Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity
Abstract
:1. Introduction
2. Materials and Methods
2.1. Healthy Volunteers
2.2. Cell Culture
2.3. Cell Viability
2.4. Flow Cytometry
2.5. Western Blots
2.6. Quantitative Polymerase Chain Reaction (qPCR)
2.7. Gene Knockdown
2.8. Enzyme-Linked Immunosorbent Assay (ELISA) for Measurement of IFNγ
2.9. RNAseq Profiling and Bioinformatics Analysis
2.10. Isolation of Peripheral Blood Mononuclear Cells (PBMCs) and CD8+ T Cells
2.11. Chromatin Immunoprecipitation (ChIP)
2.12. Statistical Analysis
3. Results
3.1. Irradiation and PBMCs Synergistically Inhibited Tumor Cell Viability and Induced Apoptosis in Lung Cancer Cells
3.2. Reactivation of Healthy CD8+ T Cells in Encountering HCC827 and A549 In Vitro
3.3. IFNγ Dominantly Phosphorylated STAT3 in the Premise of Phosphorylated EGFR
3.4. IFNγ Increased MCL1 Expression in the Premise of Phosphorylated EGFR
3.5. Irradiation Specifically Blocked IFNγ-Mediated Phosphorylations on STAT1 and STAT3 in A549 Cells
3.6. STAT3 Dominantly Determined IFNγ-Mediated Gene Expression and Knockdown of STAT3-Mediated MCL1 Augmented PBMCs against A549 Cells
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Gene | Direction | Primer Sequence |
---|---|---|
GAPDH | Forward | GAGTCAACGGATTTGGTCGT |
Reverse | TTGATTTTGGAGGGATCTCG | |
GZMB | Forward | ACTGCAGCTGGAGAGAAAGG |
Reverse | TTCGCACTTTCGATCTTCCT | |
CD274 (PD-L1) | Forward | GTACCTTGGCTTTGCCACAT |
Reverse | CCAACACCACAAGGAGGAGT | |
TNFRSF14 (HVEM) | Forward | CCACTGGGTATGGTGGTTTC |
Reverse | TCACCTTCTGCCTCCTGTCT | |
LGALS9 (Galectin-9) | Forward | CCTTTGACCTCTGCTTCCTG |
Reverse | AAACAGACAGGCTGGGAGAA | |
STAT1 | Forward | CCGTTTTCATGACCTCCTGT |
Reverse | TGAATATTCCCCGACTGAGC | |
STAT2 | Forward | GAGGCCTCAACTCAGACCAG |
Reverse | GCGTCCATCATTCCAGAGAT | |
STAT3 | Forward | TTTCACTTGGGTGGAGAAGG |
Reverse | GCTACCTGGGTCAGCTTCAG | |
PSM88 | Forward | CACGGGTAGTGGGAACACTT |
Reverse | TCACCCAACCATCTTCCTTC | |
PSMB10 | Forward | AATGTGGACGCATGTGTGAT |
Reverse | TCCAGGGTTAGTGGCTTCAC | |
NFKB2 | Forward | GAACAGCCTTGCATCTAGCC |
Reverse | TCCGAGTCGCTATCAGAGGT | |
SOCS3 | Forward | GCCACCTACTGAACCCTCCT |
Reverse | ACGGTCTTCCGACAGAGATG | |
UBE2L6 | Forward | CAACCTCCCTACCACCTGAA |
Reverse | GCAAGGCTTCCAGTTCTCAC | |
EFNA1 | Forward | GGTGACTGTCAGTGGCAAAA |
Reverse | AGTGGAAGGAGCAGCACAGT | |
PML | Forward | ACACAACGTGAGCTTCATGG |
Reverse | AAGTGGGGTGGAGACTCCTT | |
FOSL1 | Forward | AGCTGCAGAAGCAGAAGGAG |
Reverse | GGAGTTAGGGAGGGTGTGGT | |
MCL1 | Forward | AGAAAGCTGCATCGAACCAT |
Reverse | CCAGCTCCTACTCCAGCAAC | |
ISG15 | Forward | TGTCGGTGTCAGAGCTGAAG |
Reverse | GCCCTTGTTATTCCTCACCA | |
ChIP_STAT1/3_MCL1 | Forward | AAAAGGGCTCACAAATCAGGT |
Reverse | GTCTTCGGAGGCTCTGAGTG | |
ChIP_STAT1/3_PD-L1 | Forward | ACTAGCATGGCTGAGACAGTGA |
Reverse | CATACCTAGTAGAACCTGCCCTGT |
Gene | Clone ID | Targeted Sequence |
---|---|---|
Luciferase | TRCN0000072249 | GCGGTTGCCAAGAGGTTCCAT |
STAT1 | TRCN0000004266 | CGACAGTATGATGAACACAGT |
STAT3 | TRCN0000020842 | CACAATCTACGAAGAATCAA. |
MCL1 | TRCN0000005515 | GCAGAAAGTATCACAGACGTT |
PDL1 | TRCN0000056914 | CGAATTACTGTGAAAGTCAAT |
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Wang, C.-I.; Chang, Y.-F.; Sie, Z.-L.; Ho, A.-S.; Chang, J.-S.; Peng, C.-L.; Cheng, C.-C. Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity. Cells 2021, 10, 2515. https://doi.org/10.3390/cells10102515
Wang C-I, Chang Y-F, Sie Z-L, Ho A-S, Chang J-S, Peng C-L, Cheng C-C. Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity. Cells. 2021; 10(10):2515. https://doi.org/10.3390/cells10102515
Chicago/Turabian StyleWang, Chun-I., Yi-Fang Chang, Zong-Lin Sie, Ai-Sheng Ho, Jung-Shan Chang, Cheng-Liang Peng, and Chun-Chia Cheng. 2021. "Irradiation Suppresses IFNγ-Mediated PD-L1 and MCL1 Expression in EGFR-Positive Lung Cancer to Augment CD8+ T Cells Cytotoxicity" Cells 10, no. 10: 2515. https://doi.org/10.3390/cells10102515