HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition
Abstract
:1. Introduction
2. Results
2.1. Consistent Downregulation of HDAC5 in Urothelial Carcinoma Cell Lines
2.2. HDAC5 Expression Impairs Long-Term Proliferation and Clonogenic Potency of UCCs
2.3. HDAC5 Induces an Epithelial-Mesenchymal Transition in VM-Cub-1 Cells
2.4. The Proteome of VM-Cub-1 Cells is Profoundly Altered by HDAC5
2.5. Transcriptome Changes by HDAC5 Are Most Extensive in VM-Cub-1 and Hint at an Involvement of TGFβ
2.6. Effects of Added TGFβ and TNFα on Epithelial UC Cell Lines Are Not Influenced by HDAC5
2.7. Weak Effects of HDAC4 Overexpression on UC Cell Lines
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Generation of Stably Eexpressing HDAC5 and Vector Control UC Cell Lines
4.3. Clone Formation Assays
4.4. Cell Proliferation Analysis
4.5. Migration Assay
4.6. Counting of Viable Cells by Flow Cytometry
4.7. Immunoblot Analysis
4.8. RNA Isolation, Sample Preparation, and High Throughput mRNA Sequencing
4.9. Proteome Analysis by Label-Free Quantification Based Mass Spectrometry
4.10. RNA-Seq and Data Analysis
4.11. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
Abbreviations
BCA | Bicinchoninic acid |
DMEM | Dulbecco’s modified Eagle’s medium |
EMT | Epithelial-mesenchymal transition |
FDR | False discovery rate |
HDAC | Histone deacetylase |
IPA | Ingenuity Pathway analysis |
SD | Standard deviation |
TGF | Transforming growth factor |
TNF | Tumor necrosis factor |
UCC(s) | Urothelial carcinoma cell line(s) |
References
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Cell Lines | No. Genes FDR 1: p ≤ 0.05 | No. Genes Fold Change ≥2 | No. Genes Bonferroni: p ≤ 0.05 | No. Genes Fold Change ≥2 |
---|---|---|---|---|
RT112 Vector vs. HDAC5 | 3278 | 510 173 up 337 down | 1245 | 261 69 up 192 down |
VM-Cub-1 Vector vs. HDAC5 | 16,883 | 9387 3331 up 6056 down | 12,074 | 6948 2645 up 4303 down |
SW1710 Vector vs. HDAC5 | 10,941 | 2381 927 up 1454 down | 5949 | 1265 516 up 749 down |
UM-UC-3 Vector vs. HDAC5 | 8318 | 1379 542 up 837 down | 4062 | 733 233 up 500 down |
Rank | RT112 | VM-Cub-1 | SW1710 | UM-UC-3 |
---|---|---|---|---|
1 | LPS/IL-1 Mediated Inhibition of RXR Function (9.5% 21/221) 1 | Axonal Guidance Signaling (46.9% 212/452) | Axonal Guidance Signaling (18.1% 82/452) | Hepatic Fibrosis/Hepatic Stellate Cell Activation (16.9% 31/183) |
2 | Granulocyte Adhesion and Diapedesis (10.1% 18/179) | Hepatic Fibrosis/Hepatic Stellate Cell Activation (55.2% 101/183) | LPS/IL-1 Mediated Inhibition of RXR Function (18.1% 40/221) | Axonal Guidance Signaling (10.8% 49/452) |
3 | Agranulocyte Adhesion and Diapedesis (9.4% 18/191) | Role of Macrophages, Fibroblasts and Endothelial Cells in Rheumatoid Arthritis (48.2% 150/311) | Coagulation System (34.3% 12/35) | Cellular Effects of Sildenafil (Viagra) (15.3% 20/131) |
4 | Xenobiotic Metabolism Signaling (7.3% 21/287) | Colorectal Cancer Metastasis Signaling (49.6% 123/248) | LXR/RXR Activation (20.7% 25/121) | Osteoarthritis Pathway (12.9% 27/210) |
5 | Inhibition of Matrix Metalloproteases (17.9% 7/39) | IL-8 Signaling (51.8% 102/197) | Hepatic Fibrosis/Hepatic Stellate Cell Activation (18.0% 33/183) | Coagulation System (25.7% 9/35) |
Rank | RT112 | VM-Cub-1 | SW1710 | UM-UC-3 |
---|---|---|---|---|
1 | TNF (Inhibited) | TNF (Inhibited) | TNF (Activated) | TNF (Inhibited) |
2 | SMARCA4 (not given) | estrogen receptor (Inhibited) | dexamethasone (not given) | Lipopolysaccharide (Inhibited) |
3 | Tretinoin (Inhibited) | TGFB1 (not given) | TGFB1 (Activated) | TGFB1 (not given) |
4 | Lipopolysaccharide (Inhibited) | SMARCA4 (Inhibited) | SMARCA4 (not given) | IFNG (Inhibited) |
5 | beta-estradiol (not given) | beta-estradiol (not given) | IFNG (not given) | IL1B (Inhibited) |
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Jaguva Vasudevan, A.A.; Hoffmann, M.J.; Beck, M.L.C.; Poschmann, G.; Petzsch, P.; Wiek, C.; Stühler, K.; Köhrer, K.; Schulz, W.A.; Niegisch, G. HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition. Int. J. Mol. Sci. 2019, 20, 2135. https://doi.org/10.3390/ijms20092135
Jaguva Vasudevan AA, Hoffmann MJ, Beck MLC, Poschmann G, Petzsch P, Wiek C, Stühler K, Köhrer K, Schulz WA, Niegisch G. HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition. International Journal of Molecular Sciences. 2019; 20(9):2135. https://doi.org/10.3390/ijms20092135
Chicago/Turabian StyleJaguva Vasudevan, Ananda Ayyappan, Michèle J. Hoffmann, Michael L. C. Beck, Gereon Poschmann, Patrick Petzsch, Constanze Wiek, Kai Stühler, Karl Köhrer, Wolfgang A. Schulz, and Günter Niegisch. 2019. "HDAC5 Expression in Urothelial Carcinoma Cell Lines Inhibits Long-Term Proliferation but Can Promote Epithelial-to-Mesenchymal Transition" International Journal of Molecular Sciences 20, no. 9: 2135. https://doi.org/10.3390/ijms20092135