6 research outputs found
Hmga1 and Hmga2 proteins are increased in invasive areas of adenocarcinomas.
<p>Immunohistochemical staining for Hmga2 (A-C, G, H) and Hmga2 (D-F, I, J), in sections from WT small intestine (S.I.) (A, D), <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> S.I. (B, E), <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> adenoma (C, F), and <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> adenocarcinoma (AdenoCA) (G-J). An enlargement of a region containing invasive HMGA1-positive tumor cells from G (dotted yellow box) is pictured in H, while a region containing invasive HMGA2-positive tumor cells from I is likewise displayed in J. Pictures in A-F, H, and J are at same magnification (200x), with scale bar = 100 ÎĽm, while pictures in G and I are both at 40x, with scale bar = 250 ÎĽm.</p
Quantification of Let-7 target mRNA levels in intestinal epithelium crypts.
<p>A) Expression of Let-7 target mRNA levels in small intestine crypts isolated from <i>wild-type</i> (WT) and <i>Vil-Lin28b</i><sup><i>Med</i></sup> mice. B) Expression of Let-7 target mRNA levels in small intestine (jejunum) crypts isolated from <i>wild-type</i> (WT), <i>Vil-Lin28b</i><sup><i>Lo</i></sup>, <i>Let7</i><sup><i>IEC-KO</i></sup>, <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>+/-</i></sup>, and <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from <i>Vil-Lin28b</i><sup><i>Med</i></sup> mice vs. <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> 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 <i>Hprt</i> and <i>Actb</i>, 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.</p
Hmga2 mediates Lin28b effects on stem cell colony formation and enteroid proliferation.
<p>Colony formation assay of small intestine enteroids from wild-type (WT) mice (A) and <i>Vil-Lin28b</i><sup><i>Med</i></sup> mice (B). Two wells of a 24-well plate are pictured. C) Quantification of colony forming assay of WT mice and <i>Vil-Lin28b</i><sup><i>Med</i></sup> mice. D) Schematic of lentiviral vector-mediated transduction of enteroids with a “tet-on” doxycycline (dox)-inducible vector. Inducible expression of a turbo RFP reporter in the unmodified pTRIPz vector is readily observed in enteroids (H) vs. untreated cells (G). Phase contrast images of untreated and doxycycline treated enteroids are pictured in (E) and (F), respectively. Immunostaining for Hmga2 in sectioned enteroids from pTRIPz-transduced (I), pTRIPz-Hmga2-transduced (J), and pTRIPz-Hmga2-transduced plus doxycycline (K). L) Comparison of colony forming potential of cells from dissociated enteroids transduced with pTRIPz (empty), Arid3a, Hif3a, or Hmga2 vectors. Phase contrast images of colony formation assays from enteroids transduced with pTRIPz (empty) (M) or Hmga2 (N) vectors. O) Colony forming assays of non-transgenic mice (NTG) or <i>Vil-Lin28b</i><sup><i>Med</i></sup> mice with and without inactivation of one conditional Hmga2 allele using <i>Vil-Cre</i>. EdU incorporation of enteroids, as assayed by flow cytometry, transduced with pTRIPz-Hmga2 (P) or pTRIPz-Hif3a (Q) vectors. Cultures were treated with 100 ng/ml doxycycline in all experiments except in (F) and (H), which were treated with 500 ng/ml doxycycline. All experiments were performed at least in triplicate. 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 pTRIPz vector controls, unless noted otherwise.</p
Comprehensive depletion of all Let-7 miRNAs leads to the development of intestinal adenocarcinomas.
<p>A) Schematic of the intestine-specific deletion of the <i>Mirlet7c-2/Mirlet7b</i> floxed locus via <i>Villin-Cre</i> and expression of Lin28b with a <i>Villin-Lin28b-ires-tdTomato</i> 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 <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> 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 <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> mouse. F) Representative section of adenocarcinoma from a <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.</p
Let-7 and HMGA2 are associated with a stem cell signature in intestinal adenocarcinomas.
<p>A) Heat map of TCGA mRNA-seq colon and rectal adenocarcinoma dataset from UCSC Cancer Genome Browser (genome-cancer.ucsc.edu) comparing expression of Let-7 target mRNAs in normal tissue (N.T.) vs. cancer. Significant up-regulation (red) or down-regulation (green) is indicated below heatmap in plots of the-log(p-value) of Benjamini-Hochberg-corrected T-tests on the y-axis. T-test results are shown for expression in tumors vs. N.T. and in tumors associated with at least one lymph node metastases vs. tumors with no associated lymph node metastases. Inverse relationships for Let-7 and target mRNAs could be discerned by plotting miRNA-seq data against mRNA-seq data for Let-7c vs. <i>HMGA2</i> (B), Let-7a vs. <i>PLAGL2</i> (C), Let-7a vs. <i>HMGA2</i> (D), and Let-7a vs. <i>IGF2BP2</i> (E). F) Taqman QPCR for mature Let-7a and Let-7b miRNAs in a cohort of colon adenocarcinomas (N = 20) indicates that Let-7a and Let-7b are down-regulated. G) Intestinal epithelial stem cell markers <i>EPHB2</i>, <i>ASCL2</i>, and <i>LGR5</i> are significantly up-regulated in colon cancer vs. normal adjacent tissues. H) Mature Let-7a and Let-7b levels are tightly correlated in these tissue specimens. I) Let-7a and Let-7b levels are inversely proportional to mRNA levels of stem cell markers <i>EPHB2</i> and <i>LGR5</i>, suggesting that Let-7 may repress a stem cell signature. J) Expression of stem cell markers is dramatically up-regulated in <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> tumors, relative to WT jejunum, with a trend for up-regulation in <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> jejunum, relative to WT. K) Comparison of stem cell marker expression and Let-7 target mRNA expression levels in WT jejunum, <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> jejunum, and <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> tumors by linear regression yielded Pearson correlation coefficients, with <i>Arid3a</i>, <i>Hmga1</i>, and <i>Hmga2</i> correlating very highly with expression of stem cell markers. L) HMGA2 and LGR5 expression from the TCGA mRNA-seq colon and rectal adenocarcinoma dataset exhibit significant positive correlation. Expression analysis (F-K) was performed by QPCR, normalized to <i>Hprt</i> and <i>Actb</i>, with n = 3–4 for each mouse genotype with error bars representing +/–the S.E.M. Human QPCR was normalized to <i>PPIA</i> and <i>B2M</i>, with error bars representing +/–the S.E.M. 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.</p
Identification of Let-7 targets up-regulated specifically in transformed cells from intestinal adenocarcinomas.
<p>A) Schematic of experimental procedure where tumors were micro-dissected from the small intestine (S.I.) of <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> mice and cultured as epithelial tumoroid/enteroids (T/E), grown in ENR medium, and tumoroid cysts (TC), grown in medium lacking Noggin and Rspo1. B) Typical tumoroid grown in ENR medium. C) Tumoroid cysts grown in basal medium containing EGF. D) Let-7 miRNAs are repressed consistently in tumoroid/enteroids (TE) and tumoroid cysts (TC). E) Wnt (Tcf4/β-catenin) target genes Axin2, CD44 and cMyc mRNAs were up-regulated in tumors, T/E, and TC. F) Transcripts with highest expression in tumor or tumoroid, but tend to be down-regulated in tumoroid cysts. G) Transcripts that maintain high expression and/or are increased in tumoroid cysts. Note logarithmic scale where Hmga2 mRNA is induced approximately 200-fold in TC compared to wild-type S.I. Immunostaining in <i>Lin28b</i><sup><i>Lo</i></sup><i>/Let7</i><sup><i>IEC-KO</i></sup> adenomas (H, I) and adenocarcinomas (J, K) revealed that nuclear β-catenin (H, J) and Hmga2 (I, K) are often co-expressed at high levels. Expression analysis was performed by Q-RT-PCR, normalized to <i>Hprt</i> and <i>Actb</i>, with n = 3–4 for each tissue/organoid with error bars representing +/–the S.E.M. 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 wild-type small intestine. One-way ANOVA standard weighted-means analysis was also performed in D-G, with p-values < 0.05 indicated above each gene. Tukey's HSD (honest significant difference) post-test was also performed in D-G, with samples p < 0.05 (red asterisk) and p < 0.01 (†) indicated, relative to mean of WT small intestine (jej.).</p