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

Abstract

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. KRAS mRNA and protein expression were increased in cultured endometrial stromal cells of women with the KRAS variant. Increased KRAS protein was due to altered miRNA binding as demonstrated in reporter assays. Endometrial stromal cells from women with the KRAS variant showed increased proliferation and invasion. In a murine model, endometrial xenografts containing the KRAS variant demonstrated increased proliferation and decreased progesterone receptor levels. These findings suggest that an inherited polymorphism of a let-7 miRNA binding site in KRAS leads to abnormal endometrial growth and endometriosis. The LCS6 polymorphism is the first described genetic marker of endometriosis risk.

Copyright © 2012 EMBO Molecular Medicine.

Figures

Figure 1. KRAS expression in hESCs obtained from endometrium of 10 women without endometriosis, eutopic endometrium of 10 women with endometriosis carrying WT KRAS and 10 women with endometriosis carrying the variant allele of KRAS gene at the LCS6 site

1. q-RT-PCR results show comparatively low levels of KRAS mRNA in normal endometrium, increased KRAS mRNA in hESC from women with endometriosis and the WT KRAS allele (p = 0.0007) and highest expression of KRAS mRNA in hESC carrying the variant KRAS allele (p = 0.00018 when compared to normal hESC, p = 0.0049 when compared to hESC from endometriosis patients with WT allele). (*, difference is significant when compared to normal hESC; **, difference is significant when compared to hESC from women with endometriosis homozygous for the WT KRAS allele).

2. Western blot results show a 2.8-fold increase in KRAS protein in hESC with the variant allele.

Figure 2. Let-7 miRNA family expression in hESC from 10 subjects without endometriosis (normal controls), 10 subjects with endometriosis carrying WT KRAS and 10 subjects with endometriosis and the variant allele of KRAS gene

q-RT-PCR results show a trend towards decreased transcript levels of all let-7 miRNAs in hESC from subjects with endometriosis compared to normal hESC from subjects without endometriosis (normal control). Endometrial cells from subjects with endometriosis with the LCS6 variant in the KRAS gene showed lower levels of let-7a, b and e compared to hESC from endometriosis with non-variant KRAS (*p = 0.0047, 0.003 and 0.05, respectively). let-7a and b were also lower in KRAS-variant cells from women with endometriosis compared to normal hESC (‡p = 0.05 and 0.02, respectively).

Figure 3. The effect of siRNA mimicking let-7 action on luciferase expression from the reporter plasmid carrying WT or variant KRAS allele in Dual Luciferase Reporter assay

Normal hESC were co-transfected with pGL3 vector carrying the variant LCS6 of the KRAS gene and either siRNA modified to bind the variant LCS6 or a negative control RNA sequence. Luciferase activity from the reporter plasmid carrying the KRAS-variant allele was greatly increased compared to that from a reporter plasmid containing non-variant allele at baseline. There was a 70% reduction in luciferase activity when the KRAS-variant allele was co-transfected with siRNA designed to bind the LCS6 site (p = 0.045). No significant decrease in luciferase activity was obtained after co-transfecting this reporter plasmid with a control RNA sequence. The pGL3 control vector was used to assess transfection efficiency. Transfection with pGL3 carrying non-variant LCS6 of KRAS gene resulted in minimal luciferase activity likely due to inhibitory effects of endogenous let-7 miRNAs. Each experiment was carried out in duplicate in three separate experiments using cells from individual subjects. WT KRAS, hESC transfected with WT KRAS; KRAS V, hESC transfected with KRAS variant allele; KRAS V + siRNA, hESC co-transfected with KRAS variant allele and siRNA; KRAS V + siRNA NC, hESC co-transfected with KRAS variant allele and siRNA negative control; pGL3 PC, hESC transfected with pGL3 control vector.

Figure 4. The effect of the KRAS variant allele on proliferation and invasion capacity of hESC

1. The effect of the KRAS variant allele on proliferation of hESC from women with endometriosis as determined by BrdU incorporation. There was a 71% increase (*p = 0.04) in the BrdU label in hESC with the variant allele (n = 5) versus WT KRAS LCS6 (n = 5). These results indicated an increase in cell proliferation rate of hESC containing the mutant KRAS LCS6.

2. The effect of the KRAS variant allele on hESC invasion capacity. An invasion assay in which hESC from women with and without endometriosis and with the WT non-variant or the alternative KRAS allele was used to determine the ability to invade extracellular matrix. There was a significant increase in the invasion of hESC containing the variant allele (n = 9) compared to hESC without the variant allele (n = 6) (*p = 0.013). The difference between normal cells (n = 6) and cells from endometriosis with WT KRAS was not significant.

Figure 5. Morphological and molecular features of endometriotic lesions containing the WT non-variant or variant alleles of the KRAS LCS6

Cultured endometrial stromal cells were injected under the kidney capsule of immunodeficient mice.

1. 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.

2. 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.

3. 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.