Chemoresistance-Associated Silencing of miR-4454 Promotes Colorectal Cancer Aggression through the GNL3L and NF-κB Pathway
Abstract
:1. Introduction
2. Results
2.1. Generation of Drug-Resistant Cell Lines
2.2. Apoptotic Reduction and Cell Cycle Alteration Increase CPT-11-R Cell Chemoresistance
2.3. CPT-11-R Cells Show Enhanced First-Line Chemoresistance, Colony Formation, Invasion, and Migration Ability
2.4. Downregulation of miR-4454 Is Attributed to Elevated Levels of GNL3L in the CPT-11-R Cells
2.5. GNL3L Is a Functional Target of miR-4454
2.6. MiR-4454 Suppresses Colon Cancer Cell Growth, Invasion, and Migration through the GNL3L and NF-κB Pathways
2.7. Silencing GNL3L Attenuates Proliferation, Colony Formation, and Invasion/Migration
2.8. Doxycycline-Induced Expression of miR-4454 Suppresses Colon Cancer Growth
2.9. Schematic Representation of the miR-4454/GNL3L/NFκB Pathway
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium Bromide Assay
4.3. Flow Cytometry for the Annexin V Apoptosis Assay
4.4. Cell Cycle Analysis
4.5. Microarray Profiling and Target Prediction
4.6. Western Blot Analysis
4.7. RNA Extraction, microRNA First-Strand Synthesis, and qRT-PCR
4.8. Extracellular Vehicle RNA Isolation
4.9. MicroRNA and siRNA or shRNA Transfection Experiments
4.10. miR-4454 Dox-Inducible Expression Vector and Gene Reporter Plasmid Construction
4.11. Luciferase Reporter Assay
4.12. Generation of miR-4454 pSBinducer Cells
4.13. Transwell Invasion/Migration Assay
4.14. Colony Formation Assay and Hematoxylin and Eosin Stain
4.15. Immunohistochemistry
4.16. In Situ TUNEL Assay
4.17. Dox Induction In Vivo Tumor Studies
4.18. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Group | Cases (n) | GNL3L Expression | 𝝌2 | * p Value | |
---|---|---|---|---|---|
Low | High | ||||
Expression | Expression | ||||
Normal colon/rectum | 33 | 25 (76%) | 8 (24%) | ||
Cancer | 33 | 11 (33%) | 22 (67%) | 11.98 | <0.0003 |
Metastatic cancer | 33 | 5 (15%) | 28 (85%) | 24.44 | <0.0001 |
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Kannathasan, T.; Kuo, W.-W.; Chen, M.-C.; Viswanadha, V.P.; Shen, C.-Y.; Tu, C.-C.; Yeh, Y.-L.; Bharath, M.; Shibu, M.A.; Huang, C.-Y. Chemoresistance-Associated Silencing of miR-4454 Promotes Colorectal Cancer Aggression through the GNL3L and NF-κB Pathway. Cancers 2020, 12, 1231. https://doi.org/10.3390/cancers12051231
Kannathasan T, Kuo W-W, Chen M-C, Viswanadha VP, Shen C-Y, Tu C-C, Yeh Y-L, Bharath M, Shibu MA, Huang C-Y. Chemoresistance-Associated Silencing of miR-4454 Promotes Colorectal Cancer Aggression through the GNL3L and NF-κB Pathway. Cancers. 2020; 12(5):1231. https://doi.org/10.3390/cancers12051231
Chicago/Turabian StyleKannathasan, Thetchinamoorthy, Wei-Wen Kuo, Ming-Cheng Chen, Vijaya Padma Viswanadha, Chia-Yao Shen, Chuan-Chou Tu, Yu-Lan Yeh, Mahalakshmi Bharath, Marthandam Asokan Shibu, and Chih-Yang Huang. 2020. "Chemoresistance-Associated Silencing of miR-4454 Promotes Colorectal Cancer Aggression through the GNL3L and NF-κB Pathway" Cancers 12, no. 5: 1231. https://doi.org/10.3390/cancers12051231