LIN28B promotes growth and tumorigenesis of the intestinal epithelium via Let-7

Abstract

The RNA-binding proteins LIN28A and LIN28B have diverse functions in embryonic stem cells, cellular reprogramming, growth, and oncogenesis. Many of these effects occur via direct inhibition of Let-7 microRNAs (miRNAs), although Let-7-independent effects have been surmised. We report that intestine targeted expression of LIN28B causes intestinal hypertrophy, crypt expansion, and Paneth cell loss. Furthermore, LIN28B fosters intestinal polyp and adenocarcinoma formation. To examine potential Let-7-independent functions of LIN28B, we pursued ribonucleoprotein cross-linking, immunoprecipitation, and high-throughput sequencing (CLIP-seq) to identify direct RNA targets. This revealed that LIN28B bound a substantial number of mRNAs and modestly augmented protein levels of these target mRNAs in vivo. Conversely, Let-7 had a profound effect; modulation of Let-7 levels via deletion of the mirLet7c2/mirLet7b genes recapitulated effects of Lin28b overexpression. Furthermore, intestine-specific Let-7 expression could reverse hypertrophy and Paneth cell depletion caused by Lin28b. This was independent of effects on insulin-PI3K-mTOR signaling. Our study reveals that Let-7 miRNAs are critical for repressing intestinal tissue growth and promoting Paneth cell differentiation. Let-7-dependent effects of LIN28B may supersede Let-7-independent effects on intestinal tissue growth. In summary, LIN28B can definitively act as an oncogene in the absence of canonical genetic alterations.

Keywords: CLIP-seq; LIN28B; Let-7; Paneth cells; colon cancer; intestinal epithelium.

Figure 1.

LIN28B drives mucosal hypertrophy in Vil-Lin28b mice. Vil-Lin28b mice generated using the 13-kb mouse Vil1 promoter exhibit low-, medium-, and high-level mRNA (A) and protein (B–D) expression. Immunofluorescence (IFC) of adult Vil-Lin28b reveals nuclear expression in crypts (E) but mostly cytoplasmic localization in villus epithelial cells of the jejunum (F). (G) Measurement of individual Let-7 levels reveals that Let-7 is significantly reduced, except for Let-7c. (H) Estimated total levels compiled from G. (I) mRNA levels of the Let-7 target Hmga2 is significantly elevated with increasing levels of Lin28b expression. (J–L) Small intestine mass of adult wild-type (WT) and Vil-Lin28b (TG) mice. (M) Adult TG mouse small intestines exhibit increased longitudinal folds, where the mucosa has buckled inward (arrowheads); 4× magnification is shown. Outlines of intestine submucosa (black line) and epithelial layer (pink line) are shown to the right. (N) Intestine thickness (mass/unit length) of Vil-Lin28b (TG) mice. Magnification: B-F, 400×; M, 40×. The mean is plotted ±SD (bar graphs), where n ≥ 3; (*) P < 0.05; (**) P < 0.01, using a paired t-test (Student's) for littermates. Box plot whiskers represent 1.5× the interquartile range (IQR) above the third quartile or below the first quartile. The minimum and maximum outliers, when present, are represented by small triangles.

Figure 2.

Identifying RNA targets of LIN28B. (A) Procedure for CLIP-seq for unbiased isolation of RNAs within LIN28B RNPs. Enrichment was determined relative to RNA sequencing (RNA-seq) from total RNA that was depleted of 28s and 18s rRNA. (B) CLIP of endogenous LIN28B in three cell lines using a rabbit monoclonal antibody followed by visualization by near-infrared (near-IR) fluorescence of TYE705-linked RNA adapters ligated to cross-linked RNPs. (B) For CLIP-seq, LIN28B-bound targets (in cross-linked RNPs) were excised following transfer to nitrocellulose (dotted box). Cross-linked RNPs were easily identified by near-IR fluorescence (red), while “empty LIN28B” (without cross-linked RNAs) (green) migrated just below these complexes. (B) Endogenous LIN28B was examined in Caco-2 cells, whereas exogenous LIN28B was examined in DLD1 and Lovo cells in Tet-inducible stable cell lines or colonic epithelium from Vil-Lin28b^Med mice. (C) Enriched peaks mapped largely to processed mRNAs primarily in the ORF or 3′ UTR for all data sets. (C) CIMSs also mapped largely to processed mRNAs. The “other” category includes reads that mapped upstream of or downstream from genes or at splice junctions. (D,E) LIN28B does not affect steady-state levels of mRNA targets, as determined by microarray and CLIP-seq analysis (enriched transcripts [black]; enriched transcripts with CIMSs [red]) of Vil-Lin28b colonic epithelium (D) and DLD1 and Lovo cells (E) (DLD1 and Lovo microarrays from King et al. 2011a). (F) Kyoto Encyclopedia of Genes and Genomics (KEGG) (Ogata et al. 1999) and WikiPathway (Pico et al. 2008) categories enriched for CLIP-seq target sets identified from Vil-Lin28b jejunum crypts, Vil-Lin28b colon epithelium, and Caco-2, DLD1, and Lovo cells. Adjusted P-values were calculated using the Benjamini-Hochberg step-up procedure for multiple test adjustment. (G) LIN28B targets were examined by Western blot in lysates from Vil-Lin28b (low, medium, and high) jejunum epithelium. (H) CTTN, CTNND1, and EEF2 levels correlated with Lin28b levels, suggesting that Lin28b promotes the translation of these targets. (I) Six separate Caco-2 clones with an inducible shRNA against LIN28B or nonspecific (ns) shRNA were examined by Western blots for effects on target protein levels at low-level (−dox) and high-level (+dox) expression of the shRNA. (J) Quantification indicates that CTTN, CLDN1, CTNND1, PKM2, and EIF4G1 levels were reduced following LIN28B knockdown. (K) In Caco-2 cells, sensitivity to LIN28B knockdown correlates with average fold enrichment (Supplemental Table S6) and the average CIMS score (m/k) of target mRNA.

Figure 3.

Let-7 controls growth of the small intestine. (A) Targeting strategy for generating a conditional (floxed) allele of mouse mirLet7c2 and mirLet7b. (B) Southern blots of two successfully targeted ES clones. (C) PCR of genomic DNA confirms intestine-specific deletion of floxed (L/L) mirLet7c2 and mirLet7b. Mature Let-7b and Let-7c levels are depleted (>90%) in intestine-specific knockouts using Vil-Cre in jejunum (D) and colon (E) epithelium. (F) Compilation of data from D and E reveals that deletion of mirLet7c2 and mirLet7b yields an estimated ∼40% and ∼60% reduction of total Let-7 levels in the jejunum and colon, respectively. Depletion of Let-7b and Let-7c causes intestinal hypertrophy (G), with a significant increase of intestine thickness at 12–16 wk of age (H). (I) H&E-stained sections of adult small intestine reveal folds/evaginations similar to those seen in Vil-Lin28b mice. (J) Rates of crypt fission are increased in Let-7^IEC-KO mice. Magnification: I, 40×. For bar graphs, mean is plotted ±SD, where n ≥ 3.

Figure 4.

Let-7 is required for the Paneth cell lineage. (A–D) IHC for lysozyme revealed a severe depletion of Paneth cells in the small intestine of Vil-Lin28b^Hi (TG-Hi) mice (B) but not Vil-Lin28b^Lo (TG-Lo) (C) or Let7^IEC-KO mice (D). (E) Coordinated depletion of Let-7 in Vil-Lin28b^Lo/Let7^IEC-KO mice causes severe depletion of Paneth cells. (F) mRNA levels for Paneth cell lineage markers (Lyz1, Mmp7, Defa5, Kit, and Wnt3) in jejunum epithelium are significantly decreased in all adult Vil-Lin28b mice, whereas Goblet markers (Dll4 and Spdef) were unaffected. Sox9, which is required for Paneth cell differentiation, is decreased in Vil-Lin28b^Med and Vil-Lin28b^Hi mice. (G) Vil-Lin28b^Lo/Let7^IEC-KO mice have a significant depletion of lineage markers at 4.5 wk of age. (H) Schematic of a doxycycline-inducible transgene for intestine-specific expression of Let-7a (iiLet7 mice), which yields a variegated pattern of GFP expression. Crypt epithelium was sorted into GFP-negative and GFP-positive fractions for RT–PCR for Let-7a (I) and lysozyme (Lyz1) (J). (K) Quantification of Paneth cell numbers in GFP-negative and GFP-positive crypts in Vil-Lin28b^Med/iiLet7 mice and iiLet7 mice. (L–N) Rescue of Paneth cell lineage in Vil-Lin28b^Med/iiLet7 mice as revealed by IFC for GFP (green outline) and lysozyme (red). Magnification: A–E, 200×; L–N, 400×. Littermates were compared when possible, and the mean is plotted ±SD (bar graphs), where n ≥ 3; (*) P < 0.05; (**) P < 0.01, using an unpaired t-test (Student's). For Paneth cell quantification, 25–30 crypts of each type (GFP⁻ or GFP⁺) of proximal small intestine from each mouse (n ≥ 3) were counted ±S.E.M, where P < 0.01 (**), using a paired t-test for GFP⁻ and GFP⁺ pairs of crypts.

Figure 5.

LIN28B drives epithelial hyperplasia and tumorigenesis. (A–F) Vil-Lin28 mice exhibit crypt hyperplasia in the small intestine, as determined by Ki67 IHC. (G) Quantification of Ki67-positive cells indicates a significant increase in Vil-Lin28b^Hi mice. (H) Tumors/polyps (arrowheads) were found in Vil-Lin28b mice aged for 12–17 mo, almost exclusively in the small intestine. H&E sections revealed the presence of adenomas (I), flat adenomas (J), and adenocarcinomas (K). (L) RT–PCR for Wnt target genes from eight microdissected adenomas (Ad) from the small intestines of four 15- to 17-mo-old Vil-Lin28b mice compared with normal (N) adjacent tissue. (M–O) IFC for β-catenin and transgene-generated Lin28b (anti-HA) in adenomatous tissue and adenocarcinomas from Vil-Lin28b mice. (P–W) Let-7 repression is responsible for crypt hyperplasia. (P–R) Let7^IEC-KO/Vil-Lin28b^Lo mice exhibit a significant increase of BrdU incorporation in the small intestine compared with wild-type (WT) littermates (jejunum) or Let7^IEC-KO mice (colon). (S–W) Restoration of Let-7 levels in Vil-Lin28b^Med/iiLet7 mice represses crypt hyperplasia, as determined by Ki67 expression and BrdU incorporation. (V,W) Quantification of Ki67-positive or BrdU-positive cells in GFP-negative and GFP-positive crypts in Vil-Lin28b^Med/iiLet7 mice and iiLet7 mice. For BrdU labeling, mice were injected with BrdU (100 mg/kg) 4 h prior to sacrifice. Magnification: A,C,E,Q,R, 200×; M,N,S–U, 400×; I–K, 100×. Littermates were compared when possible, and the mean is plotted ±SD (bar graphs), where n ≥ 3; (*) P < 0.05; (**) P < 0.01, using Student's unpaired t-test. For Ki67 and BrdU quantification, 50–60 crypts (G,P) or 25–30 crypts of each type (GFP⁻ or GFP⁺) (V,W) of proximal small intestines from each mouse were counted ±S.E.M, where P < 0.01 (**), using a paired t-test for GFP⁻ and GFP⁺ pairs of crypts. In G and H, a mixed model ANOVA (nested ANOVA) was also performed with and without Tukey adjustment.

Figure 6.

Let-7 represses Hmga2, Igf2bp1, Igf2bp2, Hmga2, E2f5, and Acvr1c in intestinal epithelial crypts. (A,B) Microarray analysis of adult jejunum and colon epithelium from Vil-Lin28b^Lo and Vil-Lin28b^Med mice reveals up-regulation of known and predicted Let-7 target genes. (A) A three-way mixed model ANOVA (Factor = LIN28B) across all tissues and genotypes revealed that Igf2bp2 and Hmga2 are the most significantly changed mRNAs. (B) Volcano plot using P-values (pairwise t-tests) relative to fold change of mRNA levels from jejunum epithelium of Vil-Lin28b^Med mice. Predicted Let-7 targets are indicated in green (miRBase predicted), blue (TargetScan predicted), or red (predicted by both algorithms), with Igf2bp2 and Hmga2 representing the most significantly changed mRNAs. Individual Let-7 miRNAs were assayed by RT–PCR in crypts from wild-type (WT), Let7^IEC-KO, Vil-Lin28b^Lo, and Vil-Lin28b^Lo/Let7^IEC-KO mouse jejunum (C), and total Let-7 values were estimated (D). (E) RT–PCR confirmed that Hmga2, Igf2bp1, Igf2bp2, E2f5, Acvr1c, and Nr6a1 mRNAs increase with decreasing Let-7 dosage. (F–I) IHC for Hmga2 shows increasing protein expression with decreasing dosage of Let-7 in Let7^IEC-KO, Vil-Lin28b^Lo, and Vil-Lin28b^Lo/Let7^IEC-KO mouse jejunum. (J) Restoration of mRNA levels of Let-7 targets Hmga2, Igf2bp1, Igf2bp2, E2f5, and Acvr1c in GFP-sorted crypts from iiLet7/Vil-Lin28b^Med and iiLet7 mice. (K) RT–PCR for Let-7 targets in wild-type mouse intestine reveals that Hmga2, Acvr1c, and Igf2bp1 are expressed at higher levels in the jejunum crypt compartment, whereas Igf2bp2 and E2f5 are expressed in both villus and crypts, and Nr6a1 appears restricted to villus epithelium. Magnification: F–I, 200×. Box plot whiskers represent 1.5× the interquartile range (IQR) above the third quartile or below the first quartile. The minimum and maximum outliers, when present, are represented by small squares. Littermates were compared when possible, and the mean is plotted ±SEM (bar graphs), where n ≥ 3; (*) P < 0.01; (**) P < 0.001, using Student's unpaired t-test.

Figure 7.

Model: LIN28B promotes intestinal growth via effects on Let-7 in the crypt. LIN28B is found exclusively in the nucleus of crypt epithelial cells. Here, Let-7 is critical for Paneth cell differentiation and the repression of crypt fission and proliferation, likely through repression of known Let-7 targets Hmga2 and Igf2bp1. In differentiated epithelium, LIN28B is found mostly in the cytoplasm. LIN28B may promote tissue growth via effects on mRNAs associated with metabolism, translation, and cell cycle. The effective dosage of Let-7 from a large reservoir of partially redundant miRNAs may help maintain Let-7 levels within a tolerable range, which prevents hyperplasia and supports Paneth cell differentiation. Depletion below a certain threshold fails to repress key Let-7 targets, yielding a “disease” state, such as hyperplasia or carcinogenesis. Transient depletion of Let-7 across this threshold, however, could be integral to normal processes, such as wound healing. Increased LIN28B levels drive carcinogenesis primarily through inhibition of Let-7, leading to deregulation of known oncogenes such as Igf2bp1 and Hmga2. Dotted lines represent proposed effects of LIN28B on mRNAs in these pathways.