MicroRNAs (miRNAs) are 21-24-nucleotide non-coding RNAs found in diverse organisms. Although hundreds of miRNAs have been cloned or predicted, only very few miRNAs have been functionally characterized. Embryo implantation is a crucial step in mammalian reproduction. Many genes have been shown to be significantly changed in mouse uterus during embryo implantation. However, miRNA expression profiles in the mouse uterus between implantation sites and inter-implantation sites are still unknown. In this study, miRNA microarray was used to examine differential expression of miRNAs in the mouse uterus between implantation sites and inter-implantation sites. Compared with inter-implantation sites, there were 8 up-regulated miR-NAs at implantation sites, which were confirmed by both Northern blot and in situ hybridization. miR-21 was highly expressed in the subluminal stromal cells at implantation sites on day 5 of pregnancy. Because miR-21 was not detected in mouse uterus during pseudopregnancy and under delayed implantation, miR-21 expression at implantation sites was regulated by active blastocysts. Furthermore, we showed that Reck was the target gene of miR-21. Our data suggest that miR-21 may play a key role during embryo implantation.

The extensive involvement of miRNAs in cancer pathobiology has opened avenues for drug development based on oncomir inhibition. Dicer is the core enzyme in miRNA processing that cleaves the terminal loop of precursor microRNAs (pre-miRNAs) to generate mature miRNA duplexes. Using the three-dimensional structure of the Dicer binding site on the pre-miR-21 oncomir, we conducted an in silico high-throughput screen for small molecules that block miR-21 maturation. By this method, we identified a specific small-molecule inhibitor of miR-21, termed AC1MMYR2, which blocked the ability of Dicer to process pre-miR-21 to mature miR-21. AC1MMYR2 upregulated expression of PTEN, PDCD4, and RECK and reversed epithelial–mesenchymal transition via the induction of E-cadherin expression and the downregulation of mesenchymal markers, thereby suppressing proliferation, survival, and invasion in glioblastoma, breast cancer, and gastric cancer cells. As a single agent in vivo, AC1MMYR2 repressed tumor growth, invasiveness, and metastasis, increasing overall host survival with no observable tissue cytotoxicity in orthotopic models. Our results offer a novel, high-throughput method to screen for small-molecule inhibitors of miRNA maturation, presenting AC1MMYR2 as a broadly useful candidate antitumor drug. Cancer Res; 73(17); 5519–31. ©2013 AACR.

Decidualization is an essential process of maternal endometrial stromal cells to support pregnancy. Although it is known that enhanced glucose influx is critical for decidualization, the underlying mechanism in regulating glucose metabolism in decidua remains insufficiently understood. Here, we demonstrate that aerobic glycolysis-related genes and factors are all substantially induced during decidualization, indicting the existence of Warburg-like glycolysis in decidua. In vitro, progesterone activates Hif1α and c-Myc through Pi3k-Akt signaling pathway to maintain aerobic glycolysis in decidualizing cells. Knocking down of Pkm2 attenuates the induction of decidual marker gene. Decidual formation in vivo is also impaired by glycolysis inhibitor 3-bromopyruvate. Besides, lactate exporter Mct4 is induced in newly formed decidual cells, whereas lactate importer Mct1 and proliferation marker Ki-67 are complementarily located in the surrounding undifferentiated cells, which are supposed to consume lactate for proliferation. Hif1α activation is required for lactate-dependent proliferation of the undifferentiated cells. Inhibition of lactate flux leads to compromised decidualization and decelerated lactate-dependent proliferation. In summary, we reveal that Warburg-like glycolysis and local lactate shuttle are activated in decidua, and play important roles for supporting early pregnancy.

Embryo implantation into the maternal uterus is a crucial step for the successful establishment of mammalian pregnancy. Following the attachment of embryo to the uterine luminal epithelium, uterine stromal cells undergo steroid hormone-dependent decidualization, which is characterized by stromal cell proliferation and differentiation. The mechanisms underlying steroid hormone-induced stromal cell proliferation and differentiation during decidualization are still poorly understood. Ribonucleotide reductase, consisting of two subunits (RRM1 and RRM2), is a rate-limiting enzyme in deoxynucleotide production for DNA synthesis and plays an important role in cell proliferation and tumorgenicity. Based on our microarray analysis, Rrm2 expression was significantly higher at implantation sites compared with interimplantation sites in mouse uterus. However, the expression, regulation, and function of RRM2 in mouse uterus during embryo implantation and decidualization are still unknown. Here we show that although both RRM1 and RRM2 expression are markedly induced in mouse uterine stromal cells undergoing decidualization, only RRM2 is regulated by progesterone, a key regulator of decidualization. Further studies showed that the induction of progesterone on RRM2 expression in stromal cells is mediated by the AKT/c-MYC pathway. RRM2 can also be induced by replication stress and DNA damage during decidualization through the ATR/ATM-CHK1-E2F1 pathway. The weight of implantation sites and deciduoma was effectively reduced by specific inhibitors for RRM2. The expression of decidual/trophoblast prolactin-related protein (Dtprp), a reliable marker for decidualization in mice, was significantly reduced in deciduoma and steroid-induced decidual cells after HU treatment. Therefore, RRM2 may be an important effector of progesterone signaling to induce cell proliferation and decidualization in mouse uterus.