<i>miR-451</i> Deficiency Is Associated with Altered Endometrial Fibrinogen Alpha Chain Expression and Reduced Endometriotic Implant Establishment in an Experimental Mouse Model

<div><p>Endometriosis is defined as the growth of endometrial glandular and stromal components in ectopic locations and affects as many as 10% of all women of reproductive age. Despite its high prevalence, the pathogenesis of endometriosis remains poorly understood. MicroRNAs, small non-coding RNAs that post-transcriptionally regulate gene expression, are mis-expressed in endometriosis but a functional role in the disease pathogenesis remains uncertain. To examine the role of microRNA-451 (<i>miR-451</i>) in the initial development of endometriosis, we utilized a novel mouse model in which eutopic endometrial fragments used to induce endometriosis were deficient for <i>miR-451</i>. After induction of the disease, we evaluated the impact of this deficiency on implant development and survival. Loss of <i>miR-451</i> expression resulted in a lower number of ectopic lesions established <i>in vivo</i>. Analysis of differential protein profiles between <i>miR-451</i> deficient and wild-type endometrial fragments revealed that fibrinogen alpha polypeptide isoform 2 precursor was approximately 2-fold higher in the <i>miR-451</i> null donor endometrial tissue and this elevated expression of the protein was associated with altered expression of the parent fibrinogen alpha chain mRNA and protein. As this polypeptide contains RGD amino acid “cell adhesion” motifs which could impact early establishment of lesion development, we examined and confirmed using a cyclic RGD peptide antagonist, that endometrial cell adhesion and endometriosis establishment could be respectively inhibited both <i>in vitro</i> and <i>in vivo</i>. Collectively, these results suggest that the reduced <i>miR-451</i> eutopic endometrial expression does not enhance initial establishment of these fragments when displaced into the peritoneal cavity, that loss of eutopic endometrial <i>miR-451</i> expression is associated with altered expression of fibrinogen alpha chain mRNA and protein, and that RGD cyclic peptide antagonists inhibit establishment of endometriosis development in an experimental mouse model suggesting that this approach may prove useful in the prevention of endometriosis establishment and survival.</p></div

Development of experimental endometriosis is associated with the level of implant <i>miR-451</i> expression.

<p>Experimental endometriosis was induced and development assessed as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>”. A. The number of implants of each genotype that developed in wild-type host mice (N = 8/genotype). Data are presented as the mean ± SEM and were analyzed by one-way ANOVA followed by post-hoc analysis. Different letters indicate statistical significance (P<0.05). B. Elevated <i>miR-451</i> expression is associated with wild-type “implant” tissue that develops ectopically. Uterine fragments were obtained from wild-type mice at the time (0 h) of PMSG administration or 44 h later (the time of endometrial fragment harvest). <i>miR-451</i> and <i>miR-144</i> expression was determined by qRT-PCR. Different letters indicate statistical significance (P<0.05) between groups (by unpaired t-test). ND indicates that <i>miR-144</i> levels were not detectable by qRT-PCR.</p

Fibrinogen transcript, protein and active plasmin expression in endometrial fragments of wild-type and <i>miR-451</i> deficient mice.

<p>Fibrinogen alpha chain (Fga) transcript (A) and protein (B) were analyzed by qRT-PCR and Western blot expression, respectively. Data are representative of 5 observations per endpoint per genotype (N = 5). <i>Fga</i> mRNA levels were not normally distributed and were analyzed using Mann-Whitney tests. Bar graph data are displayed as the mean ± SEM. In B), black arrow indicates molecular weight (95 kDa) of fibrinogen alpha chain while white arrows indicate major Fga fragments of approximately 42 kDa and 34 kDa. C) Plasmin activity was determined in uterine fragments from wild-type and <i>miR-451</i> null mice as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>.” Data are displaed as the mean ± SEM and are representative of 6 separate observations per genotype (N = 6). P values are indicated in each figure and data were analyzed by unpaired t-tests in B and C. As Fga mRNA data (A) were not normally distributed, data was analyzed using the non-parametric t-test (Mann-Whitney).</p

Most significantly modulated proteins in uterine fragments from <i>miR-451</i> deficient mice compared to wild-type counterparts.

<p>a  =  Pir/Pid  =  reported isoelectric point/detected isoelectric point. Differences between the reported and detected isoelectric points may be due to alterations in post-translational modifications such as Phosphorylation which decreases the PI.</p><p>b  =  Fold change is expressed as level of each protein detected in the <i>miR-451</i> null/<i>miR-451</i> wild-type samples.</p

CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and <i>miR-451</i> deficient endometrial fragment proteomes.

<p>Endometrial fragments were obtained from wild-type (N = 6) and <i>miR-451</i> deficient mice (N = 6) as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>”. Wild-type samples were labeled with Cy3 (green) and <i>miR-451</i> deficient samples with Cy5 (red). Samples were then mixed and separated on analytical 2-D DIGE. The resulting gel was scanned and the merged image is shown where red proteins represent proteins whose expression is higher in the <i>miR-451</i> deficient tissue and green proteins represent proteins whose expression is higher in the wild-type tissue. Circled and numbered spots represent proteins which were most differentially expressed of which only those indicated by white (up-regulated) or yellow (down-regulated) arrows are reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#pone-0100336-t001" target="_blank">Table 1</a>. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.</p

Diagrammatic representation of experimental endometriosis induction in mice and histological representation of ectopic endometriotic implant.

<p>A) Diagram depicts donor mice (and genotype) from which endometrial tissue was dissected and separated into myometrium and endometrium (stromal and epithelial components), transfer of 10 equal size (1 mm³) endometrial fragments to recipient mice and subsequent assessment of establishment of ectopic “endometriotic implants.” B) histological representation of “endometriotic implant” with red boxed area enlarged in C) white arrow indicates epithelial component and stromal compartment and underlying peritoneum are labeled. Notice the stromal and peritoneal contact at the site of implant attachment. White scale bar  = 50 um.</p

Development of experimental endometriosis is dependent upon implant <i>miR-451</i> expression and not host expression.

<p>A. Endometriosis was induced in wild-type and <i>miR-451</i> null mice using donor tissue from both wild-type and <i>miR-451</i> null mice and the number of implants which developed was assessed as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means as determined by one-way ANOVA followed by post-hoc analysis (N = 6 per group). B. <i>miR-451</i> wild-type and not <i>miR-451</i> deficient implants develop in the same wild-type host. Endometriosis was induced as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>” in wild-type host mice using both miR-451 wild-type (expressing EGFP) and <i>miR-451</i> deficient (non-EGFP) endometrial fragments and implant establishment was assessed. Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means as determined by unpaired t-test (N = 6 per group).</p

RGD cycle peptide inhibits development of experimental endometriosis.

<p>Experimental endometriosis was induced in wild-type mice with wild-type endometrial fragments which were pre-treated with either vehicle (Veh) or RGD peptide (RGD) and development of ectopic lesions was assessed as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means between treatments as determined by unpaired t-tests (N = 8/group). B Chi square analysis of the proportion of mice which received implant tissue pre-treated with either vehicle (Veh) or RGD cyclic peptide (RGD).</p

Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.

<p>A. Protein sequence of mouse fibrinogen alpha polypeptide isoform 2 precursor. Three RGD sequences were detected in the protein sequence and are indicated in red underlined text. B. Pre-treatment of the immortalized human endometrial stromal cell line, t-HESC with cyclic RGD peptide inhibits in vitro cell spreading and survival. T-HESC cells were treated and cell adhesion, spreading and survival assessed as described under “<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100336#s2" target="_blank">Materials and Methods</a></i>.” Data are displayed as the mean ± SEM. Different letters indicate statistically significant differences among the means within each substrate as determined by one-way ANOVA followed by post-hoc analysis (N = 4 separate experiments). C. Pre-treatment with RGD cyclic peptide does not induce cell death. Data are displayed as the mean ± SEM and are representative of 4 separate experiments (N = 4 separate experiments). Means are not significantly different among the different doses of RGD peptide.</p

Rapid proteasome degradation of SMAD4 in DBA iPSCs.

<p>DBA iPSCs, and the corrected DBA cells were cultured in the iPSC medium, and treated with proteasome inhibitors MG132 (4 μM) for 12h or PS341 (20 μM) for 4h. A-B) Increased level of SMAD4 in both DBA cells after treatment with proteasome inhibitor MG132 for 12h. C) Increased level of nuclear SMAD4 in DBA iPSCs with <i>RPS19</i> mutation after PS341 treatment. D and E) Increase of nuclear and cytoplasmic TRIM33, and increase of nuclear USP9X in both DBA iPSCs. The PS341 treatment did not affect the levels of TRIM33 and USP9X.</p