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Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2.

Madison BB, Jeganathan AN, Mizuno R, Winslow MM, Castells A, Cuatrecasas M, Rustgi AK - PLoS Genet. (2015)

Bottom Line: Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2.In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b.In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri, United States of America; Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACT
Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

No MeSH data available.


Related in: MedlinePlus

Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.A) Schematic of the intestine-specific deletion of the Mirlet7c-2/Mirlet7b floxed locus via Villin-Cre and expression of Lin28b with a Villin-Lin28b-ires-tdTomato transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a Lin28bLo/Let7IEC-KO mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse. F) Representative section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.
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pgen.1005408.g001: Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.A) Schematic of the intestine-specific deletion of the Mirlet7c-2/Mirlet7b floxed locus via Villin-Cre and expression of Lin28b with a Villin-Lin28b-ires-tdTomato transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a Lin28bLo/Let7IEC-KO mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse. F) Representative section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.

Mentions: Vil-Lin28bLow mice and Let7IEC-KO mice were generated and described previously [18]. To generate compound mutant animals we used a low-expressing transgenic line (Lin28bLo), in which we could not detect measureable changes in either protein or mRNA levels of Let-7-independent Lin28b targets [18]. These compound Lin28bLo/Let7IEC-KO mice, exhibit depletion of all Let-7 miRNAs specifically in intestinal epithelial cells (IEC) achieved through deletion of the MirLet7c-2/MirLet7b locus and repression of all other Let-7 miRNAs through inhibition by Lin28b [18] (and Fig 1A). Lin28bLo/Let7IEC-KO mice thrived initially, with normal behavior and weight gain, but displayed significantly increased mortality and morbidity starting around 6 months of age, whereas neither Vil-Lin28bLo nor Let7IEC-KO age-matched mice exhibited any overt phenotype (Fig 1B). Surviving Lin28bLo/Let7IEC-KO were sacrificed between 10 and 14 months of age and exhibited a significant incidence of adenomas and adenocarcinomas, restricted to the small intestine, with an average of 2.86 lesions per mouse and 100% penetrance (S1 Table and Fig 1C, 1D and 1E). Six of seven Lin28bLo/Let7IEC-KO mice developed invasive adenocarcinoma (S1 Table and Fig 1C, 1D and 1E). Tumors from mice also displayed nuclear localization of β-catenin (Fig 1F), indicative of constitutive activation of the Wnt signaling pathway. The severity of the Lin28bLo/Let7IEC-KO phenotype was substantially more dramatic than in Vil-Lin28bLo or Vil-Lin28bMed mice (18). Vil-Lin28bMed mice express higher levels of Lin28b, have partially depleted Let-7 miRNAs and develop adenocarcinomas of the small intestine as do Lin28bLo/Let7IEC-KO mice but do not exhibit a phenotype as severe as Lin28bLo/Let7IEC-KO mice (18).


Let-7 Represses Carcinogenesis and a Stem Cell Phenotype in the Intestine via Regulation of Hmga2.

Madison BB, Jeganathan AN, Mizuno R, Winslow MM, Castells A, Cuatrecasas M, Rustgi AK - PLoS Genet. (2015)

Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.A) Schematic of the intestine-specific deletion of the Mirlet7c-2/Mirlet7b floxed locus via Villin-Cre and expression of Lin28b with a Villin-Lin28b-ires-tdTomato transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a Lin28bLo/Let7IEC-KO mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse. F) Representative section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4526516&req=5

pgen.1005408.g001: Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.A) Schematic of the intestine-specific deletion of the Mirlet7c-2/Mirlet7b floxed locus via Villin-Cre and expression of Lin28b with a Villin-Lin28b-ires-tdTomato transgene, which repress all 8 of the Let-7 clusters. Let-7 miRNA genes are shown as black hairpins while non-let-7 miRNA genes are depicted as gray hairpins. B) Kaplan-Meier plot showing survival over 10 months. C) Representative small intestine from a Lin28bLo/Let7IEC-KO mouse containing two tumors, T1 and T2 (box outline with yellow dotted lines). D) Large tumor from (C) dissected with luminal side facing outward. E) H&E stained paraffin section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse. F) Representative section of adenocarcinoma from a Lin28bLo/Let7IEC-KO mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.
Mentions: Vil-Lin28bLow mice and Let7IEC-KO mice were generated and described previously [18]. To generate compound mutant animals we used a low-expressing transgenic line (Lin28bLo), in which we could not detect measureable changes in either protein or mRNA levels of Let-7-independent Lin28b targets [18]. These compound Lin28bLo/Let7IEC-KO mice, exhibit depletion of all Let-7 miRNAs specifically in intestinal epithelial cells (IEC) achieved through deletion of the MirLet7c-2/MirLet7b locus and repression of all other Let-7 miRNAs through inhibition by Lin28b [18] (and Fig 1A). Lin28bLo/Let7IEC-KO mice thrived initially, with normal behavior and weight gain, but displayed significantly increased mortality and morbidity starting around 6 months of age, whereas neither Vil-Lin28bLo nor Let7IEC-KO age-matched mice exhibited any overt phenotype (Fig 1B). Surviving Lin28bLo/Let7IEC-KO were sacrificed between 10 and 14 months of age and exhibited a significant incidence of adenomas and adenocarcinomas, restricted to the small intestine, with an average of 2.86 lesions per mouse and 100% penetrance (S1 Table and Fig 1C, 1D and 1E). Six of seven Lin28bLo/Let7IEC-KO mice developed invasive adenocarcinoma (S1 Table and Fig 1C, 1D and 1E). Tumors from mice also displayed nuclear localization of β-catenin (Fig 1F), indicative of constitutive activation of the Wnt signaling pathway. The severity of the Lin28bLo/Let7IEC-KO phenotype was substantially more dramatic than in Vil-Lin28bLo or Vil-Lin28bMed mice (18). Vil-Lin28bMed mice express higher levels of Lin28b, have partially depleted Let-7 miRNAs and develop adenocarcinomas of the small intestine as do Lin28bLo/Let7IEC-KO mice but do not exhibit a phenotype as severe as Lin28bLo/Let7IEC-KO mice (18).

Bottom Line: Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2.In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b.In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

View Article: PubMed Central - PubMed

Affiliation: Division of Gastroenterology, Washington University School of Medicine, Saint Louis, Missouri, United States of America; Department of Medicine, Washington University School of Medicine, Saint Louis, Missouri, United States of America.

ABSTRACT
Let-7 miRNAs comprise one of the largest and most highly expressed family of miRNAs among vertebrates, and is critical for promoting differentiation, regulating metabolism, inhibiting cellular proliferation, and repressing carcinogenesis in a variety of tissues. The large size of the Let-7 family of miRNAs has complicated the development of mutant animal models. Here we describe the comprehensive repression of all Let-7 miRNAs in the intestinal epithelium via low-level tissue-specific expression of the Lin28b RNA-binding protein and a conditional knockout of the MirLet7c-2/Mirlet7b locus. This ablation of Let-7 triggers the development of intestinal adenocarcinomas concomitant with reduced survival. Analysis of both mouse and human intestinal cancer specimens reveals that stem cell markers were significantly associated with loss of Let-7 miRNA expression, and that a number of Let-7 targets were elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids revealed that Hmga2 is necessary and sufficient to mediate many characteristics of Let-7 depletion, namely accelerating cell cycle progression and enhancing a stem cell phenotype. In addition, inactivation of a single Hmga2 allele in the mouse intestine epithelium significantly represses tumorigenesis driven by Lin28b. In aggregate, we conclude that Let-7 depletion drives a stem cell phenotype and the development of intestinal cancer, primarily via Hmga2.

No MeSH data available.


Related in: MedlinePlus