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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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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 assayNormal 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.
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fig03: 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 assayNormal 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.

Mentions: To demonstrate that the increased level of KRAS protein seen in cultured hESC from subjects with the KRAS variant was in fact due to altered let-7 binding to the mutant LCS6, we introduced an siRNA construct designed to rescue let-7 activity by binding to the altered LCS6. Normal hESCs were co-transfected with a luciferase reporter construct carrying the variant LCS6, the siRNA or a siRNA negative control (Fig 3). Luciferase activity in the cells transfected with a reporter carrying the KRAS variant allele was approximately 30-fold greater than the activity from the reporter with the WT allele (relative luciferase activity: 0.1 ± 0.001 vs. 2.9 ± 0.05). The siRNA targeting the LCS6 variant RNA reduced the luciferase activity in the cells with the reporter containing the KRAS LCS6 variant by 70% (to 0.9 ± 0.13; p = 0.045).


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)

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 assayNormal 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.
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Related In: Results  -  Collection

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fig03: 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 assayNormal 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.
Mentions: To demonstrate that the increased level of KRAS protein seen in cultured hESC from subjects with the KRAS variant was in fact due to altered let-7 binding to the mutant LCS6, we introduced an siRNA construct designed to rescue let-7 activity by binding to the altered LCS6. Normal hESCs were co-transfected with a luciferase reporter construct carrying the variant LCS6, the siRNA or a siRNA negative control (Fig 3). Luciferase activity in the cells transfected with a reporter carrying the KRAS variant allele was approximately 30-fold greater than the activity from the reporter with the WT allele (relative luciferase activity: 0.1 ± 0.001 vs. 2.9 ± 0.05). The siRNA targeting the LCS6 variant RNA reduced the luciferase activity in the cells with the reporter containing the KRAS LCS6 variant by 70% (to 0.9 ± 0.13; p = 0.045).

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