牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响

doi:10.3969/j.issn.1674-8115.2024.02.002

上海交通大学学报（医学版） ›› 2024, Vol. 44 ›› Issue (2): 161-168.doi: 10.3969/j.issn.1674-8115.2024.02.002

• 论著 · 基础研究 • 上一篇

牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响

李虎虓¹(), 李笑甜¹, 赵旭日², 张桓瑜¹, 周薇², 宋忠臣¹()

^1.上海交通大学医学院附属第九人民医院牙周病科，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海市口腔医学研究所，上海 200011
^2.上海交通大学医学院附属第九人民医院口腔微生态与系统性疾病实验室，上海交通大学口腔医学院，国家口腔医学中心，国家口腔疾病临床医学研究中心，上海市口腔医学重点实验室，上海 200125

收稿日期:2023-09-20 接受日期:2023-12-26 出版日期:2024-02-28 发布日期:2024-03-25
通讯作者: 宋忠臣 E-mail:tentigerli@163.com;szhongchen@sina.com
作者简介:李虎虓（1994—），男，硕士，住院医师；电子信箱：tentigerli@163.com。
基金资助:
国家自然科学基金(82071112);上海交通大学医学院附属第九人民医院“交叉基金”(JYJC202005)

Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6

LI Huxiao¹(), LI Xiaotian¹, ZHAO Xuri², ZHANG Huanyu¹, ZHOU Wei², SONG Zhongchen¹()

^1.Department of Periodontology, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai Research Institute of Stomatology, Shanghai 200011, China
^2.Laboratory of Oral Microbiota and Systemic Disease, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine; College of Stomatology, Shanghai Jiao Tong University; National Center for Stomatology; National Clinical Research Center for Oral Diseases; Shanghai Key Laboratory of Stomatology, Shanghai 200125, China

Received:2023-09-20 Accepted:2023-12-26 Online:2024-02-28 Published:2024-03-25
Contact: SONG Zhongchen E-mail:tentigerli@163.com;szhongchen@sina.com
Supported by:
National Natural Science Foundation of China(82071112);Cross-disciplinary Research Fund of Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine(JYJC202005)

摘要/Abstract

摘要：

目的·观察牙龈素提取物对口腔鳞状细胞癌（oral squamous cell carcinoma，OSCC）细胞HN6生物学特性的影响。方法·选取人OSCC细胞系HN6，加入牙龈卟啉单胞菌（Porphyromonas gingivalis，P. gingivalis）牙龈素提取物进行培养。根据牙龈素提取物的蛋白浓度不同分为对照组（control组），及牙龈素提取物蛋白浓度3.125 μg/mL组、6.25 μg/mL组、12.5 μg/mL组、25 μg/mL组、50 μg/mL组和100 μg/mL组，培养24、48 h后，采用细胞计数试剂盒8（cell counting kit-8，CCK-8）检测牙龈素提取物对HN6细胞增殖活性的影响。后续其他实验，分为control组、25 μg/mL组和50 μg/mL组进行。通过流式细胞术检测牙龈素提取物对细胞周期的影响，划痕实验和Transwell实验检测细胞迁移和侵袭能力，实时荧光定量PCR（real-time PCR，RT-PCR）和Western blotting检测细胞E-cadherin和N-cadherin蛋白和基因的表达。结果·在牙龈素提取物刺激HN6细胞24 h时，相比较control组，牙龈素提取物蛋白浓度25 μg/mL组（P=0.025），50 μg/mL组（P=0.000）和100 μg/mL组（P=0.049）的HN6细胞增殖活性显著增加；当刺激48 h时，6.25 μg/mL组（P=0.024）、12.5 μg/mL组（P=0.006）、25 μg/mL组（P=0.000）、50 μg/mL组（P=0.000）和100 μg/mL组（P=0.000）均较control组HN6细胞增殖活性有显著增加。细胞周期检测结果显示，与control组相比，经牙龈素提取物刺激24 h，HN6细胞G1期比例下降，S＋G2期比例显著性上升（25 μg/mL组：P=0.024；50 μg/mL组：P=0.001）。细胞迁移实验结果显示，与control组相比，随着牙龈素提取物浓度升高，划痕愈合的百分比显著增加（P=0.001）。细胞Transwell侵袭实验结果显示，与control组相比，随着牙龈素提取物浓度升高，细胞穿过小室底部的数量显著性增加。RT-PCR和Western blotting实验结果显示，与control组相比，随着牙龈素提取物浓度增加，HN6细胞中N-cadherin mRNA和蛋白表达量显著性增加，E-cadherin mRNA和蛋白表达量显著性减少。结论·牙龈素提取物对OSCC细胞HN6的增殖、迁移和侵袭有促进作用。

关键词: 牙龈素, 牙周炎, 口腔鳞状细胞癌, 细胞增殖, 细胞迁移, 细胞侵袭

Abstract:

Objective ·To observe the effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6. Methods ·The HN6 cell line was selected, cultivated, and divided into different groups based on the protein concentration of gingipain extract from Porphyromonas gingivalis: control group, 3.125 μg/mL group, 6.25 μg/mL group, 12.5 μg/mL group, 25 μg/mL group, 50 μg/mL group, and 100 μg/mL group. After 24 and 48 h of cultivation, CCK-8 assay was used to detect the effects of gingipain extract on HN6 cell proliferation activity. Subsequent experiments were divided into control group, 25 μg/mL group and 50 μg/mL group. Flow cytometry was used to examine the effects of gingipain extract on cell cycle. Scratch assay and Transwell assay were performed to evaluate cell migration and invasion ability. Real-time PCR (RT-PCR) and Western blotting were used to measure the expression of E-cadherin and N-cadherin proteins and genes in cells. Results ·Stimulated with gingipain extract for 24 h, the HN6 cells showed significantly increased proliferation activity in the 25 μg/mL (P=0.025), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.049) groups compared to the control group. After 48 h, proliferation activity was significantly higher in the 6.25 μg/mL(P=0.024), 12.5 μg/mL (P=0.006), 25 μg/mL (P=0.000), 50 μg/mL (P=0.000), and 100 μg/mL (P=0.000) groups compared to the control group. Cell cycle analysis revealed that, after 24 h of gingipain stimulation, the proportion of HN6 cells in the G1 phase decreased, while the proportion in the S+G2 phase significantly increased compared to the control group (25 μg/mL group: P=0.024; 50 μg/mL group: P=0.001). Compared to the control group, the scratch assay demonstrated a significant increase in the percentage of scratch closure as the concentration of gingipain extract increased (P=0.001). Compared to the control group, the Transwell invasion assay showed a significant increase in the number of cells passing through the bottom of the chamber as the concentration of gingipain extract increased. RT-PCR and Western blotting results indicated that as the concentration of gingipain extract increased, the expression levels of N-cadherin mRNA and protein in HN6 cells significantly increased, while the expression levels of E-cadherin mRNA and protein significantly decreased compared to the control group. Conclusion ·Gingipain extract could promote proliferation, migration, and invasion of oral squamous cell carcinoma HN6 cells.

Key words: gingipain, periodontitis, oral squamous cell carcinoma (OSCC), cell proliferation, cell migration, cell invasion

中图分类号:

R781.4

李虎虓, 李笑甜, 赵旭日, 张桓瑜, 周薇, 宋忠臣. 牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响[J]. 上海交通大学学报（医学版）, 2024, 44(2): 161-168.

LI Huxiao, LI Xiaotian, ZHAO Xuri, ZHANG Huanyu, ZHOU Wei, SONG Zhongchen. Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2024, 44(2): 161-168.

图/表 7

表1 RT-PCR引物序列

Tab 1 Primer sequences for real-time PCR

Gene	Forward primer (5´→3´)	Reverse primer (5´→3´)
GAPDH	GAAGGTGAAGGTCGGTC	GAAGATGGTGATGGGATTTC
N-cadherin	CAGGGACCAGTTGAAGCACT	TGCCGTGGCCTTAAAGTTAT
E-cadherin	CGAAGATGTAAACGAAGCC	GCCATTTCCAGTGACAATC

图1 牙龈素提取物对HN6细胞增殖的影响Note： A/B. The effects of gingipain extract on HN6 cell proliferation after 24 h (A) and 48 h (B). ①P=0.025, ②P=0.000, ③P=0.049, ④P=0.024, ⑤P=0.006, compared with the control group.

Fig 1 Effects of gingipain extract on HN6 cell proliferation

图2 牙龈素提取物对HN6细胞周期的影响Note： A. The effects of gingipain extract on HN6 cell cycle detected by flow cytometry. B. The effects of gingipain extract on S+G2 of HN6 cells. ①P=0.024, ②P=0.001, compared with the control group.

Fig 2 Effects of gingipain extract on HN6 cell cycle

图3 牙龈素提取物对HN6细胞迁移作用的影响Note： A. Effects of gingipain extract on HN6 cell migration. B. The percentage of scratch closure. ①P=0.001, compared with the control group.

Fig 3 Effects of gingipain extract on HN6 cell migration

图4 牙龈素对HN6细胞侵袭的影响（×100）

Fig 4 Effects of gingipain extract on HN6 cell invasion (×100)

图5 牙龈素提取物对HN6细胞侵袭相关基因表达的影响Note：①P=0.000, ②P=0.005, ③P=0.001, compared with the control group.

Fig 5 Effects of gingipain extract on invasion-associated genes of HN6 cells

图6 牙龈素对HN6细胞侵袭相关蛋白表达的影响Note： A/B. Western blotting (A) and grayscale analysis (B) of invasion-related protein expression in HN6 cells

Fig 6 Effects of gingipain extract on invasion-associated proteins of HN6 cells

参考文献 30

1	KOMLÓS G, CSURGAY K, HORVÁTH F, et al. Periodontitis as a risk for oral cancer: a case-control study[J]. BMC Oral Health, 2021, 21(1): 640.
2	SANZ M, MARCO DEL CASTILLO A, JEPSEN S, et al. Periodontitis and cardiovascular diseases: consensus report[J]. J Clin Periodontol, 2020, 47(3): 268-288.
3	王兴. 第四次全国口腔健康流行病学调查报告[M]. 北京: 人民卫生出版社, 2018.
	WANG X. Report on the Fourth National Epidemiological Survey of Oral Health[M]. Beijing: People's Health Publishing House, 2018.
4	KAVARTHAPU A, GURUMOORTHY K. Linking chronic periodontitis and oral cancer: a review[J]. Oral Oncol, 2021, 121: 105375.
5	ALLON I, ABBA M, KAPLAN I, et al. Oral variant of acantholytic squamous cell carcinoma: histochemical and immunohistochemical features[J]. Acta Histochem, 2019, 121(8): 151443.
6	MORRISON A G, SARKAR S, UMAR S, et al. The contribution of the human oral microbiome to oral disease: a review[J]. Microorganisms, 2023, 11(2): 318.
7	LAMONT R J, FITZSIMONDS Z R, WANG H Z, et al. Role of Porphyromonas gingivalis in oral and orodigestive squamous cell carcinoma[J]. Periodontol 2000, 2022, 89(1): 154-165.
8	GENG F X, LIU J C, GUO Y, et al. Persistent exposure to Porphyromonas gingivalis promotes proliferative and invasion capabilities, and tumorigenic properties of human immortalized oral epithelial cells[J]. Front Cell Infect Microbiol, 2017, 7: 57.
9	KYLMÄ A K, SORSA T, JOUHI L, et al. Prognostic role of Porphyromonas gingivalis gingipain rgp and matrix metalloproteinase 9 in oropharyngeal squamous cell carcinoma[J]. Anticancer Res, 2022, 42(11): 5415-5430.
10	GUO K, LIU Y K, ZHOU H J, et al. Involvement of protein kinase C β-extracellular signal-regulating kinase 1/2/p38 mitogen-activated protein kinase-heat shock protein 27 activation in hepatocellular carcinoma cell motility and invasion[J]. Cancer Sci, 2008, 99(3): 486-496.
11	张桓瑜, 江旖婷, 朱晓晨, 等. 牙龈素提取物对小鼠脑神经炎症的影响[J]. 上海交通大学学报(医学版), 2022, 42(5): 570-577.
	ZHANG H Y, JIANG Y T, ZHU X C, et al. Effects of gingipain extracts on brain neuroinflammation in mice[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(5): 570-577.
12	KOLIARAKIS I, MESSARITAKIS I, NIKOLOUZAKIS T K, et al. Oral bacteria and intestinal dysbiosis in colorectal cancer[J]. Int J Mol Sci, 2019, 20(17): 4146.
13	MALINOWSKI B, WĘSIERSKA A, ZALEWSKA K, et al. The role of Tannerella forsythia and Porphyromonas gingivalis in pathogenesis of esophageal cancer[J]. Infect Agent Cancer, 2019, 14: 3.
14	TAN Q, MA X, YANG B, et al. Periodontitis pathogen Porphyromonas gingivalis promotes pancreatic tumorigenesis via neutrophil elastase from tumor-associated neutrophils[J]. Gut Microbes, 2022, 14(1): 2073785.
15	GAO S G, LI S G, MA Z K, et al. Presence of Porphyromonas gingivalis in esophagus and its association with the clinicopathological characteristics and survival in patients with esophageal cancer[J]. Infect Agent Cancer, 2016, 11: 3.
16	LIU Y W, YUAN X, CHEN K S, et al. Clinical significance and prognostic value of Porphyromonas gingivalis infection in lung cancer[J]. Transl Oncol, 2021, 14(1): 100972.
17	CHANG C R, WANG H Y, LIU J C, et al. Porphyromonas gingivalis infection promoted the proliferation of oral squamous cell carcinoma cells through the miR-21/PDCD4/AP-1 negative signaling pathway[J]. ACS Infect Dis, 2019, 5(8): 1336-1347.
18	WOO B H, KIM D J, CHOI J I, et al. Oral cancer cells sustainedly infected with Porphyromonas gingivalis exhibit resistance to Taxol and have higher metastatic potential[J]. Oncotarget, 2017, 8(29): 46981-46992.
19	LIU S Y, ZHOU X D, PENG X, et al. Porphyromonas gingivalis promotes immunoevasion of oral cancer by protecting cancer from macrophage attack[J]. J Immunol, 2020, 205(1): 282-289.
20	INABA H, SUGITA H, KUBONIWA M, et al. Porphyromonas gingivalis promotes invasion of oral squamous cell carcinoma through induction of proMMP9 and its activation[J]. Cell Microbiol, 2014, 16(1): 131-145.
21	OHSHIMA J, WANG Q, FITZSIMONDS Z R, et al. Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis[J]. Proc Natl Acad Sci USA, 2019, 116(17): 8544-8553.
22	REN J L, HAN X, LOHNER H, et al. P. gingivalis infection upregulates PD-L1 expression on dendritic cells, suppresses CD8⁺ T-cell responses, and aggravates oral cancer[J]. Cancer Immunol Res, 2023, 11(3): 290-305.
23	WHEELOCK M J, JOHNSON K R. Cadherins as modulators of cellular phenotype[J]. Annu Rev Cell Dev Biol, 2003, 19: 207-235.
24	CHRISTOFORI G. Changing neighbours, changing behaviour: cell adhesion molecule-mediated signalling during tumour progression[J]. EMBO J, 2003, 22(10): 2318-2323.
25	LAMOUILLE S, XU J, DERYNCK R. Molecular mechanisms of epithelial-mesenchymal transition[J]. Nat Rev Mol Cell Biol, 2014, 15(3): 178-196.
26	XU Z L, CHEN Z M, PENG M S, et al. MicroRNA miR-490-5p suppresses pancreatic cancer through regulating epithelial-mesenchymal transition via targeting MAGI2 antisense RNA 3[J]. Bioengineered, 2022, 13(2): 2673-2685.
27	PIERCEALL W E, WOODARD A S, MORROW J S, et al. Frequent alterations in E-cadherin and α- and β-catenin expression in human breast cancer cell lines[J]. Oncogene, 1995, 11(7): 1319-1326.
28	DERYCKE L D M, BRACKE M E. N-cadherin in the spotlight of cell-cell adhesion, differentiation, embryogenesis, invasion and signalling[J]. Int J Dev Biol, 2004, 48(5/6): 463-476.
29	GRABOWSKA M M, DAY M L. Soluble E-cadherin: more than a symptom of disease[J]. Front Biosci (Landmark Ed), 2012, 17(5): 1948-1964.
30	KATZ J, YANG Q B, ZHANG P, et al. Hydrolysis of epithelial junctional proteins by Porphyromonas gingivalis gingipains[J]. Infect Immun, 2002, 70(5): 2512-2518.

[1]	陈瑾, 傅瑶. 人角膜内皮细胞自体再生的研究进展[J]. 上海交通大学学报（医学版）, 2023, 43(6): 775-780.
[2]	谢欣宜, 周薇, 邱澈, 沈慧, 宋忠臣. 伴阿尔茨海默病牙周炎患者血清Th17/Treg相关细胞因子水平变化研究[J]. 上海交通大学学报（医学版）, 2023, 43(5): 600-605.
[3]	杨万里, 宋娟, 李兵, 劳一敏. CBX8抑制前列腺癌细胞侵袭的机制研究[J]. 上海交通大学学报（医学版）, 2023, 43(12): 1507-1519.
[4]	李露, 李雨霖, 柳燕, 段胜仲. 牙周炎加重主动脉弓缩窄诱导的小鼠心肌肥厚[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1275-1287.
[5]	杨琳, 王晶波, 黄小娟, 任继亮, 袁瑛, 陶晓峰. 荧光探针cMBP⁃ICG检测口腔鳞状细胞癌颈部淋巴结转移及包膜外侵犯的探索性研究[J]. 上海交通大学学报（医学版）, 2022, 42(9): 1296-1302.
[6]	丁贺, 曹杨琳, 何杨, 魏星, 杨剑峰. SMAD7在多发性骨髓瘤中的表达及对细胞增殖和耐药的影响[J]. 上海交通大学学报（医学版）, 2022, 42(7): 858-865.
[7]	胡哲轩, 张欣, 沃璐璐, 李静池, 王娇, 周佽想, 赵倩. TRMT61A在肝癌细胞中的功能及其机制研究[J]. 上海交通大学学报（医学版）, 2022, 42(6): 742-750.
[8]	张桓瑜, 江旖婷, 朱晓晨, 何智妍, 周薇, 宋忠臣. 牙龈素提取物对小鼠脑神经炎症的影响[J]. 上海交通大学学报（医学版）, 2022, 42(5): 570-577.
[9]	徐斐翔, 汪升, 薛明明, 童朝阳, 陈玉梅. 长链非编码RNA-B230352I09表达改变对H9C2心肌细胞增殖及周期的影响[J]. 上海交通大学学报（医学版）, 2022, 42(5): 578-582.
[10]	刘子杨, 王小文, 陈力. lncRNA GK-IT1通过调控醛缩酶A影响非小细胞肺癌细胞的恶性进展[J]. 上海交通大学学报（医学版）, 2022, 42(5): 591-601.
[11]	陈鸣, 张靖. 血根碱通过上调m⁶A甲基转移酶14对胃癌细胞增殖和侵袭的抑制作用[J]. 上海交通大学学报(医学版), 2022, 42(2): 135-141.
[12]	贺艳琪, 迟锐, 陈梦萍, 陈思, 刘春良, 刘云霞, 孙海鹏. 支链氨基酸分解代谢在肺癌细胞中的功能[J]. 上海交通大学学报(医学版), 2021, 41(7): 858-864.
[13]	鲁昱晟, 杨文艺, 马海龙, 胡镜宙. Ras相关结构域家族成员5对头颈鳞癌细胞迁移和侵袭能力的影响[J]. 上海交通大学学报(医学版), 2021, 41(6): 717-723.
[14]	丁远森, 王枫, 孙家悦, 邵正威, 邹德荣, 陆家瑜. 不同年龄2型糖尿病患者牙周健康流行病学调查[J]. 上海交通大学学报(医学版), 2021, 41(2): 217-222.
[15]	严宇青，沈超琴，陈豪燕，洪洁#，王震华#. lnc-MTBP-5在结直肠癌中的表达及其对细胞侵袭能力的影响[J]. 上海交通大学学报(医学版), 2020, 40(9): 1193-1201.

牙龈素提取物对口腔鳞癌细胞HN6生物学特性的影响

Effects of gingipain extract on the biological characteristics of oral squamous cell carcinoma cell HN6

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 30

相关文章 15

编辑推荐

Metrics