MicroRNA regulation of cyclooxygenase-2 during embryo implantation - PubMed (original) (raw)

MicroRNA regulation of cyclooxygenase-2 during embryo implantation

Anindita Chakrabarty et al. Proc Natl Acad Sci U S A. 2007.

Abstract

The implantation process is complex, requiring reciprocal interactions between implantation-competent blastocysts and the receptive uterus. Because microRNAs (miRNAs) have major roles in regulating gene expression, we speculated that they participate in directing the highly regulated spatiotemporally expressed genetic network during implantation. Here, we show that two miRNAs, mmu-miR-101a and mmu-miR-199a*, are spatiotemporally expressed in the mouse uterus during implantation coincident with expression of cyclooxygenase-2, a gene critical for implantation. More interestingly, our in vitro gain- and loss-of-function experiments show that cyclooxygenase-2 expression is posttranscriptionally regulated by these two miRNAs. We report on miRNA-mediated regulation of uterine gene expression in the context of implantation. We believe that many other critical genes related to this process are also regulated by miRNAs. Thus, elucidating the physiological roles of uterine miRNAs will help us better understand the genetic control of implantation, the gateway to a successful pregnancy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

miRNA biogenesis in periimplantation mouse uteri. (A) miRNA profiling with oligonucleotide microarray analysis of day-1 (D1) and day-4 (D4) pregnant uteri. Yellow spots along the diagonal line represent miRNAs with similar expression levels between days 1 and 4. Orange spots indicate up-regulated miRNAs in prereceptive uteri (day 1), and blue spots indicate up-regulated miRNAs in receptive uteri (day 4). (B) RT-PCR of Dicer1 and Ago1–4 in periimplantation uteri. β-Actin serves as a control. (C) Western blotting of Dicer1 protein. Mouse ovary, positive controls; Actin, loading control. IS, implantation site; IIS, interimplantation site.

Fig. 2.

Fig. 2.

Mature miR-199a* is expressed in periimplantation mouse uteri. (A) Northern hybridization with 32P-labeled probe in total uterine RNA samples on day 1 (D1) and day 4 (D4) of pregnancy. The ethidium bromide-stained gel served as a loading control. (B) In situ hybridization of uterine cross-sections with DIG-labeled LNA probe on days 1, 4, 5, and 6 of pregnancy. Arrows indicate locations of blastocysts. le, luminal epithelium; ge, glandular epithelium; s, stroma; myo, myometrium; M, mesometrial pole; AM, antimesometrial pole; IS, implantation site. (Scale bar: 400 μm.)

Fig. 3.

Fig. 3.

Mature miR-101a is expressed in periimplantation mouse uteri. (A) Northern hybridization of total uterine RNA samples from day-1 (D1) and day-4 (D4) pregnant uteri. The ethidium bromide-stained gel served as a loading control. (B) In situ hybridization of uterine cross-sections with DIG-labeled LNA probe on days 1, 4, 5, and 6 of pregnancy. Arrows indicate the location of blastocysts. Abbreviations are as in Fig. 2. (Scale bar: 400 μm.)

Fig. 4.

Fig. 4.

Uterine expression of miR-101a, miR-199a*, and Cox-2. (A) In situ hybridization of Cox-2 with 35S-labeled probe (Left) and protein by immunohistochemistry (Right) in serial cross-sections from day-5 implantation sites (IS). (B) In situ hybridization of miR-199a* with DIG-labeled LNA probe on longitudinal sections from delayed and implanting uteri. Arrows indicate the location of blastocysts. (C) Uterine Cox-2 and miR-101a levels during experimentally induced decidualization. Western blot shows Cox-2 protein levels at time points after intrauterine oil infusion (+); noninfused contralateral uterine horns serve as negative controls (−). Actin is a loading control. The ethidium bromide-stained gels served as loading control for miR-101a Northern blot. (Scale bars: 200 μm, A; 400 μm, B.)

Fig. 5.

Fig. 5.

Cross-species homology of miRNAs targeting COX-2 mRNA. (A and B) The structure and homology of the predicted interactions between Cox-2 mRNA and miR-101 (A) and miR-199a* (B). (C) The predicted annealing sites of the seed and 3′ terminus of these miRNAs are well conserved in Cox-2 mRNA.

Fig. 6.

Fig. 6.

miR-199a* and miR-101a regulate Cox-2 translation. (A) Renilla Luc-m_Cox-2_-3′ UTR reporter construct. (B) Concentration-dependent decreases in reporter activity with increasing doses of precursor miR-199a* or miR-101a in HEK293 cells. A scrambled miRNA had no effect on reporter activity (control). (C) Antisense inhibitors of miR-199a* and miR-101a relieve miRNA-mediated repression of _Cox-2_-3′ UTR-Luc reporter activity; scrambled antisense sequence (control) had no effect. Each transfection was performed in triplicate. Data are presented as relative Luc activity (mean ± SEM). (D) miR-199a* and miR-101a repress endogenous Cox-2 translation. Concentration-dependent decreases in endogenous Cox-2 protein levels in HeLa cells 72 h after transfection with miR-199a* or miR-101a precursors. Western blotting of Cox-2 (Upper) and corresponding densitometry analyses (Lower) are shown. Actin, loading control.

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