Journal: Cancer Communications

Article Title: BAALC‐AS1/G3BP2/c‐Myc feedback loop promotes cell proliferation in esophageal squamous cell carcinoma

doi: 10.1002/cac2.12127

Figure Lengend Snippet: The expression of BAALC‐AS1 in ESCC. A. In situ hybridization of BALC‐AS1 in ESCC tissue samples and their corresponding adjacent normal tissues. Scale bar: left, 100 μm; right, 200 μm. Adjacent, adjacent normal tissues; Cancer, ESCC tissues. B. Comparison of BAALC‐AS1 scores of ESCC and adjacent normal tissues. C. ESCC patients with high levels of BAALC‐AS1 expression showed reduced survival compared with those with low levels of BAALC‐AS1 expression ( P = 0.037; log‐rank test). D. Differences in the overall survival and pathological stage between patients with high BAALC‐AS1 expression level and low BAALC‐AS1 expression level were highly significant. E. Expression of BAALC‐AS1 quantified by RT‐qPCR in ESCC cell lines (YES‐2, KYSE‐30, ‐150, ‐180, ‐410, ‐450, ‐510, and COLO‐680) and normal esophageal epithelial cell line (Het‐1A). F. Analysis of BAALC‐AS1 distribution by cellular fractionation followed RT‐qPCR in KYSE‐510 cells. U6 and 18s mRNAs were served as controls for nuclear and cytoplasmic RNAs, respectively. G. RNA‐ FISH was performed to detect BAALC‐AS1 expression in KYSE‐510 cells. The nuclei were counterstained with 4′,6‐diamidino‐2‐phenylindole (DAPI). Scale bar, 10 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All of the values are expressed as the mean ± SEM of 3 experiments

Article Snippet: The normal esophageal epithelial cell line (Het‐1A) and the human ESCC cell lines (YES‐2, KYSE‐30, ‐150, ‐180, ‐410, ‐450, ‐510 and COLO‐680) were generously provided by Dr. Yutaka Shimada of Kyoto University (Kyoto, Japan) and cultured as previously described [ ].

Techniques: Expressing, In Situ Hybridization, Comparison, Quantitative RT-PCR, Cell Fractionation

Journal: Cancer Communications

Article Title: BAALC‐AS1/G3BP2/c‐Myc feedback loop promotes cell proliferation in esophageal squamous cell carcinoma

doi: 10.1002/cac2.12127

Figure Lengend Snippet: BAALC‐AS1 promotes tumor growth of ESCC in vivo . A. Images of the xenograft tumors formed in nude mice injected with BAALC‐AS1‐overexpressing (BAALC‐AS1) or ‐silencing (sh/BAALC‐AS1‐1 and ‐2) KYSE‐450 or KYSE‐410 cells. The nude mice injected with control cells (Vector and siNC) were used as control. B. Tumor weight in nude mice injected with the abovementioned cells. C. Growth curve of xenograft tumors in nude mice injected with the abovementioned cells. Five mice per group were used in the tumor formation assay. D. The body weight of the nude mice in tumor formation assay. E. Representative images of H&E staining and Ki67 IHC staining for tumor tissues harvested from nude mice in tumor formation assay. Scale bar, 50 μm. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All values are expressed as the mean ± SEM

Article Snippet: The normal esophageal epithelial cell line (Het‐1A) and the human ESCC cell lines (YES‐2, KYSE‐30, ‐150, ‐180, ‐410, ‐450, ‐510 and COLO‐680) were generously provided by Dr. Yutaka Shimada of Kyoto University (Kyoto, Japan) and cultured as previously described [ ].

Techniques: In Vivo, Injection, Control, Plasmid Preparation, Tube Formation Assay, Staining, Immunohistochemistry

Journal: Cancer Communications

Article Title: BAALC‐AS1/G3BP2/c‐Myc feedback loop promotes cell proliferation in esophageal squamous cell carcinoma

doi: 10.1002/cac2.12127

Figure Lengend Snippet: c‐Myc acts directly with the BAALC‐AS1 promoter. A. Expression of BAALC‐AS1 quantified by RT‐qPCR after c‐Myc knockdown in KYSE‐450 cells. B. Silencing c‐Myc decreased the luciferase activity controlled by BAALC‐AS1 promoter. C. Summary diagram describing how BAALC‐AS1 regulates proliferation in ESCC cells. NC, negative control; si/c‐Myc, small interfering RNA for c‐Myc; *, P < 0.05. All values are expressed as the mean ± SEM of 3 experiments

Article Snippet: The normal esophageal epithelial cell line (Het‐1A) and the human ESCC cell lines (YES‐2, KYSE‐30, ‐150, ‐180, ‐410, ‐450, ‐510 and COLO‐680) were generously provided by Dr. Yutaka Shimada of Kyoto University (Kyoto, Japan) and cultured as previously described [ ].

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Luciferase, Activity Assay, Negative Control, Small Interfering RNA

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: The most up and down-regulated mRNA expression profile of ESCC cell lines by oligonucleotide microarray analysis

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques: Expressing, Microarray

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: Characteristics of Group I and Group II of ESCC clinical specimens

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques:

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: Fibronectin assembly on the membrane correlates with miR-146a expression and ESCC cell migration. a Fibronectin assembling on cell membrane was shown by FITC conjugated anti-fibronectin antibody under fluorescent microscope (×400, red arrow). b The fluorescence signals from CE81 FN− and CE81 FN+ cells merged by flow cytometry software. The pericellular FN (periFN) in CE81 FN− and CE81 FN+ cells were quantified and represented as indicated. c The expression level of miR-146a in CE81 FN− and CE81 FN+ cells were evaluated by real-time PCR. The data were analyzed by ΔCT method and the relative expression fold was normalized to the internal control U54 gene expression. d The migration ability of CE81 FN− and CE81 FN+ cells was investigated using Transwell™ migration assay at 48 h after seeding of cells (10 6 cells/well). Scale bar: 100 μm. The migrated cell number was counted by Image J software. P values were obtained by Student’s t. test. Statistically significant difference was indicated (*P < 0.05, ***P < 0.001)

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques: Membrane, Expressing, Migration, Microscopy, Fluorescence, Flow Cytometry, Software, Real-time Polymerase Chain Reaction, Control, Gene Expression, Transwell Migration Assay

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: Predication and validation of vimentin as a target of MiR-146a gene in ESCC cells. a The expression level of miR-146a in CE81 FN+ + CON and CE81 FN+ + 146a stable cells was evaluated by real-time PCR. The data was analyzed by ΔCT method and the relative expression was normalized to the internal control U54 gene expression. b The expression levels of the six overlapped target genes of miR-146a predicted by three software programs, i.e., Targetscan, Microcosm and miRNAMap were measured in CE81 FN+ CON and CE81 FN+ 146a stable cell lines by real-time PCR. c The mRNA expression levels of vimentin in CE81 FN+ and CE81 FN− cells were determined by RT-PCR. d The protein levels of vimentin in CE81 FN+ and CE81 FN− cells were determined by Western blotting. β-actin was used as the internal control. e Vimentin was labelled by FITC conjugated anti-vimentin antibody in CE81 FN+ and CE81 FN− cells by IFA assay. The cells with fluorescent vimentin expression were shown and quantified by the fluorescent microscopy (×400). Scale bar: 100 μm. P values were obtained from the Student’s t test. Statistically significant difference was indicated (*P < 0.05, ***P < 0.001)

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques: Biomarker Discovery, Expressing, Real-time Polymerase Chain Reaction, Control, Gene Expression, Software, Stable Transfection, Reverse Transcription Polymerase Chain Reaction, Western Blot, Microscopy

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: MiR-146a inhibited ESCC cell invasion by targeting vimentin. a CE81 FN+ + CON stable cells were transiently transfected with 100 pmol/l of mimic- miR-146a or scramble microRNA (N.C.) using Lipofectamine™ for 48 h followed by measuring the expression of miR-146a by real-time PCR. b CE81 FN+ CON cells harboring transiently transfected scramble (N.C.) or miR-146a used in ( a ) were further transiently transfected with pcDNA3.1-vimentin or vector, followed by measuring the level of vimentin using Western blotting. Total lysates in the same blotted PVDF membrane stained by Coomassie blue were used as the internal control. c The invasive ability of the cells used in ( b ) was evaluated by Transwell™ invasion assay. d CE81 FN+ + 146a stable cells were transiently transfected with anti- miR-146a or anti-scramble microRNA (anti-N.C.) followed by measuring the expression of miR-146a . e In parallel, the study was CE81 FN+ miR-146a cells harboring anti-scramble (anti-N.C.) or anti- miR-146a were further transfected with si-Vimentin or si-RNA negative control (si–N.C.). The levels of vimentin and β-actin were investigated by Western blotting. Total lysates in the same blotted PVDF membrane stained by Coomassie blue were used as the internal control. f The invasive abilities of the cells used in ( e ) were measured by Transwell™ invasion assay. The numbers of the invaded cells on the bottom of the membrane were counted after incubating for 108 h. The quantitative data are shown. P values were obtained by Student’s t test, **P < 0.01, ***P < 0.001. The quantifications for ( b ) and ( e) were derived from at least three independent biological repetitions (Additional file : Figure S 7 )

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques: Transfection, Expressing, Real-time Polymerase Chain Reaction, Plasmid Preparation, Western Blot, Membrane, Staining, Control, Transwell Invasion Assay, Negative Control, Derivative Assay

Journal: Journal of Biomedical Science

Article Title: MicroRNA-146a suppresses tumor malignancy via targeting vimentin in esophageal squamous cell carcinoma cells with lower fibronectin membrane assembly

doi: 10.1186/s12929-020-00693-4

Figure Lengend Snippet: Low miR-146a or high vimentin expression levels in the tumorous ESCC tissue array correlated with poor overall survival rate. a The miR-146a levels in the tumorous and adjacent non-tumorous cells of 68 paired ESCC patient specimens in the tissue array after ISH treatment were quantified by defining regions of interest (ROI) using automated cell acquisition and quantification software (Histoquest™) (P < 0.001). b The protein levels of vimentin in the same ESCC specimens used in ( a ) were stained by IHC followed by Histoquest™ quantification (P < 0.0001). c Two representative patient specimens containing tumorous and adjacent non-tumorous cells from the 68 ESCC patients were analyzed to determine miR-146a and vimentin expression levels after ISH and IHC treatment. Hematoxylin was used to stain nucleus and AEC (3-amino-9-ethylcarbazole) for miR-146a and vimentin expression. Scale bar 20 μm. The correlation of ( d ) miR-146a level (P < 0.0001) and ( e ) vimentin expression (P < 0.05) with ESCC patient overall survival rate were assessed by Kaplan–meier analysis and Log rank test

Article Snippet: CE81 FN+ and CE81 FN− cells were sorted from a human ESCC cell line CE81T (ATCC® HTB 56TM) by flow cytometry.

Techniques: Expressing, Software, Staining

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Expression Profiles in Superficial Esophageal Squamous Cell Carcinoma before Endoscopic Submucosal Dissection: A Pilot Study

doi: 10.3390/ijms22094789

Figure Lengend Snippet: Unsupervised hierarchical clustering analysis of differentially expressed miRNAs between superficial esophageal squamous cell carcinoma (ESCC) and adjacent normal tissues. Columns represent the patients and rows represent the individual miRNAs. Red and blue indicate high and low expression levels, respectively.

Article Snippet: Five human ESCC cell lines (KYSE-150, KYSE-180, KYSE-850, and KYSE-960) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Expression Profiles in Superficial Esophageal Squamous Cell Carcinoma before Endoscopic Submucosal Dissection: A Pilot Study

doi: 10.3390/ijms22094789

Figure Lengend Snippet: Summary of significantly upregulated and downregulated miRNAs between superficial esophageal squamous cell carcinoma (ESCC) tumor and adjacent (non-tumor) tissues. Fold change (FC) > 2.0, FC < 0.5, p -value < 0.05.

Article Snippet: Five human ESCC cell lines (KYSE-150, KYSE-180, KYSE-850, and KYSE-960) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Expression Profiles in Superficial Esophageal Squamous Cell Carcinoma before Endoscopic Submucosal Dissection: A Pilot Study

doi: 10.3390/ijms22094789

Figure Lengend Snippet: Unsupervised hierarchical clustering analysis of differentially expressed miRNAs in ESCC tissue according to invasion depth and lymphovascular invasion. ( A ) Patients were assigned to two groups based on depth of invasion (cluster A): epithelium (EP, n = 5) or lamina propria, muscularis mucosae, or submucosa (LP/MM/SM1, n = 5). See text for details. ( B ) Patients were assigned to two groups based on the presence ( n = 2) or absence ( n = 8) of lymphovascular invasion (Cluster B). Columns represent the patients and rows represent the individual miRNAs. Red and blue indicate high and low miRNA expression levels, respectively.

Article Snippet: Five human ESCC cell lines (KYSE-150, KYSE-180, KYSE-850, and KYSE-960) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: MicroRNA Expression Profiles in Superficial Esophageal Squamous Cell Carcinoma before Endoscopic Submucosal Dissection: A Pilot Study

doi: 10.3390/ijms22094789

Figure Lengend Snippet: Validation of differential expression of miRNAs in ESCC tissues and cell lines. ( A , B ) qRT-PCR analysis of mi-21-5p, miR-210-3p, and miR-146b-5p in ( A ) 10 matched pairs of superficial ESCC compared with normal tissues (CON), and ( B ) ESCC cell lines compared with normal esophageal epithelial cells (Het-1A). ESCC cells were transfected with a control RNA or miR-21-5p, miR-210-3p, and miR-146b-5p mimics or inhibitors and analyzed in cell counting kit-8 (CCK-8) proliferations assays ( C ) or Transwell invasion and migration assays ( D ). Data are presented as the mean ± standard deviation of triplicates. * p < 0.05, ** p < 0.01 vs. control group.

Article Snippet: Five human ESCC cell lines (KYSE-150, KYSE-180, KYSE-850, and KYSE-960) were obtained from the Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan).

Techniques: Biomarker Discovery, Quantitative Proteomics, Quantitative RT-PCR, Transfection, Control, Cell Counting, CCK-8 Assay, Migration, Standard Deviation