The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration
Abstract
1. Introduction
2. Results
2.1. α9-nAChR Is Overexpressed in Melanoma
2.2. Correlation of α9-nAChR Levels with Clinicopathological Features in Melanoma
2.3. Correlation Between α9-nAChR and PD-L1 in Melanoma
2.4. α9-nAChR Induces PD-L1 Expression and Regulates in Proliferation and Migration
2.5. α9-nAChR Regulates Melanoma Cell Proliferation via the AKT and ERK Signaling Pathways
2.6. α9-nAChR Promotes Melanoma Cell Migration through EMT
2.7. α9-nAChR Regulates PD-L1 Expression via the STAT3 Signaling Pathway
2.8. Nicotine-Induced α9-nAChR Activity Upregulates PD-L1 Expression and Promotes Melanoma Cell Proliferation and Migration
2.9. Nicotine-Induced α9-nAChR Activity Significantly Increases Melanoma Cell Proliferation via the AKT and ERK Signaling Pathways
2.10. Nicotine-Induced α9-nAChR Activity Upregulates PD-L1 Expression via STAT3 Signaling Pathway
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Bioinformatics Analysis
4.2.1. Melanoma Cell Line Dataset Analysis
4.2.2. TCGA-SKCM Cohort Analysis
4.3. Kaplan-Meier Analysis
4.4. Nicotine Treatment
4.5. Inhibitor Treatment
4.6. Cell Proliferation and Viability Assays
4.6.1. Cell Counting Assay
4.6.2. MTT Assay
4.6.3. Soft-Agar Growth Assay
4.7. Wound healing Scratch Assay
4.8. Construction of Vectors
4.8.1. α9-nAChR siRNA
4.8.2. α9-nAChR Overexpression
4.9. Generation of Stable Cell Lines
4.10. RNA Isolation and RT-PCR Analysis
4.11. Protein Extraction, Western Blotting, and Antibody
4.12. ChIP Assay
4.13. IHC Staining and Scoring System
4.13.1. IHC Staining
4.13.2. Immunohistochemistry Score
4.14. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Parameters | CHRNA9 Low mRNA n (%) | CHRNA9 High mRNA n (%) | p-Value | |
---|---|---|---|---|
Age (year) | ≥40 | 147 (32%) | 257 (55%) | 0.33 |
<40 | 18 (4%) | 42 (9%) | ||
Sex | Female | 114 (24%) | 179 (38%) | 0.04 * |
Male | 53 (11%) | 126 (27%) | ||
Ulceration | Yes | 61 (19%) | 106 (34%) | 0.08 |
No | 40 (13%) | 107 (34%) | ||
Breslow depth (mm) | ≥2 | 76 (21%) | 145 (40%) | 0.8 |
<2 | 46 (13%) | 93 (26%) | ||
Clark level | I-III | 32 (10%) | 69 (21%) | 0.24 |
IV-V | 85 (26%) | 136 (42%) | ||
Tumor size | T1 | 14 (3%) | 27 (7%) | 0.87 |
T2 | 27 (7%) | 52 (13%) | ||
T3 | 32 (8%) | 59 (14%) | ||
T4 | 60 (15%) | 93 (23%) | ||
Unknown | 15 (4%) | 32 (8%) | ||
Lymph node status | N0 | 94 (21%) | 141 (31%) | 0.035 * |
N1 | 20 (4%) | 54 (12%) | ||
N2 | 17 (4%) | 32 (7%) | ||
N3 | 22 (5%) | 34 (8%) | ||
Unknown | 6 (1%) | 30 (7%) | ||
Distant metastasis status | M0 | 148 (33%) | 270 (61%) | 0.39 |
M1 | 10 (2%) | 15 (3%) | ||
Stage | I-II | 94 (25%) | 123 (30%) | 0.009 ** |
III-IV | 64 (15%) | 131 (32%) |
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Nguyen, H.D.; Liao, Y.-C.; Ho, Y.-S.; Chen, L.-C.; Chang, H.-W.; Cheng, T.-C.; Liu, D.; Lee, W.-R.; Shen, S.-C.; Wu, C.-H.; et al. The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration. Cancers 2019, 11, 1991. https://doi.org/10.3390/cancers11121991
Nguyen HD, Liao Y-C, Ho Y-S, Chen L-C, Chang H-W, Cheng T-C, Liu D, Lee W-R, Shen S-C, Wu C-H, et al. The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration. Cancers. 2019; 11(12):1991. https://doi.org/10.3390/cancers11121991
Chicago/Turabian StyleNguyen, Hai Duong, You-Cheng Liao, Yuan-Soon Ho, Li-Ching Chen, Hui-Wen Chang, Tzu-Chun Cheng, Donald Liu, Woan-Ruoh Lee, Shing-Chuan Shen, Chih-Hsiung Wu, and et al. 2019. "The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration" Cancers 11, no. 12: 1991. https://doi.org/10.3390/cancers11121991
APA StyleNguyen, H. D., Liao, Y.-C., Ho, Y.-S., Chen, L.-C., Chang, H.-W., Cheng, T.-C., Liu, D., Lee, W.-R., Shen, S.-C., Wu, C.-H., & Tu, S.-H. (2019). The α9 Nicotinic Acetylcholine Receptor Mediates Nicotine-Induced PD-L1 Expression and Regulates Melanoma Cell Proliferation and Migration. Cancers, 11(12), 1991. https://doi.org/10.3390/cancers11121991