Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Reagents
2.3. RNA Extraction and cDNA Synthesis
2.4. Quantitative Reverse Transcription-PCR (qRT-PCR)
2.5. Western Blot Analysis
2.6. Immunofluorescence
2.7. Wound Healing Assay
2.8. Transwell Migration Assay
2.9. Cell Viability Assay
2.10. PDL-1 Knockdown
2.11. Drug Combination Analysis
2.12. Statistical Analysis
3. Results
3.1. HCC Cells Undergo EMT upon Sorafenib Exposure
3.2. Sorafenib Treatment Induces Immune Checkpoint Molecules Expression in HCC Cells
3.3. SB431542 Inhibits TGF-β1-Mediated EMT in HCC Cells
3.4. SB431542 Attenuates TGF-β1-Induced PD-L1 Expression
3.5. Knockdown of PD-L1 in HCC Cells Can Reverse TGF-β1-Mediated EMT
3.6. HCC Cells Can Overcome Sorafenib Resistance by Combined Targeting of PD-L1 and TGF-β1 Signalling
3.7. Combining PD-L1 Knockdown with SB431542 Can Reverse Sorafenib-Induced EMT in HCC Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Primers | Sequence (5′-3′) |
---|---|
Snai1 forward | GCTGCAGGACTCTAATCCAGA |
Snai1 reverse | ATCTCCGGAGGTGGGATG |
TGF-β1 forward | TACCTGAACCCGTGTTGCTCTC |
TGF-β1 reverse | GTTGCTGAGGTATCGCCAGGAA |
TNF-α forward | CCCAGGGACCTCTCTCTAATC |
TNF-α reverse | TCTCAGCTCCACGCCATT |
TIM-3 reverse | GACTCTAGCAGACAGTGGGATC |
TIM-3 reverse | GGTGGTAAGCATCCTTGGAAAGG |
Antibodies | Cat. No. | Manufacturer | Antibody Category | Western Blot | Immunofluorescence |
---|---|---|---|---|---|
TGF-β1 | MA5-15065 | Thermofisher | Primary | 1:1000 | |
Ki67 | ab66155 | Abcam | Primary | 1:200 | |
β-Actin | 4967s | Cell Signaling | Primary | 1:2000 | |
Snai1 | 3879S | Cell Signaling | Primary | 1:1000 | |
Snai2 | 9585S | Cell Signaling | Primary | 1:1000 | |
VISTA | ab235362 | Abcam | Primary | 1:1000 | |
TIM-3 | ab241332 | Abcam | Primary | 1:1000 |
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Shrestha, R.; Prithviraj, P.; Bridle, K.R.; Crawford, D.H.G.; Jayachandran, A. Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment. J. Clin. Med. 2021, 10, 1889. https://doi.org/10.3390/jcm10091889
Shrestha R, Prithviraj P, Bridle KR, Crawford DHG, Jayachandran A. Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment. Journal of Clinical Medicine. 2021; 10(9):1889. https://doi.org/10.3390/jcm10091889
Chicago/Turabian StyleShrestha, Ritu, Prashanth Prithviraj, Kim R. Bridle, Darrell H. G. Crawford, and Aparna Jayachandran. 2021. "Combined Inhibition of TGF-β1-Induced EMT and PD-L1 Silencing Re-Sensitizes Hepatocellular Carcinoma to Sorafenib Treatment" Journal of Clinical Medicine 10, no. 9: 1889. https://doi.org/10.3390/jcm10091889