中性粒细胞胞外诱捕网调控口腔鳞状细胞癌的发展及预后的相关研究

doi:10.13591/j.cnki.kqyx.2024.07.004

摘要/Abstract

摘要：

目的探究中性粒细胞胞外诱捕网(neutrophil extracellular traps,NETs)在口腔鳞状细胞癌(oral squamous cell carcinoma,OSCC)中的表达及作用。方法通过免疫检测技术对OSCC组织中NETs的表达情况进行了确认,随后对不同NETs表达水平患者的临床病理特征及预后进行了分析。通过密度梯度离心法分离健康人外周血中性粒细胞并使用佛波酯(phorbol 12-myristate 13-acetate,PMA)诱导NETs生成,将其与OSCC细胞共培养后,通过CCK-8细胞增殖实验检测OSCC细胞增殖能力的变化,通过Transwell细胞迁移实验和细胞划痕实验检测OSCC细胞迁移能力的变化;通过蛋白免疫印迹实验(Western blotting,WB)检测NETs对OSCC上皮间充质转化(epithelial-mesenchymal transition,EMT)过程的调控。结果 NETs在OSCC中的表达水平高于正常组织(P<0.001),NETs高表达患者较NETs低表达患者预后更差(P<0.05),NETs表达水平与OSCC患者的临床分级、浸润和复发程度相关(P<0.05)。NETs可促进Cal27和HN6细胞的增殖和迁移能力(P<0.05),抑制了上皮标志物的蛋白表达,并促进了间充质标志物的蛋白表达(P<0.05),这些作用可被NETs抑制剂DNaseⅠ逆转。结论 NETs在OSCC中高表达,其可通过影响上皮间充质转化促进OSCC细胞的增殖和迁移,并影响OSCC患者预后。

关键词: 中性粒细胞胞外诱捕网, 口腔鳞状细胞癌, 上皮间充质转化

Abstract:

Objective To investigate the expression and role of neutrophil extracellular traps(NETs) in oral squamous cell carcinoma(OSCC). Methods Immunofluorescence and immunohistochemistry were used to detect the expression of NETs in OSCC. The clinicopathological characteristics and prognosis of patients with different NETs expression levels were analyzed. Density gradient centrifugation was used to isolate neutrophils from human peripheral blood. NETs were induced by phorbol 12-myristate 13-acetate(PMA) and co-cultured with OSCC cells. CCK-8 assay was used to detect changes in OSCC cell proliferation ability. OSCC cell migration ability was detected by Transwell cell migration assay and cell scratch assay. Western blotting(WB) assay was used to detect the effect of NETs on the index of epithelial-mesenchymal transition(EMT). Results NETs’ expression in OSCC was higher than that in normal tissues(P<0.001). The prognosis of patients with high NETs expression was worse than that of patients with low NETs expression(P<0.05). The expression level of NETs was correlated with the clinical grade, invasion and recurrence degree of OSCC patients(P<0.05). NETs promoted the proliferation and migration of Cal27 and HN6 cells(P<0.05), and inhibited the protein expression level of epithelial marker and promoted the protein expression level of mesenchymal markers(P<0.05), which could be reversed by the NETs inhibitor DNaseⅠ. Conclusion NETs are expressed at high levels in OSCC. NETs can promote the proliferation and migration of OSCC cells by regulating epithelial-mesenchymal transition and can affect the prognosis of OSCC patients.

Key words: neutrophil extracellular traps, oral squamous cell carcinoma, epithelial-mesenchymal transition

中图分类号:

R739.85

伊婕, 丁宇洁, 单雨菲, 顾佳麒, 孙志达. 中性粒细胞胞外诱捕网调控口腔鳞状细胞癌的发展及预后的相关研究[J]. 口腔医学, 2024, 44(7): 500-517.

YI Jie, DING Yujie, SHAN Yufei, GU Jiaqi, SUN Zhida. Neutrophil extracellular traps regulating the development and prognosis of oral squamous cell carcinomas[J]. Stomatology, 2024, 44(7): 500-517.

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参考文献 26

[1]	Tan YH, Wang ZH, Xu MT, et al. Oral squamous cell carcinomas: State of the field and emerging directions[J]. Int J Oral Sci, 2023, 15(1): 44. doi: 10.1038/s41368-023-00249-w pmid: 37736748
[2]	Li CC, Shen Z, Bavarian R, et al. Oral cancer[J]. Surg Oncol Clin N Am, 2020, 29(1): 127-144.
[3]	Chen DG, Zhang XM, Li ZJ, et al. Metabolic regulatory crosstalk between tumor microenvironment and tumor-associated macrophages[J]. Theranostics, 2021, 11(3): 1016-1030. doi: 10.7150/thno.51777 pmid: 33391518
[4]	Binnewies M, Roberts EW, Kersten K, et al. Understanding the tumor immune microenvironment (TIME) for effective therapy[J]. Nat Med, 2018, 24(5): 541-550. doi: 10.1038/s41591-018-0014-x pmid: 29686425
[5]	Kim HJ, Ji YR, Lee YM. Crosstalk between angiogenesis and immuneregulation in the tumor microenvironment[J]. Arch Pharm Res, 2022, 45(6): 401-416.
[6]	史金鹏, 吴凤英. 中性粒细胞在肿瘤微环境中的作用及研究进展[J]. 现代免疫学, 2024, 10(1): 72-76.
[7]	Cristinziano L, Modestino L, Antonelli A, et al. Neutrophil extracellular traps in cancer[J]. Semin Cancer Biol, 2022, 79: 91-104.
[8]	刘欢, 于洋, 王哲, 等. 中性粒细胞胞外诱捕网与牙周炎高凝状态的相关性研究[J]. 口腔医学, 2021, 41(9):788-792.
[9]	Wang Y, Liu F, Chen L, et al. Neutrophil extracellular traps (NETs) promote non-small cell lung cancer metastasis by suppressing lncRNA MIR503HG to activate the NF-κB/NLRP3 inflammasome pathway[J]. Front Immunol, 2022, 13: 867516.
[10]	Xiao YS, Cong M, Li JT, et al. Cathepsin C promotes breast cancer lung metastasis by modulating neutrophil infiltration and neutrophil extracellular trap formation[J]. Cancer Cell, 2021, 39(3): 423-437.e7. doi: 10.1016/j.ccell.2020.12.012 pmid: 33450198
[11]	Yang SF, Sun BS, Li JC, et al. Neutrophil extracellular traps promote angiogenesis in gastric cancer[J]. Cell Commun Signal, 2023, 21(1): 176. doi: 10.1186/s12964-023-01196-z pmid: 37480055
[12]	Zhang Y, Chandra V, Sanchez ER, et al. Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer[J]. J Exp Med, 2020, 217(12): e20190354.
[13]	Silvestre-Roig C, Fridlender ZG, Glogauer M, et al. Neutrophil diversity in health and disease[J]. Trends Immunol, 2019, 40(7): 565-583. doi: S1471-4906(19)30101-2 pmid: 31160207
[14]	Mukaida N, Sasaki SI, Baba T. Two-faced roles of tumor-associated neutrophils in cancer development and progression[J]. Int J Mol Sci, 2020, 21(10): 3457.
[15]	Chen Q, Zhang L, Li X, et al. Neutrophil extracellular traps in tumor metastasis: Pathological functions and clinical applications[J]. Cancers (Basel), 2021, 13(11): 2832.
[16]	王玉婷, 顾兵, 李华南, 等. 中性粒细胞胞外诱捕网检测方法综述[J]. 中国药理学通报, 2019, 35(12): 1646-1649.
[17]	Fousert E, Toes R, Desai J. Neutrophil extracellular traps (NETs) take the central stage in driving autoimmune responses[J]. Cells, 2020, 9(4): 915.
[18]	Burgener SS, Schroder K. Neutrophil extracellular traps in host defense[J]. Cold Spring Harb Perspect Biol, 2020, 12(7): a037028.
[19]	Hsu PP, Sabatini DM. Cancer cell metabolism: Warburg and beyond[J]. Cell, 2008, 134(5): 703-707. doi: 10.1016/j.cell.2008.08.021 pmid: 18775299
[20]	Yazdani HO, Roy E, Comerci AJ, et al. Neutrophil extracellular traps drive mitochondrial homeostasis in tumors to augment growth[J]. Cancer Res, 2019, 79(21): 5626-5639. doi: 10.1158/0008-5472.CAN-19-0800 pmid: 31519688
[21]	Masucci MT, Minopoli M, Del Vecchio S, et al. The emerging role of neutrophil extracellular traps (NETs) in tumor progression and metastasis[J]. Front Immunol, 2020, 11: 1749. doi: 10.3389/fimmu.2020.01749 pmid: 33042107
[22]	Zha CJ, Meng XQ, Li LL, et al. Neutrophil extracellular traps mediate the crosstalk between glioma progression and the tumor microenvironment via the HMGB1/RAGE/IL-8 axis[J]. Cancer Biol Med, 2020, 17(1): 154-168.
[23]	De Meo ML, Spicer JD. The role of neutrophil extracellular traps in cancer progression and metastasis[J]. Semin Immunol, 2021, 57: 101595.
[24]	Yao LL, Sheng XN, Dong XR, et al. Neutrophil extracellular traps mediate TLR9/Merlin axis to resist ferroptosis and promote triple negative breast cancer progression[J]. Apoptosis, 2023, 28(9/10): 1484-1495.
[25]	Wang CL, Wang Y, Jiang QL, et al. DNase Ⅰ and sivelestat ameliorate experimental hindlimb ischemia-reperfusion injury by eliminating neutrophil extracellular traps[J]. J Inflamm Res, 2023, 16: 707-721.
[26]	Zhang YL, Deng XL, Zhang J, et al. A potential driver of disseminated intravascular coagulation in heat stroke mice: Neutrophil extracellular traps[J]. Int J Environ Res Public Health, 2022, 19(19): 12448.

临床病理参数	例数(%)		NETs表达				P		r
临床病理参数	例数(%)		低表达		高表达		P		r
性别								1.000		0
男		26(52)		13		13
女		24(48)		12		12
年龄/岁								0.571		0.321
≤60		26(52)		14		12
>60		24(48)		11		13
肿瘤位置								0.554		4.922
颊		12(24)		6		6
舌		19(38)		7		12
牙龈		11(22)		8		3
唇		3(6)		1		2
腭		2(4)		1		1
口底		1(2)		1		0
口咽		2(4)		1		1
临床分级								0.004^**		8.333
Ⅰ+Ⅱ		20(40)		15		5
Ⅲ+Ⅳ		30(60)		10		20
T分期								0.145		2.122
T1+T2		31(62)		18		13
T3+T4		19(38)		7		12
N分期								0.074		5.219
N0		28(56)		18		10
N1		12(24)		4		8
N2		10(20)		3		7
是否浸润								<0.001^***		15.062
否		17(34)		15		2
是		33(66)		10		23
是否复发								0.011^*		6.480
否		25(50)		17		8
是		25(50)		8		17