The mir-16 family is hormonally regulated in endometrial stromal cells and altered by superovulation in a murine model

Abstract

The purpose of this study was to determine the expression and hormonal regulation of the microRNA (miRNA, miR) -16 family in endometrial stromal cells during embryo implantation. Experimental laboratory study in CD1/ICR mice. To determine the differential expression of miRNAs before and at the time of implantation, eight week old CD1/ICR female mice were mated with males of known fertility and sacrificed at 0.5 dpc (morning of vaginal plug) or 4.5 dpc. RNA from endometrial stromal cells isolated from the whole uterus at 0.5 dpc and from implantation sites at 4.5 dpc of three mice at each time point underwent microRNA microarray analysis in triplicate. A separate cohort of mice was sacrificed at 0.5, 2.5, 4.5, and 8.5 dpc to confirm the microarray results. To analyze hormonal regulation of the miRNAs, ovariectomized mice were subcutaneously injected with vehicle (sesame oil) or 1 μg estradiol-17β for 48 hr. Another cohort of mice was either injected with 10 IU PMSG followed by 10 IU hCG 48 hrs later to induce superovulation or allowed to ovulate spontaneously. Endometrial stromal cells were isolated. After RNA isolation, RT-qPCR was performed for the three members of the miR-16 family, miR-15a, -15b, and -16, as well as for their predicted targets, Indian Hedgehog (IHH), HOXA10, and PTCH1. GraphPad Prism 5 was used for statistical analysis. P value < 0.05 was considered significant. In the miRNA array, miR-16 was significantly downregulated at 4.5 dpc versus 0.5 dpc; this was confirmed by RT-qPCR. The miR-16 family members, miR-15a and -15b were also decreased at 4.5 dpc. The putative targets of the miR-16 family, IHH, HOXA10 and PTCH1, were significantly, but transiently upregulated at 2.5 dpc. miR-16 family members were increased with estradiol treatment in the ovariectomized mice. On the other hand, IHH and PTCH1 were significantly downregulated following 48 hr of estradiol treatment. Superovulation also increased miR-16 family expression and downregulated the predicted target IHH. The miR-16 family is gradually downregulated following ovulation with a maximal decrease at implantation (4.5 dpc). The coordinate increase in the miR-16 targets may serve an important role in embryo implantation. By contrast, superovulation or estradiol treatment caused rapid and profound upregulation of miR-16 family expression. Our findings suggest that aberrant induction of the miR-16 family by estradiol treatment or superovulation may alter the expression of its targets necessary for successful implantation.