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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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CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and miR-451 deficient endometrial fragment proteomes.Endometrial fragments were obtained from wild-type (N = 6) and miR-451 deficient mice (N = 6) as described under “Materials and Methods”. Wild-type samples were labeled with Cy3 (green) and miR-451 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 miR-451 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 Table 1. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.
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pone-0100336-g004: CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and miR-451 deficient endometrial fragment proteomes.Endometrial fragments were obtained from wild-type (N = 6) and miR-451 deficient mice (N = 6) as described under “Materials and Methods”. Wild-type samples were labeled with Cy3 (green) and miR-451 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 miR-451 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 Table 1. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.

Mentions: To begin to determine the mechanisms by which miR-451 deficiency may impair the establishment of ectopic endometrial tissue fragments, we isolated endometrial fragments from both miR-451+/+ and miR-451−/− donors (N = 6/genotype, prior to transfer into recipients) and assessed protein profiles using two-dimensional differential in-gel electrophoresis (DiGE). From the hundreds of proteins expressed, twenty-five were further analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS; Fig. 4 and Table S1). Of these 25 proteins, we focused on the seven most significantly differentially expressed proteins (>1.5 fold change up or down) between miR-451−/− and wild-type tissues. Included in the inclusion criteria for these proteins were purity (some protein spots may contain two proteins) and identification as a single protein (some spots were identified with C.I.% of 100% but could not be identified as a single protein). After identification of these proteins and their reported biological functions, we focused on fibrinogen alpha polypeptide isoform 2 precursor (spot #5) as this protein may potentially modulate cell adhesion based on its RGD amino acid content [26] as well as neoangiogenesis [27]; both cellular events which contribute to ectopic implant survival and establishment.


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)

CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and miR-451 deficient endometrial fragment proteomes.Endometrial fragments were obtained from wild-type (N = 6) and miR-451 deficient mice (N = 6) as described under “Materials and Methods”. Wild-type samples were labeled with Cy3 (green) and miR-451 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 miR-451 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 Table 1. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0100336-g004: CyDye switch, two dimensional fluorescence difference gel electrophoresis (2D-DIGE) analysis of wild-type and miR-451 deficient endometrial fragment proteomes.Endometrial fragments were obtained from wild-type (N = 6) and miR-451 deficient mice (N = 6) as described under “Materials and Methods”. Wild-type samples were labeled with Cy3 (green) and miR-451 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 miR-451 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 Table 1. Red arrow indicates protein #5 which was identified as fibrinogen, alpha polypeptide isoform 2 precursor.
Mentions: To begin to determine the mechanisms by which miR-451 deficiency may impair the establishment of ectopic endometrial tissue fragments, we isolated endometrial fragments from both miR-451+/+ and miR-451−/− donors (N = 6/genotype, prior to transfer into recipients) and assessed protein profiles using two-dimensional differential in-gel electrophoresis (DiGE). From the hundreds of proteins expressed, twenty-five were further analyzed by MALDI-TOF (MS) and TOF/TOF (tandem MS/MS; Fig. 4 and Table S1). Of these 25 proteins, we focused on the seven most significantly differentially expressed proteins (>1.5 fold change up or down) between miR-451−/− and wild-type tissues. Included in the inclusion criteria for these proteins were purity (some protein spots may contain two proteins) and identification as a single protein (some spots were identified with C.I.% of 100% but could not be identified as a single protein). After identification of these proteins and their reported biological functions, we focused on fibrinogen alpha polypeptide isoform 2 precursor (spot #5) as this protein may potentially modulate cell adhesion based on its RGD amino acid content [26] as well as neoangiogenesis [27]; both cellular events which contribute to ectopic implant survival and establishment.

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