Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-200c Prevents TGF-β1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1

doi: 10.1016/j.omtn.2019.05.008

Figure Lengend Snippet: miR-200c Overexpression in Mesothelial Cells and Mediators of EMT and Fibrosis (A) Human peritoneal mesothelial cells (HPMCs) and (B) Met-5A cells showing miR-200c expression in non-transfected mesothelial cells (“C”) or mesothelial cells transfected with scrambled control hairpin (lenti-scramble) or lentivector-based miR precursor construct expressing miR-200c (lenti-miR-200c) by real-time PCR. ***p < 0.001, lenti-miR-200c versus C or lenti-scramble. Data analyzed by ANOVA. Representative Westerns showing the effect of miR-200c overexpression on (C) E-cadherin, ZEB2, Notch1, Jagged2, and SNAIL expression in HPMCs and on (D) fibronectin and collagen I expression in Met-5A cells following incubation with SFM or TGF-β1 (10 ng/mL) after 24 h. The intensity of the bands was normalized to β-actin and expressed as arbitrary densitometric scan (DU) .

Article Snippet: Rabbit anti-human FSP-1, mouse anti-human E-cadherin, and rabbit anti-human ZEB2 antibodies were purchased from Novus Biologicals (Gene Company, Hong Kong).

Techniques: Over Expression, Expressing, Transfection, Control, Construct, Real-time Polymerase Chain Reaction, Incubation

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-200c Prevents TGF-β1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1

doi: 10.1016/j.omtn.2019.05.008

Figure Lengend Snippet: Effect of miR-200c Overexpression on Gene Expression of Transcription Factors Related to EMT, Fibronectin, Collagen I, and Collagen III Non-transfected Met-5A cells (C) or Met-5A cells transfected with lenti-scramble (S) or lenti-miR-200c (M) were incubated with serum-free medium (SFM) or TGF-β1 (10 ng/mL) for 24 h, and gene expression of (A) E-cadherin, (B) ZEB2, (C) Notch1, (D) Jagged2, (E) SNAIL, (F) fibronectin, (G) collagen I, and (H) collagen III were determined by real-time PCR. **p < 0.01 and ***p < 0.001, SFM versus TGF-β1 for the same transfection; § p < 0.05, §§ p < 0.01, and §§§ p < 0.001, non-transfected cells versus lenti-miR-200c; ## p < 0.01 and ### p < 0.001, lenti-scramble versus lenti-miR-200c. Data analyzed by using ANOVA.

Article Snippet: Rabbit anti-human FSP-1, mouse anti-human E-cadherin, and rabbit anti-human ZEB2 antibodies were purchased from Novus Biologicals (Gene Company, Hong Kong).

Techniques: Over Expression, Gene Expression, Transfection, Incubation, Real-time Polymerase Chain Reaction

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-200c Prevents TGF-β1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1

doi: 10.1016/j.omtn.2019.05.008

Figure Lengend Snippet: Effect of miR-200c Overexpression on Mediators of EMT and Fibrosis in Mesothelial Cells

Article Snippet: Rabbit anti-human FSP-1, mouse anti-human E-cadherin, and rabbit anti-human ZEB2 antibodies were purchased from Novus Biologicals (Gene Company, Hong Kong).

Techniques: Over Expression, Marker, Transfection

Journal: Molecular Therapy. Nucleic Acids

Article Title: miR-200c Prevents TGF-β1-Induced Epithelial-to-Mesenchymal Transition and Fibrogenesis in Mesothelial Cells by Targeting ZEB2 and Notch1

doi: 10.1016/j.omtn.2019.05.008

Figure Lengend Snippet: Luciferase Reporter Assays Identify Target Genes of miR-200c miR-200c overexpression reduced luciferase activity of (A) ZEB2, (B) Jagged2, and (C) Notch1. C: non-transfected Met-5A cells, S: Met-5A cells transfected with lenti-scramble, M: Met-5A transfected with lenti-miR-200c. *p < 0.05 and ***p < 0.001, non-transfected cells versus cells transfected with lenti-miR-200c; ### p < 0.001, lenti-scramble versus lenti-miR-200c. Red line highlights the position of the miR-200c seed sequences in the 3′ UTR of ZEB2, Notch1, and Jagged2. Luciferase reporter assays showing direct binding of miR-200c to the wild-type but not mutant sequences within the 3′ UTR seed regions of (D) ZEB2 and (E) Notch1. (F) Luciferase reporter assay demonstrated that miR-200c did not directly bind to the seed region of Jagged2. Each study was performed in triplicate and repeated at least three times. The blue line in panels (D–F) highlights the putative miR-200c binding sites that were deleted in mutant-type target gene 3′ UTR for ZEB2, Notch1, and Jagged2. *p < 0.05 and ***p < 0.001, scrambled versus miR-200c mimic; # p < 0.05 and ## p < 0.01, wild-type versus mutant for miR-200c mimic. Data analyzed by using ANOVA.

Article Snippet: Rabbit anti-human FSP-1, mouse anti-human E-cadherin, and rabbit anti-human ZEB2 antibodies were purchased from Novus Biologicals (Gene Company, Hong Kong).

Techniques: Luciferase, Over Expression, Activity Assay, Transfection, Binding Assay, Mutagenesis, Reporter Assay

Journal: Nature Communications

Article Title: Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

doi: 10.1038/s41467-021-25333-9

Figure Lengend Snippet: a Cytokines secreted by CR-CSphCs ( n = 4: #1, #8, #9, #21), S-ASCs ( n = 6: #3, #5, #6, #8, #14, #20), V-ASCs ( n = 6: #3, #5, #6, #8, #14, #18), or primary adipose tissue (AT) ( n = 4). Data are the mean of 3 independent experiments. b Cell growth of CR-CSphCs treated for 5 days with IL-6 and HGF alone or in combination. The dotted red line shows the cell number at day 0. c Colony size of CR-CSphCs treated as indicated. n represents the number of colonies. Statistical significance was calculated using the two-tailed t test. d Invasion assay of CR-CSphCs pretreated with the indicated cytokines for 48 h. For b–d data show mean ± S.D. of three independent experiments using fourdifferent CR-CSphCs (CSphC #1, #8, #9, #21). e mRNA expression levels of CSC-related genes in CMS2 CR-CSphCs (CSphC #8, 9) exposed to vehicle (Medium) or IL-6 in combination with HGF for 48 h. f Immunoblot analysis of ZEB2 in CMS2 CR-CSphCs (CSphC #8) treated as indicated. Data are mean ± S.D. of three independent experiments using two different CSphCs (CSphC #8, #9). Samples were run on the same gel and images were cropped only for the purpose of this figure. Source data are provided as a Source Data file. g Kinetic growth of CR-CSphCs treated as indicated. h Number of invading CR-CSphCs at 48 h, pretreated with V-ASC CM and the indicated neutralizing antibodies for 48 h. i Flow cytometry analysis of CD44v6 positivity in CR-CSphCs treated as indicated, for 48 h. For g–i data are mean ± S.D. of six independent experiments performed with 2 different CR-CSphC lines (#8 and #9). For (b–d and g-i) statistical significance was calculated using the unpaired two-tailed t test. j , CD44v6 expression in CD44v6 - and GFP-transduced CD44v6 + cells after 3 days of exposure to V-ASC CM. One representative of 6 independent experiments is shown. k NGF, BDNF, NTF3, and NTF4 mRNA expression levels on CD44v6 - and CD44v6 + cells. Results show mean ± S.D. of three independent experiments performed with enriched cells from two different CR-CSphC lines (CSphC #8, #9). l Lollipop plot showing NGF, BDNF, NT-3, and NT-4 production by the indicated cells treated as indicated. m , Invasion assay of RFP transduced ASCs, using the indicated cells/media as chemoattractant agents. Scale bars, 100 µm. n Number of invading ASCs in presence of the indicated cells/media as chemoattractant agents. For l–n data are mean ± SD of three independent experiments using CR-CSphCs from different patients (CSphC #1, #8, #9, #21).

Article Snippet: Membrane were pre-incubated with blocking buffer (0.1% Tween 20 and 5% nonfat dry milk in PBS) for 1 h at room temperature and then exposed to a mix made by 5% BSA 0,05% Tween-20 PBS and specific antibodies (1:1000 dilution) against ZEB2 (E-11, mouse IgG 2a , Santa Cruz), p-STAT3 (Ser727) (rabbit, CST), p-STAT3 (Tyr705) (9145, rabbit IgG, CST), STAT3 (9139, mouse IgG 2a , CST), active β-catenin (Ser33/37/Thr41) (D13A1, rabbit IgG, CST), β-catenin (D10A8, rabbit IgG, CST), vimentin (R28, rabbit, CST), or β-actin (8H10D10, mouse, CST).

Techniques: Two Tailed Test, Invasion Assay, Expressing, Western Blot, Flow Cytometry

Journal: Nature Communications

Article Title: Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

doi: 10.1038/s41467-021-25333-9

Figure Lengend Snippet: a Up- (red) and down- (blue) regulated genes and their relative top twenty significantly enriched gene sets (FDR q value ≤ 0.05), common in CMS4 CR-CSphCs (CSphC #1, #21) and CMS2 cells (CSphC #8, #9) treated with V-ASC CM, selected from all gene sets within MSigDB (H, CP Biocarta, CP Kegg, MIR, CGN, CM, BP, CC, MF, C6, C7). b Heatmap of EMT-related genes in CMS2 (CSphC #8, #9) and CMS4 (CSphC #1, #21) CR-CSphCs treated for 48 h as indicated. c , Venn diagrams of up- and downregulated genes in untreated CMS4 (CSphC #1, #21) and V-ASC CM-treated CMS2 (CSphC #8, #9) CR-CSphCs, compared to untreated CMS2 cells. d ZEB1 and ZEB2 mRNA expression levels in CMS4 and CMS2 CR-CSphCs treated as indicated. e Immunofluorescence analysis of CR-CSphCs expressing nuclear ZEB1 and ZEB2 (CMS2 #8, CMS4 #21) treated as indicated. Nuclei were counterstained with Toto-3. Scale bars, 20 µm. For d and e data represent mean ± S.D. of three independent experiments using CMS2 (#8, #9) and CMS4 (#1, #21) CR-CSphC lines. f Global gene expression profile of miRNAs in CMS4 (CSphC #1, #21) and CMS2 (#8, #9) CR-CSphCs treated as indicated. g Network of most differentially expressed miRNAs and their targets inferred from miRTarBase in CMS2 CR-CSphCs (CSphC #8, #9) treated with V-ASCs CM for 48 h. Bold colors represent miRNAs with a fold-change >8. Orange area within dashed line highlights ZEB1 and ZEB2 as direct targets of miR-200a. h Immunoblot analysis of ZEB2 in CMS4 (CSphC #1, #21) and CMS2 (CSphC #8, #9) CR-CSphCs treated as indicated. Data are mean ± S.D. of three independent experiments performed with CR-CSphCs isolated from 2 different CMS2 (CSphC #8, #9) and CMS4 (CSphC #1, #21) CRC patients. Samples were run on the same gel and images were cropped only for the purpose of this figure. Source data are provided as a Source Data file. For (d and h) statistical significance was calculated using the two-tailed t test. i Flow cytometry analysis of CD44v6 in CMS2 CR-CSphCs (CSphC #8, #9) transduced with empty vector (EV) or ZEB2 synthetic gene. Bars represent means ± S.D. of three independent experiments using two CR-CSphCs. j In vivo whole-body imaging analysis of mice ( n = 6) following intrasplenic injection of CR-CSphCs transduced as in i at 30 min and 8 weeks after splenectomy (left panel) . Luciferase signal measured as ph/s/cm 2 /sr (right panel). Data are mean ± S.D. of independent experiments performed with two CMS2 CR-CSphCs (#8, #9).

Article Snippet: Membrane were pre-incubated with blocking buffer (0.1% Tween 20 and 5% nonfat dry milk in PBS) for 1 h at room temperature and then exposed to a mix made by 5% BSA 0,05% Tween-20 PBS and specific antibodies (1:1000 dilution) against ZEB2 (E-11, mouse IgG 2a , Santa Cruz), p-STAT3 (Ser727) (rabbit, CST), p-STAT3 (Tyr705) (9145, rabbit IgG, CST), STAT3 (9139, mouse IgG 2a , CST), active β-catenin (Ser33/37/Thr41) (D13A1, rabbit IgG, CST), β-catenin (D10A8, rabbit IgG, CST), vimentin (R28, rabbit, CST), or β-actin (8H10D10, mouse, CST).

Techniques: Expressing, Immunofluorescence, Gene Expression, Western Blot, Isolation, Two Tailed Test, Flow Cytometry, Transduction, Plasmid Preparation, In Vivo, Imaging, Injection, Luciferase

Journal: Nature Communications

Article Title: Adipose stem cell niche reprograms the colorectal cancer stem cell metastatic machinery

doi: 10.1038/s41467-021-25333-9

Figure Lengend Snippet: a Growth kinetics of CMS2 CR-CSphCs treated with V-ASC CM, alone or in combination with tocilizumab (Toc) and crizotinib (Criz). b Colony-forming assay of CR-CSphCs following the indicated treatment, at 21 days. c , miR-200a expression in CMS2 CR-CSphCs treated as indicated for 48 h. U6 was used as housekeeping control gene. d ZEB1 and ZEB2 expression levels in cells treated for 72 h as indicated. GAPDH was used as housekeeping control gene. e Number of invading CMS2 CR-CSphCs pretreated as indicated for 48 h. Statistical significance between two groups was determined by unpaired Student’s t test (2-tailed). For (a-e) data are mean ± S.D. of three independent experiments using two different CR-CSphC lines (CSphC #8, #9). Statistical significance was calculated using the two-tailed t test. f Schematic model of intrasplenic injection of CR-CSphCs showing time points of treatments and in vivo bioluminescence detection. g Kinetics and whole-body imaging analysis of mice ( n = 6) following intrasplenic injection of LUC-GFP-transduced CMS2 CR-CSphCs alone or co-injected with V-ASCs untreated or treated with the indicated pharmaceutical compounds. Insets represent spleen collected 30 min after cell injection, and liver, lung, and bowel collected at the time of sacrifice. Data are mean ± S.D. of independent experiments using two different CR-CSphC lines (CSphC #8, #9), and 2 S- (#3, #6) or V-ASC (#5, #14) lines. h RFS rate of CMS2/MSS/Stage1-2 CRC patients according to ZEB2 expression levels. Statistical significance was calculated using the log-rank (Mantel–Cox) test. i Univariate and Multivariate analysis of relapse-free survival (RFS) according to regression Cox model in CRC patients as in h . Statistical significance was calculated using the Wald test. j Schematic representation of bidirectional crosstalk between ASCs and CRC cells. Visceral adipose factors enhance the expression of CD44v6; CD44v6 + -released NGF/NT-3 drives the intra-tumor recruitment of ASCs; adipose-released proteins induce EMT of CRC cells through the activation of STAT3; CD44v6 + -released VEGF promotes the endothelial transdifferentiation of ASCs. Clinically available drugs targeting HGF and IL-6 are highlighted in red.

Article Snippet: Membrane were pre-incubated with blocking buffer (0.1% Tween 20 and 5% nonfat dry milk in PBS) for 1 h at room temperature and then exposed to a mix made by 5% BSA 0,05% Tween-20 PBS and specific antibodies (1:1000 dilution) against ZEB2 (E-11, mouse IgG 2a , Santa Cruz), p-STAT3 (Ser727) (rabbit, CST), p-STAT3 (Tyr705) (9145, rabbit IgG, CST), STAT3 (9139, mouse IgG 2a , CST), active β-catenin (Ser33/37/Thr41) (D13A1, rabbit IgG, CST), β-catenin (D10A8, rabbit IgG, CST), vimentin (R28, rabbit, CST), or β-actin (8H10D10, mouse, CST).

Techniques: Expressing, Control, Two Tailed Test, Injection, In Vivo, Imaging, Activation Assay

Journal: Clinical Cancer Research

Article Title: Overexpression of ZEB2 at the Invasion Front of Colorectal Cancer Is an Independent Prognostic Marker and Regulates Tumor Invasion In Vitro

doi: 10.1158/1078-0432.ccr-10-2816

Figure Lengend Snippet: Figure 1. Immunohistochemical analysis of ZEB2 in colorectal liver metastases (A–D) and primary colorectal cancer (E–H). Figures represent invasion front-specific overexpression of ZEB2 in paraffin- embedded specimens. Original magnification 200 (left) and corresponding areas (boxed areas) with lower magnification 80.

Article Snippet: Primary antibodies against ZEB2 (Rabbit polyclonal, Novus Biologicals, 1:200) and IgG-negative control (Mouse IgG, BD Pharmingen, 1:200) were incubated at 4 C overnight.

Techniques: Immunohistochemical staining, Over Expression

Journal: Clinical Cancer Research

Article Title: Overexpression of ZEB2 at the Invasion Front of Colorectal Cancer Is an Independent Prognostic Marker and Regulates Tumor Invasion In Vitro

doi: 10.1158/1078-0432.ccr-10-2816

Figure Lengend Snippet: Figure 2. DLD-1 cells were transfected with ZEB2 siRNA or scrambled control siRNA. Transfection efficiency was evaluated by qRT-PCR and reached more than 90% (A). DLD-1 cells were subjected to migration assays (B) and invasion assays (C). ZEB2 siRNA–transfected DLD-1 cells displayed an impaired migration capacity of 25% (P ¼ 0.018) and a reduced invasion capacity of 26% (P ¼ 0.001) compared with control. All assays were carried out 3 times in quadruplicates.

Article Snippet: Primary antibodies against ZEB2 (Rabbit polyclonal, Novus Biologicals, 1:200) and IgG-negative control (Mouse IgG, BD Pharmingen, 1:200) were incubated at 4 C overnight.

Techniques: Transfection, Control, Quantitative RT-PCR, Migration

Journal: Clinical Cancer Research

Article Title: Overexpression of ZEB2 at the Invasion Front of Colorectal Cancer Is an Independent Prognostic Marker and Regulates Tumor Invasion In Vitro

doi: 10.1158/1078-0432.ccr-10-2816

Figure Lengend Snippet: Figure 3. Kaplan–Meier curves display tumor-specific overall survival in patients with primary colorectal cancer (n ¼ 175). Solid line: negative/low expression of ZEB2; dashed line: high expression of ZEB2. A, overexpression of ZEB2 at the invasion front is significantly associated with a shortened tumor- specific survival (log-rank test, P 0.0001). B, overexpression of ZEB2 in the tumor center (Tc) correlated also with a shortened survival, but failed slightly to be statistically significant (log-rank test, P ¼ 0.06).

Article Snippet: Primary antibodies against ZEB2 (Rabbit polyclonal, Novus Biologicals, 1:200) and IgG-negative control (Mouse IgG, BD Pharmingen, 1:200) were incubated at 4 C overnight.

Techniques: Expressing, Over Expression

Journal: Oncology Letters

Article Title: miR-215 functions as a tumor suppressor and directly targets ZEB2 in human non-small cell lung cancer

doi: 10.3892/ol.2015.3587

Figure Lengend Snippet: Expression of miR-215 and ZEB2 in NSCLC tissues and cell lines. (A) miR-215 expression was significantly reduced in NSCLC tissues compared with that of the corresponding non-cancerous tissues. miR-215 expression levels were calculated using the 2 −∆Ct method and normalized to U6 small nuclear RNA. (B) miR-215 expression was downregulated in NSCLC cell lines A549, H460, 95D and HCC827, compared with that of NLECs. (C) Relative ZEB2 protein levels in NSCLC and corresponding non-cancerous tissues. ZEB2 protein levels were measured by western blot analysis and normalized to β-actin. (D) ZEB2 protein levels in NSCLC cells were increased compared with NLECs. (E) The inverse correlation of ZEB2 protein levels with miR-215 expression was examined by Pearson correlation analysis. Values are expressed as the mean ± standard deviation. *P<0.05; **P<0.01 vs. NLEC. miR-215, microRNA-215; NSCLC, non-small cell lung cancer; NLEC, normal lung epithelial cells, ZEB2, zinc finger E-box-binding homeobox 2.

Article Snippet: Following blocking in 5% non-fat milk in 1X Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, the membranes were incubated with purified rabbit anti-ZEB2 antisera (cat. no. LS-C160768; dilution, 1:1,000; LifeSpan BioSciences, Inc., Seattle, WA, USA) at 4°C overnight.

Techniques: Expressing, Western Blot, Standard Deviation, Binding Assay

Journal: Oncology Letters

Article Title: miR-215 functions as a tumor suppressor and directly targets ZEB2 in human non-small cell lung cancer

doi: 10.3892/ol.2015.3587

Figure Lengend Snippet: ZEB2 is a direct target of miR-215. (A) miR-215-binding sites in the ZEB2 3′UTR region. ZEB2-mut indicates the ZEB2 3′UTR with a mutation in miR-215-binding sites. (B) Western blot analysis demonstrated that transfection of miR-215 reduced ZEB2 protein expression. (C) Relative luciferase assay comparing the pGL3-ZEB2 and pGL3-ZEB2-Mut vectors in A549 cells. Firefly luciferase activity was normalized to Renilla luciferase activity. Values are expressed as the mean ± standard deviation. *P<0.05 vs. control. ZEB2, zinc finger E-box-binding homeobox 2; mut, mutant; Hsa, Homo sapien .

Article Snippet: Following blocking in 5% non-fat milk in 1X Tris-buffered saline (pH 7.4) containing 0.05% Tween-20, the membranes were incubated with purified rabbit anti-ZEB2 antisera (cat. no. LS-C160768; dilution, 1:1,000; LifeSpan BioSciences, Inc., Seattle, WA, USA) at 4°C overnight.

Techniques: Binding Assay, Mutagenesis, Western Blot, Transfection, Expressing, Luciferase, Activity Assay, Standard Deviation