基于生物信息学和体外实验筛选头颈鳞癌生物标志物

doi:10.19438/j.cjoms.2024.04.003

摘要/Abstract

摘要： 目的: 筛选头颈部鳞状细胞癌 (head and neck squamous cell carcinoma,HNSCC)潜在的分子生物标志物,并确定其功能和临床意义。方法: 从基因综合表达 (Gene Expression Omnibus,GEO) 数据库下载GSE58911数据集,筛选正常样本和头颈鳞癌样本中的差异表达基因 (differentially expressed genes,DEGs)。使用GeneCards和比较毒理学基因组 (Comparative Toxicogenomics Database,CTD) 数据库,进一步筛选HNSCC潜在生物标志物,针对HNSCC潜在生物标志物进行生物信息学分析。利用癌症基因组图谱 (The Cancer Genome Atlas,TCGA) 数据库作为外部验证,并用ssGSEA评估免疫细胞浸润情况。通过CCK-8、集落形成实验及实时荧光定量PCR(RT-PCR)验证数据库分析结果的准确性。结果: 在GSE58911数据集中得到605个DEGs,从中开发并验证了SERPINE1、PLAU、PLAUR和SERPINB2为HNSCC核心基因。与正常对照相比,HNSCC中SERPINE1,PLAU和PLAUR表达量显著上调,而SERPINB2表达量显著下调。核心基因与免疫细胞浸润关系,进一步提高对HNSCC免疫治疗的理解。RT-PCR结果与4个核心基因在数据集中的结果一致,体外实验结果表明,SERPINE1能促进HNSCC的进展。结论: SERPINE1、PLAU、PLAUR和SERPINB2可作为诊断HNSCC的潜在生物标志物,但需进一步体外和体内研究,明确其在HNSCC中的作用及具体机制。

关键词: 头颈部鳞状细胞癌, 生物信息学, 生物标志物, 核心基因

Abstract: PURPOSE: This study was aimed to identify potential molecular biomarkers in head and neck squamous cell carcinoma (HNSCC) and to determine their functional and clinical significance. METHODS: The GSE58911 dataset was downloaded from Gene Expression Omnibus (GEO) database to screen differentially expressed genes (DEGs) in normal and HNSCC samples. GeneCards and Comparative Toxicogenomics Database(CTD) were used to further determine potential biomarkers of HNSCC. A series of bioinformatic analyses were performed for potential biomarkers of HNSCC. The Cancer Genome Atlas(TCGA) database was then used as an external validation and the infiltration of immune cells was assessed using ssGSEA. Finally, the accuracy of the database analysis results was verified by cell CCK-8, flat panel cloning and RT-PCR experiments. RESULTS: In total, 605 DEGs were screened out of the GSE58911 microarray dataset. Out of these DEGs, SERPINE1, PLAU, PLAUR, and SERPINB2 were developed and validated as hub genes for HNSCC. Compared with normal controls, SERPINE1, PLAU, and PLAUR expression levels were significantly up-regulated, while SERPINB2 expression level was significantly down-regulated in HNSCC. Moreover, the relationship between hub genes and immune cell infiltration may improve the understanding of HNSCC immunotherapy. In addition, RT-PCR results were consistent with the results of the four hub genes in the dataset. In vitro results also showed that SERPINE1 could promote the progression of HNSCC. CONCLUSIONS: SERPINE1, PLAU, PLAUR and SERPINB2 can be used as potential biomarkers for the diagnosis of HNSCC, but further in vitro and in vivo studies are needed to clarify their roles and specific mechanisms in HNSCC.

Key words: Head and neck squamous cell carcinoma, Bioinformatics, Biomarkers, Hub genes

中图分类号:

R739.8

赵慧, 舒欣, 张帆, 任伟伟, 刘姣, 朱珠. 基于生物信息学和体外实验筛选头颈鳞癌生物标志物[J]. 中国口腔颌面外科杂志, 2024, 22(4): 329-338.

ZHAO Hui, SHU Xin, ZHANG Fan, REN Wei-wei, LIU Jiao, ZHU Zhu. Comprehensive bioinformatics analysis combined with experimental validation to screen biomarkers for head and neck squamous cell carcinoma[J]. China J Oral Maxillofac Surg, 2024, 22(4): 329-338.

参考文献

[1] Sung H, Ferlay J, Siegel RL, et al.Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA Cancer J Clin, 2021, 71(3): 209-249.
[2] Fuller CD, Wang SJ, Thomas Jr CR, et al.Conditional survival in head and neck squamous cell carcinoma: results from the SEER dataset 1973-1998[J]. Cancer, 2007, 109(7): 1331-1343.
[3] Wood DE, White JR, Georgiadis A, et al. A machine learning approach for somatic mutation discovery[J]. Sci Transl Med, 2018, 10(457): eaar7939.
[4] Cheng AJ, You GR, Lee CJ, et al.Systemic investigation identifying salivary miR-196b as a promising biomarker for early detection of head-neck cancer and oral precancer lesions[J]. Diagnostics(Basel), 2021, 11(8): 1411-1423.
[5] Zhang R, Gao Y.Identification of NUTF2 as a candidate diagnostic and prognostic biomarker associated with immune infiltration in head and neck squamous cell carcinoma[J]. Onco Targets Ther, 2021, 14: 5455-5467.
[6] Ghosh RD, Majumder SG.Circulating Long non-coding RNAs could be the Potential prognostic biomarker for liquid biopsy for the clinical management of oral squamous cell carcinoma[J]. Cancers(Basel), 2022, 14(22): 5590.
[7] Walther C, Tayebwa J, Lilljebjörn H, et al.A novel SERPINE1-FOSB fusion gene results in transcriptional up-regulation of FOSB in pseudomyogenic haemangioendothelioma[J]. J Pathol, 2014, 231(5): 534-540.
[8] Chen SJ, Li YQ, Zhu YH, et al.SERPINE1 overexpression promotes malignant progression and poor prognosis of gastric cancer[J]. J Oncol, 2022: 2647825.
[9] Liang ZR, Pan RL, Meng X, et al.Transcriptome study of oleanolic acid in the inhibition of breast tumor growth based on high-throughput sequencing[J]. Aging (Albany NY), 2021, 13(19): 22883-22897.
[10] Seker F, Cingoz A, Sur-Erdem I, et al.Identification of SERPINE1 as a regulator of glioblastoma cell dispersal with transcriptome profiling[J]. Cancers(Basel), 2019, 11(11): 1651-1671.
[11] Hanekom GS, Stubbings HM, Kidson SH.The active fraction of plasmatic plasminogen activator inhibitor type 1 as a possible indicator of increased risk for metastatic melanoma[J]. Cancer Detect Prev, 2002, 26(1): 50-59.
[12] Mekkawy AH, Pourgholami MH, Morris DL.Involvement of urokinase-type plasminogen activator system in cancer: an overview[J]. Med Res Rev, 2014, 34(5): 918-956.
[13] Novak CM, Horst EN, Taylor CC, et al.Fluid shear stress stimulates breast cancer cells to display invasive and chemoresistant phenotypes while upregulating PLAU in a 3D bioreactor[J]. Biotechnol Bioeng, 2019, 116(11): 3084-3097.
[14] Chen GJ, Sun JW, Xie MR, et al.PLAU promotes cell proliferation and epithelial-mesenchymal transition in head and neck squamous cell carcinoma[J]. Front Genet, 2021, 12: 651882.
[15] Montuori N, Cosimato V, Rinaldi L, et al.uPAR regulates pericellular proteolysis through a mechanism involving integrins and fMLF-receptors[J]. Thromb Haemost, 2013, 109(2):309-318.
[16] Tang CH, Wei Y.The urokinase receptor and integrins in cancer progression[J]. Cell Mol Life Sci, 2008, 65(12): 1916-1932.
[17] Madsen DH, Engelholm L, Ingvarsen S, et al.Extracellular collagenases and the endocytic receptor, urokinase plasminogen activator receptor-associated protein/endo180, cooperate in fibroblast-mediated collagen degradation[J]. J Biol Chem, 2007, 282(37): 27037-27045.
[18] Smith HW, Marshall CJ.Regulation of cell signalling by uPAR[J]. Nat Rev Mol Cell Biol, 2010, 11(1): 23-36.
[19] Jacobsen B, Ploug M.The urokinase receptor and its structural homologue C4.4A in human cancer: expression, prognosis and pharmacological inhibition[J]. Curr Med Chem, 2008, 15(25): 2559-2573.
[20] Béné MC, Castoldi G, Knapp W, et al.CD87(urokinase-type plasminogen activator receptor), function and pathology in hematological disorders: a review[J]. Leukemia, 2004, 18(3): 394-400.
[21] Astedt B, Lecander I, Brodin T, et al.Purification of a specific placental plasminogen activator inhibitor by monoclonal antibody and its complex formation with plasminogen activator[J]. Thromb Haemost, 1985, 53(1): 122-125.
[22] Kruithof EK, Baker MS, Bunn CL.Biological and clinical aspects of plasminogen activator inhibitor type 2[J]. Blood, 1995, 86(11): 4007-4024.
[23] Schroder WA, Le TT, Gardner J, et al.SerpinB2 deficiency in mice reduces bleeding times via dysregulated platelet activation[J]. Platelets, 2019, 30(5): 658-663.
[24] Sen P, Helmke A, Liao CM, et al.SerpinB2 regulates immune response in kidney injury and aging[J]. J Am Soc Nephrol, 2020, 31(5): 983-995.
[25] Elsafadi M, Shinwari T, Al-Malki S, et al.Convergence of TGFbeta and BMP signaling in regulating human bone marrow stromal cell differentiation[J]. Sci Rep, 2019, 9: 4977.
[26] Nie X, Wei J, Hao Y, et al.Consistent biomarkers and related pathogenesis underlying asthma revealed by systems biology approach[J]. Int J Mol Sci, 2019, 20(16): 4037-4055.
[27] Champelovier P, Boucard N, Levacher G, et al.Plasminogen-and colony-stimulating factor-1-associated markers in bladder carcinoma: diagnostic value of urokinase plasminogen activator receptor and plasminogen activator inhibitor type-2 using immunocytochemical analysis[J]. Urol Res, 2022, 30(5): 301-309.
[28] Nordengren J, Lidebring MF, Bendahl PO, et al.High tumor tissue concentration of plasminogen activator inhibitor 2 (PAI-2) is an independent marker for shorter progression-free survival in patients with early stage endometrial cancer[J]. Int J Cancer, 2021, 97(3): 379-385.
[29] Hanahan D, Coussens LM.Accessories to the crime: functions of cells recruited to the tumor microenvironment[J]. Cancer Cell, 2012, 21(3): 309-322.
[30] Balermpas P, Rödel F, Weiss C, et al.Tumor-infiltrating lymphocytes favor the response to chemoradiotherapy of head and neck cancer[J]. Oncoimmunology, 2014, 3(1): e27403.
[31] Eckert AW, Wickenhauser C, Salins PC, et al.Clinical relevance of the tumor microenvironment and immune escape of oral squamous cell carcinoma[J]. J Transl Med, 2016, 14(1): 85-98.
[32] Grimm M, Feyen O, Coy JF, et al.Analysis of circulating CD14⁺/CD16⁺ monocyte-derived macrophages (MDMs) in the peripheral blood of patients with oral squamous cell carcinoma[J]. Oral Surg Oral Med Oral Pathol Oral Radio, 2016, 121(3): 301-306.
[33] Iida M, Takayama E, Naganawa K, et al.Increase of peripheral blood CD57+ T-cells in patients with oral squamous cell carcinoma[J]. Anticancer Res, 2014, 34(10): 5729-5734.
[34] Bankur R, Rodrigues C, Anjaly D, et al.Quantitative analysis of tumor-associated tissue eosinophilia in different histological grades of oral squamous cell carcinoma[J]. Indian J Dent Res, 2016, 27(5): 463-467.
[35] De Paz D, Chang KP, Kao HK, et al.Clinical implications of tumor-associated tissue eosinophilia in tongue squamous cell carcinoma[J]. Laryngoscope, 2019, 129(5): 1123-1129.
[36] Hu, C, Zhang Y, Wu, C, et al. Heterogeneity of cancer-associated fibroblasts in head and neck squamous cell carcinoma: opportunities and challenges[J]. Cell Death Discov, 2023, 9(1): 124-133.
[37] Sakamoto H, Koma YI, Higashino N, et al.PAI-1 derived from cancer-associated fibroblasts in esophageal squamous cell carcinoma promotes the invasion of cancer cells and the migration of macrophages[J]. Lab Invest, 2021, 101(3): 353-368.
[38] Ciavarella, S, Laurenzana, A, De Summa, S, et al. u-PAR expression in cancer associated fibroblast: new acquisitions in multiple myeloma progression[J]. BMC Cancer, 2017, 17(1):215-226.