Skip to content
2000
image of GPR65 as a Laryngeal Cancer Risk Gene Identified through Single-Cell Transcriptomics, Mendelian Randomization Analysis, and Experimental Validation

Abstract

Introduction

Laryngeal cancer is a common malignant tumor of the head and neck worldwide. This study aimed to identify potential risk genes, with a particular focus on GPR65, and to investigate its functional mechanism in pathogenesis of laryngeal cancer.

Materials and Methods

Comprehensive analyses, including scRNA-seq analysis, genome-wide association study (GWAS), eQTL, and TCGA data, were conducted to identify risk genes for laryngeal cancer and characterize the function of these risk genes. Next, qRT-PCR, immunohistochemistry, cell proliferation, cell migration, and invasion assays were employed to verify the expression of GPR65 and its function in laryngeal squamous cell carcinoma (LSCC) .

Results

Single-cell analysis screened 416 highly expressed genes in CD8+ central memory T cells (CD8_CM). Mendelian randomization (MR) analysis identified GPR65 as a crucial gene in the development of laryngeal cancer. GPR65 expression was significantly elevated in the tumor tissues compared to normal tissues, with particularly high levels observed in stage IV HNSCC. , LSCC cell lines (TU686 and Hep-2) exhibited marked upregulation of GPR65 relative to normal epithelial cells, and siRNA-mediated silencing of GPR65 suppressed the proliferation, migration, and invasion of LSCC cells. Furthermore, GPR65 expression showed a positive correlation with immune cell infiltration, particularly CD8+ T cells and M1 macrophages.

Discussion

This study identified GPR65 as a potential risk gene for laryngeal cancer through single-cell transcriptomics and MR analyses and provided novel evidence of its involvement in the development of the cancer.

Conclusion

The present findings showed that highly expressed GPR65 was a tumor-promoting gene in laryngeal cancer, showing its clinical value as a potential therapeutic target.

© 2025 Bentham Science Publishers
Loading

Article metrics loading...

/content/journals/acamc/10.2174/0118715206414176250905114930
2025-10-09
2025-11-07

  • From This Site

    /content/journals/acamc/10.2174/0118715206414176250905114930
    dcterms_title,dcterms_subject,pub_keyword
    -contentType:Contributor -contentType:Concept -contentType:Institution
    10
    5
Loading full text...

Full text loading...

References

  1. Lan, L.L.; Liu S.H.; Fan Z.T.; Wang X.X. Wang J.T. Wang K.X. Zhao R-L. SET domain containing protein 8 (SET8) promotes tumour progression and indicates poor prognosis in patients with laryngeal squamous cell carcinoma. Oncologie 2023 25 1 61 69 10.1515/oncologie‑2023‑0019
    [Google Scholar]
  2. Gao T. Ren X. Li J. Shen N. miR‐30a‐3p inhibits the proliferation of laryngeal cancer cells by targeting DNMT3a through regulating DNA methylation of PTEN. Oncologie 2024 26 1 141 150 10.1515/oncologie‑2023‑0226
    [Google Scholar]
  3. Yao Z. Ma H. Liu L. Zhao Q. Qin L. Ren X. Wu C. Sun K. Novel defined N7-methylguanosine modification-related lncRNAs for predicting the prognosis of laryngeal squamous cell carcinoma. Biocell 2023 47 9 1965 1975 10.32604/biocell.2023.030796
    [Google Scholar]
  4. Bray F. Laversanne M. Sung H. Ferlay J. Siegel R.L. Soerjomataram I. Jemal A. Global cancer statistics 2022: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin. 2024 74 3 229 263 10.3322/caac.21834 38572751
    [Google Scholar]
  5. Qian L. Ni T. Fei B. Sun H. Ni H. An immune-related lncRNA pairs signature to identify the prognosis and predict the immune landscape of laryngeal squamous cell carcinoma. BMC Cancer 2022 22 1 545 10.1186/s12885‑022‑09524‑1 35568824
    [Google Scholar]
  6. Kim D.H. Kim S.W. Han J.S. Kim G.J. Basurrah M.A. Hwang S.H. The prognostic utilities of various risk factors for laryngeal squamous cell carcinoma: A systematic review and meta-analysis. Medicina 2023 59 3 497 10.3390/medicina59030497 36984498
    [Google Scholar]
  7. Tian Y. Bai F. Zhang D. New target DDR1: A “double-edged sword” in solid tumors. Biochim. Biophys. Acta Rev. Cancer 2023 1878 1 188829 10.1016/j.bbcan.2022.188829 36356724
    [Google Scholar]
  8. Stuart T. Butler A. Hoffman P. Hafemeister C. Papalexi E. Mauck W.M. Hao Y. Stoeckius M. Smibert P. Satija R. Comprehensive integration of single-cell data. Cell 2019 177 7 1888 1902.e21 10.1016/j.cell.2019.05.031 31178118
    [Google Scholar]
  9. Chen J. Lin A. Luo P. Advancing pharmaceutical research: A comprehensive review of cutting-edge tools and technologies. Curr. Pharm. Anal. 2024 21 1 1 19 10.1016/j.cpan.2024.11.001
    [Google Scholar]
  10. Qu S. Gong M. Deng Y. Xiang Y. Ye D. Research progress and application of single-cell sequencing in head and neck malignant tumors. Cancer Gene Ther. 2024 31 1 18 27 10.1038/s41417‑023‑00691‑2 37968342
    [Google Scholar]
  11. Sun Y. Chen S. Lu Y. Xu Z. Fu W. Yan W. Single-cell transcriptomic analyses of tumor microenvironment and molecular reprograming landscape of metastatic laryngeal squamous cell carcinoma. Commun. Biol. 2024 7 1 63 10.1038/s42003‑024‑05765‑x 38191598
    [Google Scholar]
  12. Emdin C.A. Khera A.V. Kathiresan S. Mendelian randomization. JAMA 2017 318 19 1925 1926 10.1001/jama.2017.17219 29164242
    [Google Scholar]
  13. Mei Y. Zhang X. Lin S. Fan H. Zhou M. Mao J. Wang X. Mechanism of Yangxuetongmai Decoction in treating essential hypotension: A network pharmacology, Mendelian randomization and molecular docking strategy analysis. Lett. Drug Des. Discov. 2025 22 1 100008 10.1016/j.lddd.2025.100008
    [Google Scholar]
  14. Thakral A. Lee J.J.W. Hou T. Hueniken K. Dudding T. Gormley M. Virani S. Olshan A. Diergaarde B. Ness A.R. Waterboer T. Smith-Byrne K. Brennan P. Hayes D.N. Sanderson E. Brown M.C. Huang S. Bratman S.V. Spreafico A. De Almeida J. Davies J.C. Bierut L. Macfarlane G.J. Lagiou P. Lagiou A. Polesel J. Agudo A. Alemany L. Ahrens W. Healy C.M. Conway D.I. Nygard M. Canova C. Holcatova I. Richiardi L. Znaor A. Goldstein D.P. Hung R.J. Xu W. Liu G. Espin-Garcia O. Smoking and alcohol by HPV status in head and neck cancer: A Mendelian randomization study. Nat. Commun. 2024 15 1 7835 10.1038/s41467‑024‑51679‑x 39244563
    [Google Scholar]
  15. Gui L. He X. Tang L. Yao J. Pi J. Obesity and head and neck cancer risk: A mendelian randomization study. BMC Med. Genomics 2023 16 1 200 10.1186/s12920‑023‑01634‑4 37620971
    [Google Scholar]
  16. Ihara Y. Kihara Y. Hamano F. Yanagida K. Morishita Y. Kunita A. Yamori T. Fukayama M. Aburatani H. Shimizu T. Ishii S. The G protein-coupled receptor T-cell death-associated gene 8 (TDAG8) facilitates tumor development by serving as an extracellular pH sensor. Proc. Natl. Acad. Sci. USA 2010 107 40 17309 17314 10.1073/pnas.1001165107 20855608
    [Google Scholar]
  17. Wang L. Sun L. Sun H. Xing Y. Zhou S. An G. Li J. Ren K. Sun J. GPR65 as a potential immune checkpoint regulates the immune microenvironment according to pan-cancer analysis. Heliyon 2023 9 2 e13617 10.1016/j.heliyon.2023.e13617 36852075
    [Google Scholar]
  18. Li S. Melchiore F. Kantari-Mimoun C. Mouton A. Knockaert S. Philippon W. Chanrion B. Bourgeois C. Lefebvre C. Elhmouzi-Younes J. Blanc V. Ramon Olayo F. Laugel B. In silico and pharmacological evaluation of GPR65 as a cancer immunotherapy target regulating T-cell functions. Front. Immunol. 2024 15 1483258 10.3389/fimmu.2024.1483258 39483470
    [Google Scholar]
  19. Qi J. Liu S. Zhang Z. What role does GPR65 play in the progression of osteosarcoma? Its mechanism and clinical significance. Cancer Cell Int. 2024 24 1 31 10.1186/s12935‑024‑03216‑5 38218960
    [Google Scholar]
  20. Fang F. Tai R. Yang F. Dong R. Zhang Y. Bioinformatic methods uncover 5 diagnostic biomarkers associated with drug resistance and metastasis for gastrointestinal stromal tumor. Curr. Pharm. Anal. 2025 21 2 67 76 10.1016/j.cpan.2025.01.003
    [Google Scholar]
  21. Shang Z. Xi S. Lai Y. Cheng H. Single-cell transcriptomics and Mendelian randomization reveal LUCAT1’s role in right-sided colorectal cancer risk. Front. Genet. 2024 15 1357704 10.3389/fgene.2024.1357704 38711918
    [Google Scholar]
  22. Aran D. Looney A.P. Liu L. Wu E. Fong V. Hsu A. Chak S. Naikawadi R.P. Wolters P.J. Abate A.R. Butte A.J. Bhattacharya M. Reference-based analysis of lung single-cell sequencing reveals a transitional profibrotic macrophage. Nat. Immunol. 2019 20 2 163 172 10.1038/s41590‑018‑0276‑y 30643263
    [Google Scholar]
  23. Street K. Risso D. Fletcher R.B. Das D. Ngai J. Yosef N. Purdom E. Dudoit S. Slingshot: Cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 2018 19 1 477 10.1186/s12864‑018‑4772‑0 29914354
    [Google Scholar]
  24. Jin S. Guerrero-Juarez C.F. Zhang L. Chang I. Ramos R. Kuan C.H. Myung P. Plikus M.V. Nie Q. Inference and analysis of cell-cell communication using CellChat. Nat. Commun. 2021 12 1 1088 10.1038/s41467‑021‑21246‑9 33597522
    [Google Scholar]
  25. Zhou Y. Zhou B. Pache L. Chang M. Khodabakhshi A.H. Tanaseichuk O. Benner C. Chanda S.K. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat. Commun. 2019 10 1 1523 10.1038/s41467‑019‑09234‑6 30944313
    [Google Scholar]
  26. You W. Lin Y. Liu M. Lin Z. Ye R. Zhang C. Zeng R. Investigating potential novel therapeutic targets and biomarkers for ankylosing spondylitis using plasma protein screening. Front. Immunol. 2024 15 1406041 10.3389/fimmu.2024.1406041 39185422
    [Google Scholar]
  27. Cao E.Y. Ouyang J.F. Rackham O.J.L. GeneSwitches: Ordering gene expression and functional events in single-cell experiments. Bioinformatics 2020 36 10 3273 3275 10.1093/bioinformatics/btaa099 32058565
    [Google Scholar]
  28. Cao J. Spielmann M. Qiu X. Huang X. Ibrahim D.M. Hill A.J. Zhang F. Mundlos S. Christiansen L. Steemers F.J. Trapnell C. Shendure J. The single-cell transcriptional landscape of mammalian organogenesis. Nature 2019 566 7745 496 502 10.1038/s41586‑019‑0969‑x 30787437
    [Google Scholar]
  29. Wu Y. Yang S. Ma J. Chen Z. Song G. Rao D. Cheng Y. Huang S. Liu Y. Jiang S. Liu J. Huang X. Wang X. Qiu S. Xu J. Xi R. Bai F. Zhou J. Fan J. Zhang X. Gao Q. Spatiotemporal immune landscape of colorectal cancer liver metastasis at single-cell level. Cancer Discov. 2022 12 1 134 153 10.1158/2159‑8290.CD‑21‑0316 34417225
    [Google Scholar]
  30. Liao C. Wang X. TCGAplot: An R package for integrative pan-cancer analysis and visualization of TCGA multi-omics data. BMC Bioinformatics 2023 24 1 483 10.1186/s12859‑023‑05615‑3 38105215
    [Google Scholar]
  31. Bai Y. Liu X. Qi X. Liu X. Peng F. Li H. Fu H. Pei S. Chen L. Chi X. Zhang L. Zhu X. Song Y. Wang Y. Meng S. Jiang T. Shao S. PDIA6 modulates apoptosis and autophagy of non-small cell lung cancer cells via the MAP4K1/JNK signaling pathway. EBioMedicine 2019 42 311 325 10.1016/j.ebiom.2019.03.045 30922965
    [Google Scholar]
  32. Yu S.F. Cheng T.T. Huang G.K. Hsu C.Y. Kao Y.H. Chung Y.H. Targeting FGFR3 is a useful therapeutic strategy for rheumatoid arthritis treatment. Curr. Mol. Pharmacol. 2023 17 1 e18761429261684 10.2174/0118761429261684231002062505 37982287
    [Google Scholar]
  33. Livak K.J. Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method. Methods 2001 25 4 402 408 10.1006/meth.2001.1262 11846609
    [Google Scholar]
  34. Wu Y. Zhang Y. Zheng X. Dai F. Lu Y. Dai L. Niu M. Guo H. Li W. Xue X. Bo Y. Guo Y. Qin J. Qin Y. Liu H. Zhang Y. Yang T. Li L. Zhang L. Hou R. Wen S. An C. Li H. Xu W. Gao W. Circular RNA circCORO1C promotes laryngeal squamous cell carcinoma progression by modulating the let-7c-5p/PBX3 axis. Mol. Cancer 2020 19 1 99 10.1186/s12943‑020‑01215‑4 32487167
    [Google Scholar]
  35. Ge G. Jiang X. Tian X. Zhou Y. Cao G. The role of Salvia miltiorrhiza compounds in hepatocellular carcinoma: A preliminary investigation based on computational analysis and liquid chromatography-tandem mass spectrometry. Curr. Pharm. Anal. 2025 21 4 249 264 10.1016/j.cpan.2025.04.001
    [Google Scholar]
  36. Han J. Khatwani N. Searles T.G. Turk M.J. Angeles C.V. Memory CD8+ T cell responses to cancer. Semin. Immunol. 2020 49 101435 10.1016/j.smim.2020.101435 33272898
    [Google Scholar]
  37. Zhang K. Zhang M.X. Meng X.X. Zhu J. Wang J.J. He Y.F. Li Y.H. Zhao S.C. Shi Z.M. Zheng L.N. Han T. Hong W. Targeting GPR65 alleviates hepatic inflammation and fibrosis by suppressing the JNK and NF-κB pathways. Mil. Med. Res. 2023 10 1 56 10.1186/s40779‑023‑00494‑4 38001521
    [Google Scholar]
  38. Lin R. Wu W. Chen H. Gao H. Wu X. Li G. He Q. Lu H. Sun M. Liu Z. GPR65 promotes intestinal mucosal Th1 and Th17 cell differentiation and gut inflammation through downregulating NUAK2. Clin. Transl. Med. 2022 12 3 e771 10.1002/ctm2.771 35343079
    [Google Scholar]
  39. Marie M.A. Sanderlin E.J. Satturwar S. Hong H. Lertpiriyapong K. Donthi D. Yang L.V. GPR65 (TDAG8) inhibits intestinal inflammation and colitis-associated colorectal cancer development in experimental mouse models. Biochim. Biophys. Acta Mol. Basis Dis. 2022 1868 1 166288 10.1016/j.bbadis.2021.166288 34628032
    [Google Scholar]
  40. Yan C. Yang Z. Chen P. Yeh Y. Sun C. Xie T. Huang W. Zhang X. GPR65 sensing tumor-derived lactate induces HMGB1 release from TAM via the cAMP/PKA/CREB pathway to promote glioma progression. J. Exp. Clin. Cancer Res. 2024 43 1 105 10.1186/s13046‑024‑03025‑8 38576043
    [Google Scholar]
  41. Xu H. Chen X. Huang J. Deng W. Zhong Q. Yue C. Wang P. Huang Z. Identification of GPR65, a novel regulator of matrix metalloproteinases using high through-put screening. Biochem. Biophys. Res. Commun. 2013 436 1 96 103 10.1016/j.bbrc.2013.05.065 23707809
    [Google Scholar]
  42. Rosko A.E. McColl K.S. Zhong F. Ryder C.B. Chang M.J. Sattar A. Caimi P.F. Hill B.T. Al-Harbi S. Almasan A. Distelhorst C.W. Acidosis sensing receptor gpr65 correlates with anti-apoptotic Bcl-2 family member expression in CLL cells: potential implications for the CLL microenvironment. J. Leuk. 2014 2 5 160 25984552
    [Google Scholar]
  43. Chen Y. Yu D. Qian H. Shi Y. Tao Z. CD8+ T cell-based cancer immunotherapy. J. Transl. Med. 2024 22 1 394 10.1186/s12967‑024‑05134‑6 38685033
    [Google Scholar]
  44. Zhang Y. Li L. Zheng W. Zhang L. Yao N. CD8+ T-cell exhaustion in the tumor microenvironment of head and neck squamous cell carcinoma determines poor prognosis. Ann. Transl. Med. 2022 10 6 273 10.21037/atm‑22‑867 35434003
    [Google Scholar]
  45. Wang S. Wu Z.Z. Zhu S.W. Wan S.C. Zhang M.J. Zhang B.X. CTLA-4 blockade induces tumor pyroptosis via CD8(+) T cells in head and neck squamous cell carcinoma. Mol. Ther. 2023 31 7 2154 2168 10.1016/j.ymthe.2023.02.023
    [Google Scholar]
  46. Chatzopoulos K. Kotoula V. Manoussou K. Markou K. Vlachtsis K. Angouridakis N. Nikolaou A. Vassilakopoulou M. Psyrri A. Fountzilas G. Tumor infiltrating lymphocytes and CD8+ T cell subsets as prognostic markers in patients with surgically treated laryngeal squamous cell carcinoma. Head Neck Pathol. 2020 14 3 689 700 10.1007/s12105‑019‑01101‑6 31749124
    [Google Scholar]
/content/journals/acamc/10.2174/0118715206414176250905114930
Loading
GPR65 as a Laryngeal Cancer Risk Gene Identified through Single-Cell Transcriptomics, Mendelian Randomization Analysis, and Experimental Validation
Bentham Science Publishers ; https://doi.org/10.2174/0118715206414176250905114930
/content/journals/acamc/10.2174/0118715206414176250905114930
/content/journals/acamc/10.2174/0118715206414176250905114930
Loading

Data & Media loading...

Supplements

Supplementary material is available on the publisher’s website along with the published article.

file format download Download

  • Article Type:     Research Article
Keywords: Mendelian randomization (MR) analysis ; Laryngeal cancer ; Genome-wide association study ; central memory CD8+ T cells ; single-cell transcriptomic sequencing ; GPR65

Most Cited Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error
Please enter a valid_number test