Stress responsive miR-31 is a major modulator of mouse intestinal stem cells during regeneration and tumorigenesis
- China Agricultural University, China
- Vanderbilt University Medical Center, United States
- University of California, Irvine, United States
- Shandong Academy of Agricultural Sciences, China
- Xiamen University, China
- University of Pennsylvania, United States
- Imperial College London, United Kingdom
Figures
Figure 1 with 8 supplements
MiR-31 promotes turnover of intestinal epithelial cells.
(A) Schematic picture of intestinal crypt showing Lgr5+ CBCs and Hopx+ cells. qRT-PCR for miR-31 in Lgr5-GFPhigh, Lgr5-GFPlow, Lgr5-GFPneg, Hopx- and Hopx+ sorted intestinal epithelial cells. n = 4 …
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Figure 1—source data 1
Source data for Figure 1C,E,F,G,H and I.
- https://doi.org/10.7554/eLife.29538.012
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Figure 1—source data 2
Source data for Figure 1—figure supplements 1–3.
- https://doi.org/10.7554/eLife.29538.013
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Figure 1—source data 3
Source data for Figure 1—figure supplements 4–7.
- https://doi.org/10.7554/eLife.29538.014
Figure 1—figure supplement 1
Generation of inducible TRE-miR-31 transgenic mice, constitutive miR-31 KO and conditional miR-31 KO mice.
(A) Schematic maps of constructs used to generate Rosa26-rtTA;TRE-miR31 (TRE-miR31) double transgenic mice. (B) qRT-PCR analysis for miR-31 in intestinal tissues from M2rtTA and TRE-miR31 mice at …
Figure 1—figure supplement 2
MiR-31 induction promotes crypt expansion.
(A) Quantification of length of villi and cypt/villus in M2rtTA and TRE-miR31 mice following 2 weeks of Dox induction. n = 3 biological replicates. ***p<0.001. (B) Representative histologic images …
Figure 1—figure supplement 3
MiR-31 induction promotes cell proliferation in crypts, and apoptosis at the top of villi.
(A) Immunohistochemistry for Ki67 and Cleaved Caspase 3 in jejunum from M2rtTA and TRE-miR31 mice following 7, 10 and 365 days of Dox induction. Scale bar: 100 μm. (B) Quantification of Ki67+ cells …
Figure 1—figure supplement 4
MiR-31 induction impairs cell differentiation.
(A) Immunostaining for ChgA, Mucin2 and Lysozyme in jejunum from M2rtTA and TRE-miR31 mice following 7, 10, 14 days and 1 year of Dox induction. n = 3 biological replicates at each time points. …
Figure 1—figure supplement 5
Loss of miR-31 led to shortened crypt.
(A) Quantification of body weight and intestinal length from miR-31+/− (Control) and miR-31−/− mice at 2 weeks, and 2 and 8 months of ages. n = 6 biological replicates for 2 weeks and 2 months; n = 4…
Figure 1—figure supplement 6
Loss of miR-31 does not affect cell differentiation.
(A) Immunofluorescence for ChgA, Mucin2 and Lysozyme in jejunum from miR-31+/− and miR-31−/− mice at indicated timepoints. Scale bar: 50 μm. (B) Quantification of ChgA+, Mucin2+ and Lysozyme+ cells …
Figure 1—figure supplement 7
Conditional deletion of miR-31 resulted in shortened crypt, reduced proliferation and enhanced apoptosis.
(A) Representative histologic images of jejunum from Vil-Cre and miR-31 cKO mice, and quantification of crypt height. Brackets mark crypts. Scale bar: 50 μm. n = 4 biological replicates. ***p<0.001. …
Figure 1—figure supplement 8
MiR-31 promotes cell turnover from crypt to villi.
Immunofluorescence for BrdU in M2rtTA, TRE-miR31, miR-31+/− and miR-31−/− intestinal crypts at indicated time points post 1 dose of BrdU pulse. The dashed lines marked the top of villi, middle line …
Figure 2 with 1 supplement
MiR-31 promotes expansion of Lgr5+ CBC stem cells.
(A) Representative FACS profiles and quantification of GFP positive intestinal epithelial cells (Lgr5-GFP+ cells) from an Lgr5-eGFP-CreERT reporter mice crossed with M2rtTA (control) and TRE-miR31 …
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Figure 2—source data 1
Source data for Figure 2.
- https://doi.org/10.7554/eLife.29538.017
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Figure 2—source data 2
Source data for Figure 2—figure supplement 1.
- https://doi.org/10.7554/eLife.29538.018
Figure 2—figure supplement 1
MiR-31 promotes ISC expansion.
(A) Immunohistochemistry for GFP (Lgr5-GFP) in intestines from Lgr5-eGFP-CreERT reporter mice crossed with M2rtTA (control), TRE-miR31, miR-31+/− (control) and miR-31−/− mice. Scale bar: 100 μm. The …
Figure 3 with 1 supplement
Loss of miR-31 abrogates epithelial regeneration following irradiation.
(A) Representative images of H&E and/or Ki67 immunohistochemistry from jejunum of irradiated Vil-Cre and cKO mice 2 hrs and 4 days post 12 Gy γ-IR. Quantification of Ki67+ regenerative foci per 1400 …
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Figure 3—source data 1
Source data for Figure 3.
- https://doi.org/10.7554/eLife.29538.021
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Figure 3—source data 2
Source data for Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.29538.022
Figure 3—figure supplement 1
MiR-31 is required for intestinal epithelial regeneration in response to γ-IR.
(A) Representative images of H&E from jejunum of irradiated M2rtTA, TRE-miR31, miR-31+/− and miR-31−/− mice 2 hrs post 12 Gy irradiation. n = 3 biological replicates. Scale bar: 200 μm. (B) …
Figure 4 with 1 supplement
MiR-31 activates Wnt pathway activity.
(A) Wnt activity was evaluated by Axin2-LacZ reporter activity in M2rtTA and TRE-miR31 intestine following 2 week Dox induction, and in miR-31+/− and miR-31−/− intestine. Blue, LacZ signals. n = 3 …
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Figure 4—source data 1
Source data for Figure 4.
- https://doi.org/10.7554/eLife.29538.025
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Figure 4—source data 2
Source data for Figure 4—figure supplement 1.
- https://doi.org/10.7554/eLife.29538.026
Figure 4—figure supplement 1
MiR-31 activates Wnt signaling pathway.
(A) Immunohistochemistry for β-Catenin in jejunum from miR-31+/− and miR-31−/− mice at 2 and 4 months of ages. Quantification of nuclear β-Catenin positive cells in miR-31+/− and miR-31−/− crypts. …
Figure 5 with 1 supplement
MiR-31 represses BMP/TGFβ signaling pathways.
(A) Western blotting for p-Smad1/5/8 and p-Smad2/3 in miR-31+/−, miR-31−/−, M2rtTA and TRE-miR31 intestine. Both M2rtTA and TRE-miR31 mice were treated with DOX for 2 weeks. β-Tubulin was used as a …
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Figure 5—source data 1
Source data for Figure 5.
- https://doi.org/10.7554/eLife.29538.029
Figure 5—figure supplement 1
MiR-31 represses BMP and TGFβ signaling pathways.
(A) Immunohistochemistry for p-Smad1/5/8 and p-Smad2/3 in jejunum from miR-31+/− and miR-31−/− mice at 2 months of age. Scale bar: 25 μm. (B) Immunohistochemistry for p-Smad1/5/8 and p-Smad2/3 in …
Figure 6 with 3 supplements
Identification of miR-31 target genes in intestinal epithelium.
(A, B) qRT-PCR analysis for Axin1, Gsk3b, Dkk1, Smad3, Bmpr1a, Smad4 and Tgfbr2 in miR-31+/− and miR-31−/− intestine (A), as well as M2rtTA and TRE-miR31 intestine following 2 weeks of Dox induction …
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Figure 6—source data 1
Source data for Figure 6.
- https://doi.org/10.7554/eLife.29538.034
Figure 6—figure supplement 1
Identification of miR-31 target genes.
(A) MiR-31 binding sites in 3’UTR of these putative target genes. (B) Mutant binding sites of genes, Axin1, Bmpr1a, Dkk1, Gsk3b, Smad3 and Smad4.
Figure 6—figure supplement 2
Identification of miR-31 target genes.
(A) Immunohistochemistry for Dkk1, Gsk3β, Axin1 and Bmpr1a in jejunum from miR-31+/− and miR-31−/− mice at 2 months of age. Scale bar: 25 μm. (B) Immunohistochemistry for Dkk1, Gsk3β, Axin1 and …
Figure 6—figure supplement 3
Identification of miR-31 target genes.
(A) Western blotting for Gsk3β, Dkk1, Axin1, Smad4 and Bmpr1a in cultured organoids from Vil-Cre and miR-31 cKO crypts. n = 4 technical replicates. β-Tubulin was used as a loading control. (B) …
Figure 7 with 1 supplement
MiR-31 promotes tumor growth in vivo.
(A) Gross appearance of tumors of HCT116 colorectal cancer cell xenograft 30 days post transplantation. HCT116 colorectal cancer cells were transfected with mimics-NC or miR-31 mimics, and …
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Figure 7—source data 1
Source data for Figure 7.
- https://doi.org/10.7554/eLife.29538.037
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Figure 7—source data 2
Source data for Figure 7—figure supplement 1.
- https://doi.org/10.7554/eLife.29538.038
Figure 7—figure supplement 1
MiR-31 promotes tumor growth.
(A) In vitro MTT proliferation assay of human colorectal cancer cell lines, HCT116, SW480 and LOVO upon transfection of miR-31 inhibitor (anti-miR-31), and inhibitor-NC, as well as miR-31 mimics and …
Figure 8
The STAT3 pathway mediates the induction of miR-31 caused by γ-IR.
(A) The schematic diagram showed two potential p65 binding sites and one p-STAT3 binding site in the miR-31 promoter. (B) qRT-PCR analysis for Rela, Ikk-b, IL-1, IL-6, IL-18, Tnf and Stat3 in the …
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Figure 8—source data 1
Source data for Figure 8.
- https://doi.org/10.7554/eLife.29538.040
Figure 9
The miR-31 working model in intestinal epithelial regeneration and tumorigenesis.
https://doi.org/10.7554/eLife.29538.041
Author response image 1
qRT-PCR analysis for miR-31 in HCT116, LOVO, COCA2, HT29 and SW480 colorectal cancer cells.
Author response image 2
H&E and immunohistochemistry for Ki67 in intestines from M2rtTA control and TRE-miR31 mutant mice 2 and 4 days post 6 Gy irradiation.
Author response image 3
Transcriptome profiling of miR-31 overexpressing intestines.
Author response image 4
Immunohistochemistry for β-Catenin (A), p-Smad1/5/8 (B), and p-Smad2/3 (C) in intestinal villi from M2rtTA and TRE-miR31 mice, as well as miR-31+/- and miR-31-/- mice.
Author response image 5
Immunohistochemistry for Dkk1, Axin1, Gsk3β and Bmpr1a in intestinal villi from M2rtTA and TRE-miR31 mice, as well as miR-31+/- and miR-31-/- mice.
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29538.042
