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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

Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from wild-type (WT) and Vil-Lin28bMed mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from wild-type (WT), Vil-Lin28bLo, Let7IEC-KO, Lin28bLo/Let7+/-, and Lin28bLo/Let7IEC-KO mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from Vil-Lin28bMed mice vs. Lin28bLo/Let7IEC-KO mice reveals similar expression changes in each model of Let-7 depletion, with significant correlation (Pearson correlation shown). Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3 mice for each genotype at 12 weeks of age with error bars representing +/–the S.E.M. D) Identification of conserved Let-7 target genes in ten of eleven Let-7 target genes based upon TargetScan.org prediction. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to WT small intestine. One-way ANOVA standard weighted-means analysis was also performed in B, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in B, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine.
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pgen.1005408.g002: Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from wild-type (WT) and Vil-Lin28bMed mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from wild-type (WT), Vil-Lin28bLo, Let7IEC-KO, Lin28bLo/Let7+/-, and Lin28bLo/Let7IEC-KO mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from Vil-Lin28bMed mice vs. Lin28bLo/Let7IEC-KO mice reveals similar expression changes in each model of Let-7 depletion, with significant correlation (Pearson correlation shown). Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3 mice for each genotype at 12 weeks of age with error bars representing +/–the S.E.M. D) Identification of conserved Let-7 target genes in ten of eleven Let-7 target genes based upon TargetScan.org prediction. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to WT small intestine. One-way ANOVA standard weighted-means analysis was also performed in B, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in B, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine.

Mentions: Let-7 targets were examined in small intestine crypts from Vil-Lin28b and Lin28bLo/Let7IEC-KO mice. RNA microarray expression analysis was previously performed on Vil-Lin28bMed total small intestine epithelia and we verified elevation of Hmga1, Hmga2, Igf2bp1, Igf2bp2, E2f5, Acvr1c, Nr6a1, Hif3a, Arid3a, Plagl2, Trim6, Ddx19a, and Mycn (Fig 2A and [18]). We also observed significant elevation of mRNAs for these Let-7 targets in crypts from small intestine epithelia from Lin28bLo/Let7IEC-KO (Fig 2B). Expression of all Let-7 targets also correlated significantly between Lin28bLo/Let7IEC-KO and Vil-Lin28bMed intestine crypts, with Hmga2, Igf2bp2, Hif3a, Arid3a, and E2f5 being the most highly induced targets in both models (Fig 2C). All targets contained conserved Let-7 sites in the 3’UTR or coding sequence, except for Trim6, for which only the mouse mRNA possesses Let-7 sites (Fig 2D). In addition to our findings for HMGA2, IGF2BP1, and IGF2BP2, there is experimental evidence that HMGA1, E2F5, and ARID3A are also direct targets of Let-7 [6,31,32].


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)

Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from wild-type (WT) and Vil-Lin28bMed mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from wild-type (WT), Vil-Lin28bLo, Let7IEC-KO, Lin28bLo/Let7+/-, and Lin28bLo/Let7IEC-KO mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from Vil-Lin28bMed mice vs. Lin28bLo/Let7IEC-KO mice reveals similar expression changes in each model of Let-7 depletion, with significant correlation (Pearson correlation shown). Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3 mice for each genotype at 12 weeks of age with error bars representing +/–the S.E.M. D) Identification of conserved Let-7 target genes in ten of eleven Let-7 target genes based upon TargetScan.org prediction. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to WT small intestine. One-way ANOVA standard weighted-means analysis was also performed in B, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in B, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine.
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pgen.1005408.g002: Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from wild-type (WT) and Vil-Lin28bMed mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from wild-type (WT), Vil-Lin28bLo, Let7IEC-KO, Lin28bLo/Let7+/-, and Lin28bLo/Let7IEC-KO mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from Vil-Lin28bMed mice vs. Lin28bLo/Let7IEC-KO mice reveals similar expression changes in each model of Let-7 depletion, with significant correlation (Pearson correlation shown). Expression analysis was performed by Q-RT-PCR, normalized to Hprt and Actb, with n = 3 mice for each genotype at 12 weeks of age with error bars representing +/–the S.E.M. D) Identification of conserved Let-7 target genes in ten of eleven Let-7 target genes based upon TargetScan.org prediction. Student’s two-tailed T-tests were performed to determine significance with * p-value < 0.05, ** p-value < 0.01, and *** p-value < 0.001, relative to WT small intestine. One-way ANOVA standard weighted-means analysis was also performed in B, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in B, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine.
Mentions: Let-7 targets were examined in small intestine crypts from Vil-Lin28b and Lin28bLo/Let7IEC-KO mice. RNA microarray expression analysis was previously performed on Vil-Lin28bMed total small intestine epithelia and we verified elevation of Hmga1, Hmga2, Igf2bp1, Igf2bp2, E2f5, Acvr1c, Nr6a1, Hif3a, Arid3a, Plagl2, Trim6, Ddx19a, and Mycn (Fig 2A and [18]). We also observed significant elevation of mRNAs for these Let-7 targets in crypts from small intestine epithelia from Lin28bLo/Let7IEC-KO (Fig 2B). Expression of all Let-7 targets also correlated significantly between Lin28bLo/Let7IEC-KO and Vil-Lin28bMed intestine crypts, with Hmga2, Igf2bp2, Hif3a, Arid3a, and E2f5 being the most highly induced targets in both models (Fig 2C). All targets contained conserved Let-7 sites in the 3’UTR or coding sequence, except for Trim6, for which only the mouse mRNA possesses Let-7 sites (Fig 2D). In addition to our findings for HMGA2, IGF2BP1, and IGF2BP2, there is experimental evidence that HMGA1, E2F5, and ARID3A are also direct targets of Let-7 [6,31,32].

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