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MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells

An Author Correction to this article was published on 28 February 2019

An Erratum to this article was published on 01 April 2014

This article has been updated

Abstract

Asymmetrical cell division (ACD) maintains the proper number of stem cells to ensure self-renewal. In cancer cells, the deregulation of ACD disrupts the homeostasis of the stem cell pool and promotes tumour growth. However, this mechanism is unclear. Here, we show a reduction of ACD in spheroid-derived colorectal cancer stem cells (CRCSCs) compared with differentiated cancer cells. The epithelial–mesenchymal transition (EMT) inducer Snail is responsible for the ACD-to-symmetrical cell division (SCD) switch in CRCSCs. Mechanistically, Snail induces the expression of microRNA-146a (miR-146a) through the β-catenin–TCF4 complex. miR-146a targets Numb to stabilize β-catenin, which forms a feedback circuit to maintain Wnt activity and directs SCD. Interference with the Snail–miR-146a–β-catenin loop by inhibiting the MEK or Wnt activity reduces the symmetrical division of CRCSCs and attenuates tumorigenicity. In colorectal cancer patients, the SnailHighNumbLow profile is correlated with cetuximab resistance and a poorer prognosis. This study elucidates a unique mechanism of EMT-induced CRCSC expansion.

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Figure 1: SCD predominantly occurs in CRCSCs.
Figure 2: Snail is critical in mediating tumour-initiating capability and SCD and induces miR-146a expression.
Figure 3: miR-146a is critical in maintaining tumour-initiating capability and symmetric division.
Figure 4: Snail activates MIR146A transcription through the β-catenin–TCF4 complex.
Figure 5: Repression of Numb by miR-146a promotes stemness and symmetric division.
Figure 6: Numb promotes β-catenin degradation to direct asymmetric division.
Figure 7: The miR-146a–Numb axis is not correlated with the Notch pathway.
Figure 8: Significance of the Snail–miR-146a–Numb axis in CRC.

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Change history

  • 26 February 2014

    In the version of this Article originally published, the labels of the key in the upper panel of Fig. 7h should have read 'HT29-Vec' (black) and 'HT29-146a' (grey). This error has now been corrected in the online versions of the Article.

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Acknowledgements

We thank the National Yang-Ming University-VGH Genome Research Center for technical support. We thank K.-J. Wu (National Yang-Ming University) for providing the wild-type and mutant β-catenin constructs. This work was supported by the National Science Council (NSC102-2321-B-010-006 and 102-2314-B-010-044 to M-H.Y.; 101-2321-B-010-011, 102-2314-B-010-045, 101-2320-B-010-059-MY3 and 101-2627-B-010-003 to H-W.W.; 100-2321-B-075-004 and 101-2321-B-075-001 to J-K.J.), the National Health Research Institutes (NHRI-EX102-10037BI to M-H.Y. and NHRI-EX102-10254SI to H-W.W.), Taipei City Hospital (10201-62-070 to H-W.W.), a grant from the Ministry of Education, Aim for the Top University Plan (to M-H.Y. and H-W.W.), a grant from the Department of Health, Center of Excellence for Cancer Research (DOH102-TD-C-111-007 to M-H.Y. and H-W.W.), and the UST-UCSD International Center for Excellence in Advanced Bioengineering sponsored by the Taiwan NSC I-RiCE (NSC101-2911-I-009-101 to H-W.W.).

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Authors

Contributions

W-L.H., H-W.W. and M-H.Y. conceived and designed the experiments. W-L.H. and H-Y.L. performed the experiments with the assistance of Y-P.T. for a portion of the plasmid construction. W-L.H., H-W.W. and M-H.Y. analysed the data with the assistance of J-K.J. for the clinical data analysis. T-S.H. and C-Y.Y. helped with the analysis of bioinformatic data. W-L.H. and M-H.Y. wrote the paper with assistance from H-W.W. and C-H.L. The sample collection and treatment of CRC patients were performed by J-K.J., S-H.Y. and H-W.T.

Corresponding authors

Correspondence to Hsei-Wei Wang or Muh-Hwa Yang.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Validation of the stem-like properties of the sphere-derived cancer stem cells (SDCSCs).

(a) RT-qPCR for the stemness genes (LGR5, ASCL2, and OLFM4) in SDCSCs, sphere-derived adherent cells (SDACs), and parental HT29 (upper) and HCT15 (lower) cells. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (b) RT-qPCR for the differentiated genes (BMP4, CDX2) in SDCSCs, SDACs, and parental HT29 (upper) and HCT15 (lower) cells. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (c) Immunocytochemistry for investigation of the nuclear β-catenin in colorectal SDCSCs versus parental cells. Left: the representative pictures of β-catenin staining in HT29 parental cells and SDCSCs. Brown colour, β-catenin staining; blue colour, nuclear hematoxylin staining. Blue arrow, membranous β-catenin; black arrow, nuclear β-catenin. Scale = 10 μm. Right: percentages of nuclear β-catenin in HT29 and HCT15 parental cells versus SDCSCs. Data represent mean ± s.d.n = 3 independent experiments. (d) TOP/FOP reporter activity assay. n = 2 independent experiments (each experiment contains 3 technical replicates). (e) A table summarizing the results of limiting dilution xenotransplantation assay. (f) A schema for showing the experimental procedure of serial transplantation assay. SCM, stem cell medium; FBS, differentiating medium containing 10% FBS. The SDCSCs were obtained after culturing in SCM for 14 days, and adherent cells were obtained after differentiating in FBS for 14 days. Scale bar = 100 μm. (g) A table summarizing the results of serial transplantation assay. (h) Representative photos for the serial transplanted tumours from HT29-SDCSCs. P < 0.05,P < 0.01. P value was estimated by a Students t test in (a), (b), (c); and χ2 test in (e), (g). The numbers of counted cells for each independent experiment in (c) and the original data of the reporter assay in (d) are shown in Supplementary Table 14 as Statistical Source Data.

Supplementary Figure 2 The BrdU pulse-chase assay, and using CD44 as a stem cell marker and CK20 as a differentiation marker for colorectal cancer cells.

(a) A flow chart for showing the schedule of BrdU labeling, cells synchronization, and analysis of paired cells in BrdU pulse-chase assay. (b) A representative result of the cell cycle analysis of HT29-SDCSCs for validating the synchronization of cells in different stages of BrdU pulse-chase assay. (c) Flow cytometry for confirming the BrdU-labeling efficiency. (d) Cell cycle analysis of HT29-SDCSCs pre-labeled with BrdU or control (PBS). (e) Cell viability assay of HT29-SDCSCs pre-labeled with BrdU or control (PBS) cultured under stem cell medium (SCM) or serum-containing medium (10% FBS) condition. The initial viability was confirmed after plating dissociated SDCSCs (Day 0). n = 1 independent experiment (each experiment contains 8 technical replicates). The result of another independent experiment is shown in the Statistical Source Data (Supplementary Table 14). (f) A table summarizing the result of in vitro limiting dilution assay for estimating the sphere-forming ability of HT29-SDCSCs pre-labeled with BrdU or control (PBS). The result of another independent experiment is shown in the Statistical Source Data (Supplementary Table 14). (g)–(h) Flow cytometry for analyzing the expression of Lgr5 (g) or CD44 (h) in HT29-SDCSCs/HCT15-SDCSCs cultured in stem cell medium (SCM) or serum exposure for 48 hr (FBS). Isotype was a control for antibody staining. The percentage of positive cells of each condition is shown in right upper quadrant of each panel. (i) The CD44 pair-cell assay in HT29-SDCSCs and HCT15-SDCSCs cultured in SCM or FBS. n (total counted cells from 2 independent experiments) = 132, 72, 67, and 41 for HT29-SDCSCs(SCM), HT29-SDCSCs(FBS), HCT15-SDCSCs(SCM), and HCT15-SDCSCs(FBS), respectively. (j) Representative images of BrdU and CK20 in HT29-SDCSCs undergoing asymmetric cell division (Asym). Green, BrdU; white, CK20. Scale bar = 10 μm. (k) Percentages of cells with or co-expression (CE) or inverse expression (IE) of BrdU and CK20 in daughter cells. Data represent mean ± s.d.n = 3 independent experiments. The total cell counts of each independent experiment in (k) are shown in Statistical Source Data (Supplementary Table 14). P < 0.01. P value was estimated by Students t test in k, χ2 test in (f), (i).

Supplementary Figure 3 Snail is the major EMT regulator contributing to the stem-like properties of colorectal cancer stem cells and induces miR-146a expression.

(a) Western blot of epithelial marker (E-cadherin), mesenchymal marker (vimentin), and EMT regulators (Snail, Slug, Twist1, Zeb1, and SIP1) in HT29 (left) and HCT15 (right) parental cells and SDCSCs. The fold changes of corresponding proteins are labeled. (b) Immunofluorescent images for visualizing the expression of Snail (red), CD44 (green), and DNA (blue) in HT29-SDCSCs and HT29-SDACs. Scale bar = 50 μm. (c) Western blot of Snail to show the knockdown efficiency in HT29-SDCSCs, HCT15-SDCSCs, and a primary SDCSCs (CRC1-SDCSCs) receiving shRNA against SNAI1 or a scrambled sequence. (d) A table summarizing the results of limiting dilution xenotransplantation assay of HT29-SDCSCs receiving shRNA against SNAI1 or a scrambled sequence. n = 6 mice/group. (e) Pair-cell assay in CRC1-SDCSCs receiving shRNA against SNAI1 or a scrambled sequence. n (total counted cells from 2 independent experiments) = 109 and 168 for CRC1-SDCSCs-scr and CRC1-SDCSCs-shSNAI1, respectively. (f) The sphere-forming ability (upper), SNAI1 (middle) and miR-146a (lower) level among six colorectal cancer cell lines. A positive correlation between these three indicators is shown in the sphere-forming CRC cells (highlighted in yellow). For sphere formation assay, n = 2 independent experiments (each experiment contains 5 technical replicates). The original data of sphere formation assay are shown in Statistical Source Data (Supplementary Table 14). For RT-qPCR, n = 3 independent experiments (each experiment contains 2 technical replicates). Data represent mean ± s.d. (g) The CT value for showing the endogenous level of miR-146a in 6 CRC cell lines and 3 SDCSCs. n = 3 independent experiments (each experiment contains 2 technical replicates). Data represent mean ± s.d. (h) RT-qPCR for primary and mature miR-146a, SNAI1, CDH1, and CD44 in SDCSCs, SDACs, and parental HT29 (left) or HCT15 (right) cells. n = 3 independent experiments (each experiment contains 2 technical replicates). Data represent mean ± s.d. (i) RT-qPCR for primary and mature miR-146a, SNAI1, CDH1 and CD44 in e SW480-Snail versus SW480-vector (left) and HCT15-Snail versus HCT15-vector (right). n = 3 independent experiments (each experiment contains 2 technical replicates). Data represent mean ± s.d.P < 0.05,P < 0.01. P value was estimated by a Students t test in (h), (i); and 2 test in (d), (e).

Supplementary Figure 4 miR-146a promotes stemness without affecting cellular proliferation and viability or inducing a full EMT.

(a) Cell proliferation assay. Z146a, an anti-miR146a vector; Zc, a control vector. n = 1 independent experiment (each experiments contains 6 technical replicates). (b) Cell viability assay. n = 1 independent experiment (each experiments contains 6 technical replicates). (c)–(e) Tail-vein metastasis assay. (c) Representative photos. The arrows indicate the metastatic tumours. (d) H & E stain of the lungs. The metastatic tumour was shown in mice receiving HT29-SDCSCs injection. Scale bar = 100 μm. (e) A stable summarizing the results of tail-vein assay. n = 5 mice/group. (f) RT-qPCR for miR-146a. Data represent mean ± s.d.n = 3 independent experiments (each experiments contains 2 technical replicates). (g) Sphere formation assay. n = 2 independent experiment (each experiments contains 5 technical replicates). (h) Clonogenic assay. Data represent mean ± s.d.n = 3 independent experiments (each experiments contains 2 technical replicates). (i) Soft agar assay. n=2 independent experiments (each experiments contains 3 technical replicates). (j) RT-qPCR for miR-146a. Data represent mean ± s.d.n = 3 independent experiment (each experiments contains 2 technical replicates). (k) Sphere formation assay. Upper: representative photos. Scale bar = 100 μm. Lower: quantification. n = 2 independent experiment (each experiments contains 5 technical replicates). (l) Clonogenic assay. Upper: representative photos. Lower: quantification. n = 3 independent experiments (each experiments contains 2 technical replicates). (m) Soft agar assay. n = 2 independent experiments (each experiments contains 3 technical replicates). (n)–(p) Xenotransplantation assay. (n) A representative photo. Scale bar = 1 cm. (o) A table summarizing the result. n = 9 mice/group. (p) The fold change of tumour weights. n = 2 for Vec, 7 for miR-146a. (q) Cell proliferation assay. No serum group (0% FBS) was a negative control. n = 1 independent experiment (each experiments contains 6 technical replicates). (r) Migration assay. n = 1 independent experiment (each experiment contains 2 technical replicates). (s) Western blot of E-cadherin and vimentin in HT29 and HCT15 cells transduced with miR-146a or control. P < 0.05,P < 0.01. P value was estimated by a Students t test in (f), (h), (j), (l); and 2 test in (e), (o). The results of the other independent experiments of (a), (b), (q), (r) and original data of (g), (i), (k), (m), and (p) are shown in the Statistical Source Data (Supplementary Table 14).

Supplementary Figure 5 A reciprocal regulation between β-catenin and miR-146a in Snail-dominant CRCSCs, and miR146a represses Numb to elicit asymmetrical division and Wnt activation.

(a) Schematic representation of the MIR146A promoter and primers for ChIP experiments. E1-2, E3-4, E5, and E6-9 amplicons contain the Snail-binding sequence (E-box). TSS, transcription start site (+1). (b) ChIP assay for investigating the occupancy of Snail on MIR146A promoter. Amplifying the fragment of CDH1 promoter containing the Snail-binding sequence was a positive control. Input, 10% of total input lysate. (c) Western blot of β-catenin and Snail in nuclear/cytoplasmic extracts from cells transfected with Snail or a control vector (Vec). Histone H3 or β-actin was a control for nuclear or cytoplasmic fractionation, respectively. C, cytosolic extracts; N, nuclear extracts. (d) ChIP assay performed by antibodies against different histone marks. (e) Immunofluorescent images of HCT15 cells transduced with miR-146a or Vec. β-catenin, green; E-cadherin, red; DNA, blue. Scale bar = 10 μm. (f) Percentages of nuclear β-catenin.n = 3 independent experiments. Data represent mean ± s.d. (g) 3’-UTR reporter assay. n = 2 independent experiments (each experiment contains 3 technical replicates). (h) Schematic representations of four human Numb isoforms. The blue boxes indicate exons. PTB, phosphotyrosine-binding domain; PRR, proline-rich region; PRRL, proline-rich region-contained long isoform; PRRS, proline-rich region-deleted short isoform. The location of the primers and the PCR amplicons of each isoform are indicated. (i) RT-PCR for analyzing the expression of Numb isoforms among CRC cell lines. The cDNA were first amplified by primer 1 & 2 to obtain the PRRL transcript, and then the second-run PCR amplification by primer 1 & 3 was used to validate the presence of exon 3 in the amplicons. The 120-bp PCR amplicon includes exon 3, and the 87-bp amplicon excludes exon 3. (j) Pair-cell assay in HT29 cells upon different transfections. n (total counted cells from 2 independent experiments) = 63, 50, 41 and 24 for HT29-Vec, HT29-miR146a, HCT29-miR-146a/puro-Vec, and HT29-miR146a/puro-Numb, respectively. (k) Upper: TOP/FOP reporter assay. n = 2 independent experiments (each experiment contains 3 technical replicates). Lower: a representative western blot of Numb and β-catenin.P < 0.05,P < 0.01. P value was estimated by a Students t test in (f); and χ2 test in (j). The numbers of counted cells of each independent experiment in (f) and the original data of reporter assay in (g) and (k) are provided in Statistical Source Data (Supplementary Figure 14).

Supplementary Figure 6 Numb interacts with β-catenin to promote its degradation, and the impact of Numb on β-catenin localization and asymmetrical division.

(a)–(b) Western blot of β-catenin (a) and RT-qPCR of CTNNB1 (b) in 293T cells transfected with Numb or a control vector. In (b), data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (c) Immunoprecipitation-western blot to show the polyubiquitylated β-catenin in HCT15 cells under different transfections. MG132 treatment: 20 μm for 6 hours since 48 hours after transfection. WCL: whole cell lysates. (d) Upper: western blot of β-catenin in HT29 cells transduced with different vectors. Cyclohexamide (CHX) 50 μg/ml was added as the indicated time periods for inhibiting the de novo protein synthesis. Lower: relative optical density of β-catenin. (e) Immunoprecipitation-western blots to show the presence of Numb in the β-catenin/β-Trcp complex. MG132 treatment: 20 μm for 6 h since 48 h after transfection. WCL, whole cell lysates. (f)–(g) Mapping the interacting domain on Numb (f) and β-catenin (g). Upper: schematic representation of the constructs. The interacting fragments are indicated as +. PTB, phosphotyrosine-binding domain; PRR, proline-rich region. Lower: immunoprecipitation-western blot to show the interaction. Red arrows indicate the expressed proteins upon transient transfection. (h) Left: representative results of the immunocytochemical staining of β-catenin in HCT15-SDCSCs transduced with a vector control (Vec; upper), a full-length Numb (Numb(FL); middle) and a β-catenin non-interacting Numb (FLAG-Numb(162-400); lower). Scale bar = 10 μm. Right: quantification. n = 130 (total counted cells over 2 independent experiments). (i) Immunofluorescent images in HT29-SDCSCs and HT29-SDACs. Numb, red; CD44, green; DNA, blue. Scale bar = 50 μm. (j)-(l) Pair-cell assay in HT29-SDCSCs. (j), representative images. BrdU, green; Numb, white. Scale bar = 10 μm. (k), percentage of asymmetric Numb in symmetric or asymmetric BrdU pair-cells cultured in FBS-containing medium. Sym, symmetric; Asym, asymmetric. n (total counted cells from 2 independent experiments) = 18 and 32 for BrdU-Asym and BrdU-Sym, respectively. (l) Percentages of BrdU and Numb co-expression (CE) or inverse expression (IE) in daughter cells of HT29-SDCSCs undergoing asymmetric division. Data represent mean ± s.d.n = 3 independent experiments. The total cell counts of each independent experiment are shown in Statistical Source Data (Supplementary Table 14). (m) Upper: percentage of BrdU symmetry/asymmetry in HCT15-SDCSCs transduced with indicated vectors under stem cell medium. n (total counted cells from 2 independent experiments) = 43, 33, 59, and 76 for HCT15-SDCSCs transduced with Vec, FLAG-Numb(162-400), Numb(FL), and Numb(FL)/unphosphorylatable β-catenin mutant (β-catenin(mut)), respectively. Lower: Western blot analysis of HCT15-SDCSCs transduced with indicated vectors. P < 0.05,P < 0.01. P value was estimated by a Students t test in (b), (l); and 2 test in (h), (k), (m).

Supplementary Figure 7 Inhibiting MEK/Wnt activity or knockdown of Snail represses the Snail-miR146a-β-catenin signal circuit, and disruption of the circuit circumvents cetuximab resistance.

(a)–(b) Western blot of Numb and β-catenin (left) and RT-qPCR of miR-146a and CCND1 (right) in HCT15-SDCSCs treated with 20 μm PD98059 (a) or 3 μm IWR (b) versus a vehicle control (DMSO). For RT-qPCR, data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (c)–(d) Pair-cell assay. Percentages of BrdU asymmetry/symmetry in HCT15-SDCSCs treated with PD98059 (c) or IWR (d) versus a control vehicle are shown. n (total counted cells from 2 independent experiments) for (c) = 50 and 78 for HCT15 (DMSO) and HCT15 (PD98059), respectively; n for (d) = 83 and 40 for HCT15 (DMSO) and HCT15 (IWR), respectively. (e)–(f) Western blot of Numb, β-catenin, and Snail and RT-qPCR of miR-146a and CCND1 in HT29-SDCSCs (e) and HCT15-SDCSCs (f) receiving shRNA against SNAI1 (shSNAI1) or a control sequence (Scr). For RT-qPCR, data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (g)–(h) Luciferase reporter assay in 293T cells. (g) RT-qPCR of CTNNB1 and SNAI1 for confirming the knockdown effect. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (h) The TOP/FOP (upper) or miR-146a reporter (lower) assay. n = 2 independent experiments (each experiment contains 3 technical replicates). The original data are shown in the Statistical Source Data (Supplementary Table 14). (i) Relative viability of SDCSCs derived from CaCo2 (left), HT29 (middle), and HCT15 (right) versus parent cells. (j) Relative viability of HT29-SDCSCs (left), HCT15-SDCSCs (middle), and SDCSCs from a primary sample (CRC1-SDCSCs)(right) receiving shSNAI1 versus control. (k) Relative viability of SDCSCs from a primary sample (CRC-1) transfected with a dominant-negative TCF4 vector (dnTCF4) or a control vector (Vec). (l) RT-qPCR for validating the expression of miR-146a and Wnt target genes (CCND1, JUN, and CD44) in the primary SDCSCs transfected with dnTCF4 versus Vec. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (m)–(n) Relative viability of HT29-SDCSCs (left) and HCT15-SDCSCs (right) treated with 20M PD98059 (m) or 3 μm IWR (n) versus a control vehicle. (i), (j), (k), (m), (n): the cells were treated with different concentration of cetuximab (Ctx) as indicated. n = 1 independent experiment for each concentration (each experiment contains 8 replicates). The results of the other independent experiments of the cell viability assay are shown as the Statistical Source Data in Supplementary Table 14. P < 0.05,P < 0.01. P value was estimated by a Students t test in (a), (b), (e), (f), (g), (l); and 2 test in (c), (d).

Supplementary Figure 8 Confirmation of the Snail knockdown effect in SDCSCs by an independent shRNA sequence, validation of the Snail-miR146a axis in clinical samples and public database, and tissue-specific cancer stem cell signal in HNSCC versus CRC.

(a) Western blot of Numb, β-catenin, and Snail (left) and RT-qPCR of miR-146a and CCND1 (right) in HCT15-SDCSCs receiving an independent shRNA against SNAI1 (shSNAI1#2) or a control sequence (Scr). Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (b) Pair-cell assay. Percentages of BrdU asymmetry/symmetry in HCT15-SDCSCs transfected with Scr or shSNAI1#2 are shown. n (total counted cells from 2 independent experiments) = 97 and 77 for HCT15-SDCSCs-scr and HCT15-SDCSCs-shSNAI1#2, respectively. (c) Relative viability of HCT15-SDCSCs cells transfected with shSNAI1#2 versus Scr and treated with different concentration of cetuximab (Ctx). n = 1 independent experiments (each experiment contains 8 technical replicates). The result of another independent experiment is shown in the Statistical Source Data (Supplementary Table 14). (d) A heatmap showing the expression pattern of CDH1, SNAI1, NUMB and miR-146a in CRC cases (n = 53 patients). “Snail on” indicates that SNAI1 inversely correlated with CDH1 expression. Red, upregulation; Blue, downregulation. (e) The correlation between miR-146a, SNAI1 and NUMB in Snail on CRC cases (n = 20 patients). (f) The level miR-146aCt in CRC patients with SNAIHighNUMBLow versus SNAILowNUMBHig profile (n = 15 patients). The box plots represent sample maximum (upper end of whisker), upper quartile (top of the box), median (band in the box), lower quartile (bottom of the box), and sample minimum (lower end of whisker). (g) Heatmaps showing the expressions of SNAI1 and NUMB in cells populations displaying different levels of EphB2. Top: data from mouse small intestine cells (GSE27605). Bottom: data from normal human colonic cells (GSE31255). (h) Differential expression ranking of SNAI1 (top panel) and NUMB (bottom panel) from 6 independent TCGA datasets (group1, cecum adenocarcinoma versus normal; group 2, colon adenocarcinoma versus Normal; group 3, colon mucinous adenocarcinoma versus normal; group 4, rectal adenocarcinoma versus nomal; group 5, rectal mucinous adencarcinoma versus normal; group 6 rectosigmoid adenocarcinoma versus normal). The median rank for a gene is given for that gene across each of the analyses. Red, increased gene expression in cancerous tissue and ranked in top 25%. Blue, decreased expression in cancerous tissue and ranked in top 25%. (i) RT-qPCR for examining the Twist1-BMI1-let-7i axis in HT29-SDCSCs. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (j) Heatmaps showing the expression of Twist1-BMI1-let-7i axis-related genes (Twist1, BMI1, NEDD9, and DOCK3) in intestinal cell populations with different EphB2 expression. The data was from GSE27605 and GSE31255. (k) RT-qPCR analysis for examining the Snail-miR-146a (left) and Twist1-BMI1-let-7i (right) signal axes in FaDu-SDCSCs versus FaDu parental cells. Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). (l) Left: RT-qPCR for examining the expression of Snail-miR-146a signal axis-associated genes in primary HNSCC cells receiving shRNA against a scrambled sequence (Scr) or SNAI1 (shSNAI1). Right: RT-qPCR for examining the expression of Twist1-BMI1-let-7i signal axis-associated genes in primary HNSCC cells receiving shRNA against a scramble sequence (Scr) or TWIST1 (shTWIST1). Data represent mean ± s.d.n = 3 independent experiments (each experiment contains 2 technical replicates). P < 0.05,P < 0.01. P value was estimated by a Students t test in (a), (f), (i), (k), (l); t-statistics in (h), χ2 test in (b). The r value of (e) and (g) was estimated by Pearsons correlation test.

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Hwang, WL., Jiang, JK., Yang, SH. et al. MicroRNA-146a directs the symmetric division of Snail-dominant colorectal cancer stem cells. Nat Cell Biol 16, 268–280 (2014). https://doi.org/10.1038/ncb2910

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