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A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Grechukhina O, Petracco R, Popkhadze S, Massasa E, Paranjape T, Chan E, Flores I, Weidhaas JB, Taylor HS - EMBO Mol Med (2012)

Bottom Line: Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease.Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women.We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.

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Morphological and molecular features of endometriotic lesions containing the WT non-variant or variant alleles of the KRAS LCS6Cultured endometrial stromal cells were injected under the kidney capsule of immunodeficient mice.Morphological appearance of the lesions under the kidney capsule of immune deficient mice 1 month after transplantation of hESC either with or without the variant KRAS. In all cases the transplanted endometrial cells formed endometriosis lesions with both glandular and stromal components.Proliferation marker expression in endometriotic lesions in mice. Nuclear staining for PCNA was more prominent in epithelium and stroma of the lesions formed by KRAS variant positive cells (54 ± 5% and 56 ± 6% in epithelium and stroma, respectively), compared to those derived from normal cells (8 ± 4% and 34 ± 6% in epithelium and stroma, respectively; p = 0.02 and p = 0.043) indicating higher proliferation levels in these cells.PR expression in endometriotic lesions with WT non-variant or variant KRAS allele in mice. Lesions created by hESC carrying KRAS variant allele were characterized by a smaller number of nuclei stained positively for PR in both glandular and stromal cells. The epithelium of the lesions with variant cells was found to have only 35 ± 5% of nuclei positively stained compared to 75 ± 3%, in the lesions with WT KRAS (p = 0.02). Only 13 ± 8% of nuclei of stromal cells in KRAS variant lesions were found to express PR compared to 78 ± 7% in the non-variant lesions (p = 0.028). Scale bar represents 25 µm.
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fig05: Morphological and molecular features of endometriotic lesions containing the WT non-variant or variant alleles of the KRAS LCS6Cultured endometrial stromal cells were injected under the kidney capsule of immunodeficient mice.Morphological appearance of the lesions under the kidney capsule of immune deficient mice 1 month after transplantation of hESC either with or without the variant KRAS. In all cases the transplanted endometrial cells formed endometriosis lesions with both glandular and stromal components.Proliferation marker expression in endometriotic lesions in mice. Nuclear staining for PCNA was more prominent in epithelium and stroma of the lesions formed by KRAS variant positive cells (54 ± 5% and 56 ± 6% in epithelium and stroma, respectively), compared to those derived from normal cells (8 ± 4% and 34 ± 6% in epithelium and stroma, respectively; p = 0.02 and p = 0.043) indicating higher proliferation levels in these cells.PR expression in endometriotic lesions with WT non-variant or variant KRAS allele in mice. Lesions created by hESC carrying KRAS variant allele were characterized by a smaller number of nuclei stained positively for PR in both glandular and stromal cells. The epithelium of the lesions with variant cells was found to have only 35 ± 5% of nuclei positively stained compared to 75 ± 3%, in the lesions with WT KRAS (p = 0.02). Only 13 ± 8% of nuclei of stromal cells in KRAS variant lesions were found to express PR compared to 78 ± 7% in the non-variant lesions (p = 0.028). Scale bar represents 25 µm.

Mentions: To evaluate differences in growth parameters in vivo, capacity for endometriotic lesion formation as well as histopathological and molecular characteristics of cultured endometrial stromal cells containing non-variant and variant alleles of the KRAS gene, a mouse model of endometriosis was used. We transplanted hESC obtained from subjects with and without the LCS6 variant under the kidney capsule of immune-deficient mice. Both non-variant and LCS6 variant cells formed endometriosis-like lesions with both glandular and stromal components (Fig 5a). The glandular component likely originated from progenitor stem cells in the culture as recently described (Cervello et al, 2011). Glandular cells are not identified in these cultures by the third passage, however, we cannot exclude a small number of contaminants (Taylor et al, 1998). Analysis of proliferation marker PCNA showed more cells (both epithelial and stromal) with stained nuclei in the lesion derived from LCS6 variant cells compared to those derived from non-variant cells. The percentage of stained nuclei in epithelium of lesions carrying variant KRAS allele was 54 ± 5% vs. 8 ± 1% in lesions created by non-variant hESC (p = 0.02). Stromal cells from lesions with the SNP exhibited 56 ± 5% of nuclei staining while in the lesions with WT KRAS, the percentage of positively stained nuclei was 34 ± 6% (p = 0.043; Fig 5b). These results suggest increased proliferation of cells harbouring the variant allele and were consistent with the results of the in vitro proliferation experiment. Apoptosis was assessed using cleaved caspase 3, and the amount of cleaved caspase 3 was equivalent in lesions derived from cells with either non-variant or LCS6 variant alleles (data not shown). Proliferation was increased without an increase in apoptosis in the LCS6 variant lesions.


A polymorphism in a let-7 microRNA binding site of KRAS in women with endometriosis.

Grechukhina O, Petracco R, Popkhadze S, Massasa E, Paranjape T, Chan E, Flores I, Weidhaas JB, Taylor HS - EMBO Mol Med (2012)

Morphological and molecular features of endometriotic lesions containing the WT non-variant or variant alleles of the KRAS LCS6Cultured endometrial stromal cells were injected under the kidney capsule of immunodeficient mice.Morphological appearance of the lesions under the kidney capsule of immune deficient mice 1 month after transplantation of hESC either with or without the variant KRAS. In all cases the transplanted endometrial cells formed endometriosis lesions with both glandular and stromal components.Proliferation marker expression in endometriotic lesions in mice. Nuclear staining for PCNA was more prominent in epithelium and stroma of the lesions formed by KRAS variant positive cells (54 ± 5% and 56 ± 6% in epithelium and stroma, respectively), compared to those derived from normal cells (8 ± 4% and 34 ± 6% in epithelium and stroma, respectively; p = 0.02 and p = 0.043) indicating higher proliferation levels in these cells.PR expression in endometriotic lesions with WT non-variant or variant KRAS allele in mice. Lesions created by hESC carrying KRAS variant allele were characterized by a smaller number of nuclei stained positively for PR in both glandular and stromal cells. The epithelium of the lesions with variant cells was found to have only 35 ± 5% of nuclei positively stained compared to 75 ± 3%, in the lesions with WT KRAS (p = 0.02). Only 13 ± 8% of nuclei of stromal cells in KRAS variant lesions were found to express PR compared to 78 ± 7% in the non-variant lesions (p = 0.028). Scale bar represents 25 µm.
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Related In: Results  -  Collection

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fig05: Morphological and molecular features of endometriotic lesions containing the WT non-variant or variant alleles of the KRAS LCS6Cultured endometrial stromal cells were injected under the kidney capsule of immunodeficient mice.Morphological appearance of the lesions under the kidney capsule of immune deficient mice 1 month after transplantation of hESC either with or without the variant KRAS. In all cases the transplanted endometrial cells formed endometriosis lesions with both glandular and stromal components.Proliferation marker expression in endometriotic lesions in mice. Nuclear staining for PCNA was more prominent in epithelium and stroma of the lesions formed by KRAS variant positive cells (54 ± 5% and 56 ± 6% in epithelium and stroma, respectively), compared to those derived from normal cells (8 ± 4% and 34 ± 6% in epithelium and stroma, respectively; p = 0.02 and p = 0.043) indicating higher proliferation levels in these cells.PR expression in endometriotic lesions with WT non-variant or variant KRAS allele in mice. Lesions created by hESC carrying KRAS variant allele were characterized by a smaller number of nuclei stained positively for PR in both glandular and stromal cells. The epithelium of the lesions with variant cells was found to have only 35 ± 5% of nuclei positively stained compared to 75 ± 3%, in the lesions with WT KRAS (p = 0.02). Only 13 ± 8% of nuclei of stromal cells in KRAS variant lesions were found to express PR compared to 78 ± 7% in the non-variant lesions (p = 0.028). Scale bar represents 25 µm.
Mentions: To evaluate differences in growth parameters in vivo, capacity for endometriotic lesion formation as well as histopathological and molecular characteristics of cultured endometrial stromal cells containing non-variant and variant alleles of the KRAS gene, a mouse model of endometriosis was used. We transplanted hESC obtained from subjects with and without the LCS6 variant under the kidney capsule of immune-deficient mice. Both non-variant and LCS6 variant cells formed endometriosis-like lesions with both glandular and stromal components (Fig 5a). The glandular component likely originated from progenitor stem cells in the culture as recently described (Cervello et al, 2011). Glandular cells are not identified in these cultures by the third passage, however, we cannot exclude a small number of contaminants (Taylor et al, 1998). Analysis of proliferation marker PCNA showed more cells (both epithelial and stromal) with stained nuclei in the lesion derived from LCS6 variant cells compared to those derived from non-variant cells. The percentage of stained nuclei in epithelium of lesions carrying variant KRAS allele was 54 ± 5% vs. 8 ± 1% in lesions created by non-variant hESC (p = 0.02). Stromal cells from lesions with the SNP exhibited 56 ± 5% of nuclei staining while in the lesions with WT KRAS, the percentage of positively stained nuclei was 34 ± 6% (p = 0.043; Fig 5b). These results suggest increased proliferation of cells harbouring the variant allele and were consistent with the results of the in vitro proliferation experiment. Apoptosis was assessed using cleaved caspase 3, and the amount of cleaved caspase 3 was equivalent in lesions derived from cells with either non-variant or LCS6 variant alleles (data not shown). Proliferation was increased without an increase in apoptosis in the LCS6 variant lesions.

Bottom Line: Endometriosis is found in 5-15% of women of reproductive age and is more frequent in relatives of women with the disease.Activation of KRAS results in de novo endometriosis in mice, however, activating KRAS mutations have not been identified in women.We screened 150 women with endometriosis for a polymorphism in a let-7 microRNA (miRNA) binding site in the 3'-UTR of KRAS and detected a KRAS variant allele in 31% of women with endometriosis as opposed to 5% of a large diverse control population.

View Article: PubMed Central - PubMed

Affiliation: Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.

Show MeSH
Related in: MedlinePlus