pTRIPZ empty

Let-7 miRNA is critical for promoting differentiation, regulating metabolism, inhibiting cell proliferation, and inhibiting carcinogenesis in various tissues. Here, we describe global suppression of all Let-7 miRNAs in the intestinal epithelium by low-level tissue-specific expression of the Lin28b RNA-binding protein and conditional knockout of the MirLet7c-2/Mirlet7b locus. Ablation of Let-7 triggers the development of intestinal adenocarcinoma and is associated with reduced survival. Analysis of mouse and human intestinal cancer samples showed that stem cell markers were significantly associated with the loss of Let-7 miRNA expression and that expression of a number of Let-7 targets was elevated, including Hmga1 and Hmga2. Functional studies in 3-D enteroids indicate that Hmga2 is necessary and sufficient to mediate many features of Let-7 depletion, namely accelerated cell cycle progression and enhanced stem cell phenotype. Furthermore, inactivation of a single Hmga2 allele in mouse intestinal epithelium significantly inhibited Lin28b-driven tumorigenesis.

Figure 1. Hmga2 mediates the effects of Lin28b on stem cell colony formation and enteroid proliferation. Colony formation assay of small intestinal enteroids from wild-type (WT) mice (A) and Vil-Lin28bMed mice (B). C) Quantification of colony formation assay in WT mice and Vil-Lin28bMed mice. D) Schematic representation of lentiviral vector-mediated enteroid transduction using "tet-on" doxycycline (dox)-inducible vectors. Induced expression of the Turbo RFP reporter gene in the unmodified pTRIPz vector was readily observed in enteroid cells (H) compared with untreated cells (G). Phase contrast images of untreated and doxycycline-treated intestines are shown in (E) and (F), respectively. Hmga2 immunostaining of enteroid sections from pTRIPz transduction (I), pTRIPz-Hmga2 transduction (J), and pTRIPz-Hmga2 transduction plus doxycycline (K). L) Comparison of colony-forming potential of dissociated enteroid cells transduced with pTRIPz (empty), Arid3a, Hif3a, or Hmga2 vectors. Phase contrast images of colony formation assay of enteroids transduced with pTRIPz (empty) (M) or Hmga2 (N) vectors. O) Colony formation assay using Vil-Cre in non-transgenic (NTG) or Vil-Lin28bMed mice with or without inactivation of one conditional Hmga2 allele. EdU incorporation into enteroids transduced with pTRIPz-Hmga2 (P) or pTRIPz-Hif3a (Q) vectors was determined by flow cytometry. (Madison, Blair B., et al., 2015)