Bestrophin-4 relays HES4 and interacts with TWIST1 to suppress epithelial-to-mesenchymal transition in colorectal cancer cells
- Department of Gastroenterology and Hepatology, Sichuan University-University of Oxford Huaxi Joint Centre for Gastrointestinal Cancer, West China Hospital, Sichuan University, China
- Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, United Kingdom
- College of Acupuncture and Moxibustion, Fujian University of Traditional Chinese Medicine, China
- TCM Regulating Metabolic Diseases Key Laboratory of Sichuan Province, Hospital of Chengdu University of Traditional Chinese Medicine, China
Figures
Figure 1 with 1 supplement
BEST4 inhibits colorectal cancer (CRC) cell proliferation, clonogenesis, migration, and invasion in vitro.
Overexpression of BEST4 decreased proliferation of HCT 116 (A) and Caco2 (B) cells as determined by the IncuCyte confluence assay. (C) Viability of three individual BEST4-deleted clones as …
Figure 1—figure supplement 1
Construction of BEST4-overexpressing /BEST4-depetion colorectal cancer (CRC) cell lines, and cell proliferation measured by CCK-8 assay.
(A) Hemagglutinin (HA) protein expression in BEST4-HA-transfected GFP+ HCT116 individual clonal lines. (B) HA protein expression in BEST4-HA-transfected GFP+ Caco2 individual clonal lines. (C) BEST4 …
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Figure 1—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
PDF file containing original western blots for Figure 1—figure supplement 1, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig1-figsupp1-data2-v1.zip
Figure 2 with 1 supplement
BEST4 deters epithelial-to-mesenchymal transition (EMT) in colorectal cancer (CRC) in vitro and in vivo.
(A) Expression of mRNAs encoding EMT-related genes as determined by quantitative polymerase chain reaction (qPCR) in HCT116 cells transfected with BEST4-HA or empty vector (EV). (B) As determined by …
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Figure 2—source data 1
Original files for western blot analysis displayed in Figure 2C, D, and I.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig2-data1-v1.zip
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Figure 2—source data 2
PDF file containing original western blots for Figure 2C, D, and I, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig2-data2-v1.zip
Figure 2—figure supplement 1
Western blot signals were quantified using ImageJ software.
(A–D) Each band’s mean pixel intensities were determined and normalised to mean β-actin pixel intensity. *p<0.05, **p<0.01. Data from at least three independent experiments are presented as the mean …
Figure 3 with 1 supplement
Molecular regulation of BEST4 expression by HES4.
(A) Endogenous BEST4 mRNA and protein levels in HES4-overexpressing HCT116 cells were detected by quantitative polymerase chain reaction (qPCR) and western blotting, respectively. (B) Endogenous BEST…
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Figure 3—source data 1
Original files for western blot analysis displayed in Figure 3A, B, and F.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig3-data1-v1.zip
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Figure 3—source data 2
PDF file containing original western blots for Figure 3A, B, and F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig3-data2-v1.zip
Figure 3—figure supplement 1
BEST4 interacted with HES4.
(A) HES4 mRNA level in each colorectal cancer (CRC) cell line. (B) Western blot for BEST4 expression of Figure 3A is quantified by ImageJ, each band’s mean pixel intensities were determined and …
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Figure 3—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 3—figure supplement 1C and E.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig3-figsupp1-data1-v1.zip
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Figure 3—figure supplement 1—source data 2
PDF file containing original western blots for Figure 3—figure supplement 1C and E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig3-figsupp1-data2-v1.zip
Figure 4 with 2 supplements
BEST4 relays the HES4 signal and downregulates TWIST1 expression to counteract epithelial-to-mesenchymal transition (EMT) induction in colorectal cancer (CRC).
(A) Detection of TWIST1 promoter activity in HES4-overexpressing or BEST4-overexpressing HCT116 cells through a dual-luciferase reporter assay. (B) Detection of TWIST1 promoter activity in HES4-overe…
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Figure 4—source data 1
Original files for western blot analysis displayed in Figure 4D–I.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig4-data1-v1.zip
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Figure 4—source data 2
PDF file containing original western blots for Figure 4D–I, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig4-data2-v1.zip
Figure 4—figure supplement 1
BEST4 interacted with HES4 to inhibit TWIST1.
(A) BEST4 mRNA levels in HCT116 and LS174T cells transfected with BEST4-specific RNAs silencing (siRNAs) as detected by real-time polymerase chain reaction (PCR). (B) Co-immunoprecipitation of …
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Figure 4—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 4—figure supplement 1B and C.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
PDF file containing original western blots for Figure 4—figure supplement 1B and C, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig4-figsupp1-data2-v1.zip
Figure 4—figure supplement 2
Western blot signals were quantified using ImageJ software.
(A–D) Each band’s mean pixel intensities were determined and normalised to mean β-actin pixel intensity. *p<0.05, **p<0.01. Data from at least three independent experiments are presented as the mean …
Figure 5 with 1 supplement
Correlation of the BEST4 mRNA expression with clinicopathology and outcomes of patients diagnosed with CRC.
(A) Heatmap based on the top 100 differentially expressed genes (downregulated and upregulated) in CRCs compared with ANTs. (B) Volcano plots of RNA-seq results showing all differentially expressed …
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Figure 5—source data 1
Original files for western blot analysis displayed in Figure 5E.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig5-data1-v1.zip
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Figure 5—source data 2
PDF file containing original western blots for Figure 5E, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig5-data2-v1.zip
Figure 5—figure supplement 1
BEST4 suppresses epithelial-to-mesenchymal transition (EMT) independently of its channel functions.
(A–C) Incubation of HCT116 cell lines expressed BEST4 or empty vector (EV) with DIDS (300 μM), a gradient increased CaCCinh-A01 and ionomycin (1 μM), respectively, overnight. The levels of chloride …
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Figure 5—figure supplement 1—source data 1
Original files for western blot analysis displayed in Figure 5—figure supplement 1D, E, and F.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
PDF file containing original western blots for Figure 5—figure supplement 1D, E, and F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/88879/elife-88879-fig5-figsupp1-data2-v1.zip
Figure 6
BEST4 mRNA expression in colorectal tumours and matched non-tumour tissues and the clinical relationship between BEST4, HES4, and epithelial-to-mesenchymal transition (EMT) markers.
(A) Correlation between BEST4 and CDH1 in normal and colorectal cancer (CRC) samples analysed from the The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression (GTEx) databases using the Gene …
Tables
Table 1
Correlation of BEST4 mRNA level with clinicopathological factors.
| Parameter | n | BEST4 expression | p-Value | |
|---|---|---|---|---|
| Low | High | |||
| Age (year) | 0.66 | |||
| <60 | 58 | 30 | 28 | |
| ≥60 | 67 | 32 | 35 | |
| Sex | 0.022* | |||
| Male | 74 | 43 | 31 | |
| Female | 51 | 19 | 32 | |
| Tumour differentiation | 0.258 | |||
| Poor | 10 | 3 | 7 | |
| Moderate | 114 | 59 | 55 | |
| Well | 1 | 0 | 1 | |
| Tumour stage (TNM)† | ||||
| I | 23 | 10 | 13 | 0.02* |
| II | 50 | 18 | 32 | |
| III | 43 | 27 | 16 | |
| IV | 9 | 7 | 2 | |
| Lymph node metastasis | 0.032* | |||
| N0 | 75 | 30 | 45 | |
| N1 | 41 | 26 | 15 | |
| N2 | 9 | 6 | 3 | |
| Distant metastasis | 0.08 | |||
| Absent | 116 | 55 | 61 | |
| Present | 9 | 7 | 2 | |
| Location | 0.114 | |||
| Colon | 32 | 12 | 20 | |
| Rectum | 93 | 50 | 43 | |
| CEA level | 0.13 | |||
| Low | 63 | 27 | 26 | |
| High | 62 | 35 | 27 | |
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*
Statistically significant.
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†
Staging was performed according to the eighth edition of AJCC (Amin, 2017).
Table 2
Univariate and multivariate analysis of prognostic factors for overall survival of patients with colorectal cancer (CRC).
| Variable | Comparison | Univariate analysis, p (log-rank test) | Multivariateanalysis | ||
|---|---|---|---|---|---|
| HR | 95% CI | p | |||
| Age (year) | ≥60 vs <60 | 0.001* | 2.713 | 1.524–4.810 | 0.001* |
| Sex | Female vs male | 0.681 | NA | NA | NA |
| Clinical stage | II, III, IV vs I | 0.1 | NA | NA | NA |
| Tumour differentiation | Moderate, well vs poor | 0.08 | NA | NA | NA |
| Lymph node metastasis | N1, N2 vs N0 | <0.001* | 2.190 | 1.291–3.717 | 0.004* |
| Distant metastasis | Present vs absent | <0.001* | 2.185 | 1.103–4.328 | 0.025* |
| Location | Rectum vs colon | 0.22 | NA | NA | NA |
| CEA level | High vs low | 0.002* | 1.697 | 0.953–2.960 | 0.073 |
| BEST4 level | High vs low | <0.001* | 0.285 | 0.157–0.606 | <0.001* |
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*
Statistically significant.
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HR, hazard ratio; CI, confidence interval; NA, not applicable.
Additional files
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Supplementary file 1
Table S1.
The target sequence of short hairpin RNAs (shRNAs), RNAs silencing (siRNAs), and primers used in this study.
- https://cdn.elifesciences.org/articles/88879/elife-88879-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/88879/elife-88879-mdarchecklist1-v1.docx
