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miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model.

Nothnick WB, Graham A, Holbert J, Weiss MJ - PLoS ONE (2014)

Bottom Line: 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.After induction of the disease, we evaluated the impact of this deficiency on implant development and survival.Loss of miR-451 expression resulted in a lower number of ectopic lesions established in vivo.

View Article: PubMed Central - PubMed

Affiliation: University of Kansas Medical Center, Department of Molecular & Integrative Physiology, Kansas City, Kansas, United States of America.

ABSTRACT
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 (miR-451) in the initial development of endometriosis, we utilized a novel mouse model in which eutopic endometrial fragments used to induce endometriosis were deficient for miR-451. After induction of the disease, we evaluated the impact of this deficiency on implant development and survival. Loss of miR-451 expression resulted in a lower number of ectopic lesions established in vivo. Analysis of differential protein profiles between miR-451 deficient and wild-type endometrial fragments revealed that fibrinogen alpha polypeptide isoform 2 precursor was approximately 2-fold higher in the miR-451 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 in vitro and in vivo. Collectively, these results suggest that the reduced miR-451 eutopic endometrial expression does not enhance initial establishment of these fragments when displaced into the peritoneal cavity, that loss of eutopic endometrial miR-451 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.

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Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.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 “Materials and Methods.” 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.
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pone-0100336-g006: Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.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 “Materials and Methods.” 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.

Mentions: We postulated that the reduced ability of miR-451 deficient tissue to establish ectopically may be due to elevated expression of fibrinogen alpha chain precursor as this protein contains three RGD cell adhesion motifs (Fig. 6A) which in theory would impede cell to cell adhesion and/or viability necessary for ectopic implant establishment and survival. To begin to assess this possibility, we first elected to assess endometrial stromal cell adhesion/spreading in vitro to determine if the RGD motif could prevent the stromal cells from binding with vitronectin and/or fibronectin; extracellular matrix proteins which also contains the RGD sequence and are postulated to promote cell to cell adhesion via integrin αvβ3 [28] and/or α1β5; integrins, which are expressed on endometrial tissue/cells [28], [29]. Pre-incubation of stromal cells with RGD peptide for 1 h resulted in a significant, dose-dependent decrease in stromal cell attachment and spreading on vitronectin-coated tissue culture plates as well as on fibronectin-coated tissue culture plates (to a much lesser degree compared to vitronectin) but not to bovine serum albumin-coated tissue culture plates (Fig. 6B). This reduction in spreading was not associated with an initial decrease in cell viability after the pre-incubation with RGD peptide (Fig. 6C).


miR-451 deficiency is associated with altered endometrial fibrinogen alpha chain expression and reduced endometriotic implant establishment in an experimental mouse model.

Nothnick WB, Graham A, Holbert J, Weiss MJ - PLoS ONE (2014)

Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.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 “Materials and Methods.” 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4061076&req=5

pone-0100336-g006: Fibrinogen alpha polypeptide isoform 2 precursor contains multiple cell adhesion motifs which modulate cell adhesion in vitro.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 “Materials and Methods.” 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.
Mentions: We postulated that the reduced ability of miR-451 deficient tissue to establish ectopically may be due to elevated expression of fibrinogen alpha chain precursor as this protein contains three RGD cell adhesion motifs (Fig. 6A) which in theory would impede cell to cell adhesion and/or viability necessary for ectopic implant establishment and survival. To begin to assess this possibility, we first elected to assess endometrial stromal cell adhesion/spreading in vitro to determine if the RGD motif could prevent the stromal cells from binding with vitronectin and/or fibronectin; extracellular matrix proteins which also contains the RGD sequence and are postulated to promote cell to cell adhesion via integrin αvβ3 [28] and/or α1β5; integrins, which are expressed on endometrial tissue/cells [28], [29]. Pre-incubation of stromal cells with RGD peptide for 1 h resulted in a significant, dose-dependent decrease in stromal cell attachment and spreading on vitronectin-coated tissue culture plates as well as on fibronectin-coated tissue culture plates (to a much lesser degree compared to vitronectin) but not to bovine serum albumin-coated tissue culture plates (Fig. 6B). This reduction in spreading was not associated with an initial decrease in cell viability after the pre-incubation with RGD peptide (Fig. 6C).

Bottom Line: 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.After induction of the disease, we evaluated the impact of this deficiency on implant development and survival.Loss of miR-451 expression resulted in a lower number of ectopic lesions established in vivo.

View Article: PubMed Central - PubMed

Affiliation: University of Kansas Medical Center, Department of Molecular & Integrative Physiology, Kansas City, Kansas, United States of America.

ABSTRACT
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 (miR-451) in the initial development of endometriosis, we utilized a novel mouse model in which eutopic endometrial fragments used to induce endometriosis were deficient for miR-451. After induction of the disease, we evaluated the impact of this deficiency on implant development and survival. Loss of miR-451 expression resulted in a lower number of ectopic lesions established in vivo. Analysis of differential protein profiles between miR-451 deficient and wild-type endometrial fragments revealed that fibrinogen alpha polypeptide isoform 2 precursor was approximately 2-fold higher in the miR-451 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 in vitro and in vivo. Collectively, these results suggest that the reduced miR-451 eutopic endometrial expression does not enhance initial establishment of these fragments when displaced into the peritoneal cavity, that loss of eutopic endometrial miR-451 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.

Show MeSH
Related in: MedlinePlus