Univariate and multivariate analysis of miR-148a expression and DFS in stage II/III CRC patients.

a<p>Proximal colon, located above splenic flexture; distal colon, located in splenic flexture or below.</p>b<p>p<0.05.</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>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} 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>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse. F) Representative section of adenocarcinoma from a <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mouse immunostained for β-catenin, showing a nuclear pattern of localization. Scale bars in (E) and (F) = 100 μm.</p

The Clinical Significance of MiR-148a as a Predictive Biomarker in Patients with Advanced Colorectal Cancer

<div><h3>Aim</h3><p>Development of robust prognostic and/or predictive biomarkers in patients with colorectal cancer (CRC) is imperative for advancing treatment strategies for this disease. We aimed to determine whether expression status of certain miRNAs might have prognostic/predictive value in CRC patients treated with conventional cytotoxic chemotherapies.</p> <h3>Methods</h3><p>We studied a cohort of 273 CRC specimens from stage II/III patients treated with 5-fluorouracil-based adjuvant chemotherapy and stage IV patients subjected to 5-fluorouracil and oxaliplatin-based chemotherapy. In a screening set (n = 44), 13 of 21 candidate miRNAs were successfully quantified by multiplex quantitative RT-PCR. In the validation set comprising of the entire patient cohort, miR-148a expression status was assessed by quantitative RT-PCR, and its promoter methylation was quantified by bisulfite pyrosequencing. Lastly, we analyzed the associations between miR-148a expression and patient survival.</p> <h3>Results</h3><p>Among the candidate miRNAs studied, miR-148a expression was most significantly down-regulated in advanced CRC tissues. In stage III and IV CRC, low miR-148a expression was associated with significantly shorter disease free-survival (DFS), a worse therapeutic response, and poor overall survival (OS). Furthermore, miR-148a methylation status correlated inversely with its expression, and was associated with worse survival in stage IV CRC. In multivariate analysis, miR-148a expression was an independent prognostic/predictive biomarker for advanced CRC patients (DFS in stage III, low vs. high expression, HR 2.11; OS in stage IV, HR 1.93).</p> <h3>Discussion</h3><p>MiR-148a status has a prognostic/predictive value in advanced CRC patients treated with conventional chemotherapy, which has important clinical implications in improving therapeutic strategies and personalized management of this malignancy.</p> </div

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>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} S.I. (B, E), <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} adenoma (C, F), and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} 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 μm, while pictures in G and I are both at 40x, with scale bar = 250 μm.</p

MiR-148 expression status and clinicopathologic characteristics of CRC patients.

a<p>The difference was analyzed by Mann-Whitney U test.</p>b<p>The difference was analyzed by Fisher's exact test.</p>c<p>Proximal colon, located above splenic flexure; distal colon, located in splenic flexure or below.</p>d<p>The difference was analyzed by the chi-square test.</p

Associations between miR-148a status and therapeutic response or survival in stage IV CRC patients treated with 5-FU and oxaliplatin.

<p><b>A</b>. Therapeutic response according to miR-148a expression. Complete response, CR; partial response, PR; stable disease, SD; progressive disease; PD. <b>B</b>. Kaplan-Meyer curves for progression-free survival (PFS, left panel) and OS (right panel) in stage IV patients according to miR-148a expression. <b>C</b>. Kaplan-Meyer curves for PFS (left panel) and OS (right panel) in stage IV patients according to miR-148a methylation.</p

Univariate and multivariate analysis of miR-148a expression, methylation and overall survival in stage IV MSS CRC patients.

a<p>p<0.05.</p

Supplementary Methods and Figure Legend from ZEB1 Promotes Invasiveness of Colorectal Carcinoma Cells through the Opposing Regulation of uPA and PAI-1

PDF file - 195K</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>^<i>Med</i> 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>^<i>Med</i> 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>^<i>Med</i> 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

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>^<i>Med</i> 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>^<i>Lo</i>, <i>Let7</i>^{<i>IEC-KO</i>}, <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^<i>+/-</i>, and <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} mice. C) Comparison of Let-7 target mRNA changes in small intestine crypts from <i>Vil-Lin28b</i>^<i>Med</i> mice vs. <i>Lin28b</i>^<i>Lo</i><i>/Let7</i>^{<i>IEC-KO</i>} 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