cell-specific transcriptional factor Oct-4 (fold change, 1.451 ± 0.12), the pro-apoptotic gene Bax (1.418 ± 0.12), and Caspase 3 (1.314 ± 0.19) were significantly increased in microinjected blastocysts. In addition, treatment of 2-cell embryos with 600 µm H₂O₂ induced apoptosis and increased the expression level of miR-16 at the blastocyst stage (P < 0.05). Taken together, the changes in the expression levels of miR-15a, -16, and -21 in various embryonic developmental stages indicate a possible role for them in early embryogenesis. Furthermore, the high expression levels of miR-15a and miR-16 seem to be linked to apoptosis in blastocyst-stage embryos; this may be due to an increase in the expression of pro-apoptotic genes.