Endocrine Abstracts

The multiple endocrine neoplasia type 1 (MEN1) gene, which when mutated gives rise to parathyroid, pancreatic and pituitary tumours, has been shown to have a role in embryogenesis, as Men1-null mice (Men1^−/−) are embryonic lethal by 12.5 days post coitum (dpc). MicroRNAs (miRNAs) are emerging as potent regulators of early mammalian embryogenesis, and we therefore undertook expression profiling of miRNAs in Men1^+/+ and Men1^−/− embryos at 11.5 dpc. Our studies identified differential expression of two developmentally important miRNAs, the mammalian embryonic stem cell-specific miR-302 cluster and the evolutionarily conserved pro-differentiation let-7 family. Validation of these findings using quantitative reverse transcriptase-PCR (qRT-PCR) showed 3.7-fold up-regulation of miR-302a, and 2.2-fold down-regulation of let-7a, in Men1^−/− embryos compared to Men1^+/+ (P<0.05). We next used qRT-PCR to investigate for alterations in miR-302 and let-7 known target gene mRNAs, which are CyclinD1 and Tgfbr2, and Hmga2 and Lin28b, respectively. CyclinD1 and Tgfbr2 mRNAs were not significantly different in Men1^+/+ and Men1^−/− embryos, but Hmga2 and Lin28b mRNAs were significantly increased in Men1^−/− embryos when compared to Men1^+/+, by 1.5- and 2.1-fold respectively, consistent with the reduction of let-7 miRNAs. Furthermore, a strong inverse correlation between let-7a and Lin28b mRNA was observed in Men1^+/+ and Men1^−/− embryos (R²=0.88). These results suggest that let-7a directly suppresses the expression of Lin28b, an oncofoetal protein, in the developing Men1^+/+ embryo, and that this regulation is lost in Men1^−/− embryos. In conclusion, our results, which show high levels of miR-302 and reduced levels of let-7, indicate that Men1^−/− embryos have a miRNA profile consistent with a pro-pluripotent, undifferentiated state. Thus, our results indicate that miRNAs are a facet of the embryopathy observed in Men1-deficient embryos and that a characterization of their function will provide insight into the role of the Men1 gene during mammalian embryogenesis.