茶黄素调节Snail/Slug信号通路对口腔鳞癌细胞生物学行为的影响

doi:10.11958/20230202

天津医药 ›› 2023, Vol. 51 ›› Issue (11): 1164-1169.doi: 10.11958/20230202

茶黄素调节Snail/Slug信号通路对口腔鳞癌细胞生物学行为的影响

孙绪高¹(), 杨文超², 刘彦杰¹^,^△(), 杨旭³

1.漯河医学高等专科学校口腔系（邮编462000）
2.漯河医学高等专科学校第一附属口腔医院口腔科
3.漯河医学高等专科学校口腔颌面外科学教研室

收稿日期:2023-02-17 修回日期:2023-05-12 出版日期:2023-11-15 发布日期:2023-11-07
通讯作者: ^△E-mail：526774603@qq.com
作者简介:孙绪高（1984），男，讲师，主要从事口腔医学方面研究。E-mail：oetzd62@163.com
基金资助:
河南省高等教育教学改革研究与实践立项项目(2019SJGLX751)

Impacts of theaflavin on biological behavior of oral squamous cell carcinoma cells by regulating Snail/Slug signaling pathway

SUN Xugao¹(), YANG Wenchao², LIU Yanjie¹^,^△(), YANG Xu³

1. Department of Stomatology, Luohe Medical College, Henan 462000, China
2. Department of Stomatology, First Affiliated Hospital of Luohe Medical College
3. Oral and Maxillofacial Surgery Teaching and Research Section of Luohe Medical College

Received:2023-02-17 Revised:2023-05-12 Published:2023-11-15 Online:2023-11-07
Contact: ^△E-mail：526774603@qq.com

孙绪高, 杨文超, 刘彦杰, 杨旭. 茶黄素调节Snail/Slug信号通路对口腔鳞癌细胞生物学行为的影响[J]. 天津医药, 2023, 51(11): 1164-1169.

SUN Xugao, YANG Wenchao, LIU Yanjie, YANG Xu. Impacts of theaflavin on biological behavior of oral squamous cell carcinoma cells by regulating Snail/Slug signaling pathway[J]. Tianjin Medical Journal, 2023, 51(11): 1164-1169.

摘要/Abstract

摘要：

目的研究茶黄素调节Snail/Slug信号通路对口腔鳞癌（OSCC）细胞增殖、凋亡和上皮间质转化的影响。方法采用MTT法检测25、50、100、150、175 mg/L茶黄素处理的人OSCC细胞SCC-25存活率，筛选出茶黄素的合适作用浓度。体外培养SCC-25细胞并构建OSCC移植瘤模型，分为对照组、茶黄素低剂量组、茶黄素高剂量组、茶黄素高剂量+空载组、茶黄素高剂量+Snail过表达组。采用实时荧光定量PCR与免疫印迹检测各组细胞Snail、Slug表达；采用MTT法与流式细胞术分别检测各组细胞增殖、凋亡；采用细胞划痕与Transwell侵袭实验分别检测各组SCC-25细胞迁移、侵袭；采用免疫印迹检测各组SCC-25细胞凋亡及上皮间质转化相关蛋白表达；检测各组裸鼠肿瘤体积、肿瘤质量。结果与对照组相比，茶黄素低剂量组、茶黄素高剂量组细胞存活率、迁移率、侵袭数、Snail mRNA及蛋白表达、Slug mRNA及蛋白表达、Bcl-2、N-cadherin蛋白表达、裸鼠肿瘤质量及体积均降低（P<0.05），细胞凋亡率、Bax、ZO-1及E-cadherin蛋白表达均升高（P<0.05），高剂量茶黄素作用更强。与茶黄素高剂量组相比，茶黄素高剂量+空载组各指标差异无统计学意义（P>0.05）；过表达Snail可逆转茶黄素对细胞及裸鼠各指标的影响。结论茶黄素可下调Snail/Slug通路蛋白表达，从而抑制OSCC细胞的上皮间质转化、增殖、迁移及侵袭，促使其凋亡，并可延缓裸鼠移植瘤的生长。

关键词: 茶黄素, 口腔肿瘤, 癌, 鳞状细胞, Snail家族转录因子类, 细胞增殖, 细胞凋亡, 上皮-间质转化

Abstract:

Objective To study the impact of theaflavin on the proliferation, apoptosis and epithelial mesenchymal transformation of oral squamous cell carcinoma (OSCC) cells by regulating Snail/Slug signaling pathway. Methods MTT method was used to detect the survival rate of human OSCC cells SCC-25 treated with 25, 50, 100, 150 and 175 mg/L theaflavin, and the appropriate concentration of theaflavin was selected. SCC-25 cells were cultured in vitro and OSCC transplanted tumor model was constructed. Cells were randomly divide into the control group, the low dose theaflavin group, the high dose theaflavin group, the high dose theaxanthin+empty group and the high dose theaflavin+Snail overexpression group. The expression levels of Snail and Slug in cells of each group were detected by real-time fluorescent quantitative PCR and Western blot assay. MTT assay and flow cytometry were applied to detect cell proliferation and apoptosis. The migration and invasion of SCC-25 cells in each group were detected by cell scratch and Transwell invasion tests. The apoptosis of SCC-25 cells and the expression of epithelial mesenchymal transformation related proteins were detected by Western blot assay. The tumor volume and tumor weight of nude mice in each group were detected. Results Compared with the control group, the cell survival rate, migration rate, invasion number, Snail mRNA and protein expression, Slug mRNA and protein expression, Bcl-2, N-cadherin protein expression, tumor weight and volume of nude mice were decreased in the low dose theaflavin group and the high dose theaxanthin group (P<0.05), and the apoptosis rate, Bax, ZO-1 and E-cadherin protein expression increased (P<0.05). There was no significant difference in each index between the high-dose theaxanthin group and the high-dose theaflavin+no-load group (P>0.05). Overexpression of Snail can reverse the effect of theaflavin on cells and nude mice. Conclusion Theaflavin can down regulate the expression of Snail/Slug pathway, thus inhibit the epithelial mesenchymal transformation, proliferation, migration and invasion of OSCC cells, promote their apoptosis, and delay the growth of transplanted tumors in nude mice.

Key words: theaflavins, mouth neoplasms, carcinoma, squamous cell, Snail family transcription factors, cell proliferation, apoptosis, epithelial-mesenchymal transition

中图分类号:

R739.8

图/表 12

表1 qPCR引物序列

Tab.1 Primer sequences for qPCR

基因名称	引物序列（5′→3′）	产物大小/bp
Snail	上游：TCGGAAGCCTAACTACAGCGA	160
Snail	下游：AGATGAGCATTGGCAGCGAG	160
Slug	上游：TGTGACAAGGAATATGTGAGCC	123
Slug	下游：TGAGCCCTCAGATTTGACCTG	123
β-actin	上游：CTCGCCTTTGCCGATCC	145
β-actin	下游：GGGGTACTTCAGGGTGAGGA	145

图1 不同质量浓度茶黄素对SCC-25细胞存活率的影响 n=6，F=52.040，P<0.01；a与0 mg/L茶黄素比较，P<0.05。

Fig.1 Effects of different concentrations of ttheaflavin on survival rate of SCC-25 cells

图2 免疫印迹检测各组SCC-25细胞Snail、Slug蛋白表达 A：对照组；B：茶黄素低剂量组；C：茶黄素高剂量组；D：茶黄素高剂量+空载组；E：茶黄素高剂量+Snail过表达组。

Fig.2 The expression of Snail and Slug proteins in SCC-25 cells in each group detected by immunoblotting

表2 各组SCC-25细胞Snail、Slug相对表达水平比较（n=6，$\bar{x}±s$）

Tab.2 Comparison of relative expression levels of Snail and Slug in SCC-25 cells between the five groups

组别	mRNA		蛋白
组别	Snail	Slug	Snail	Slug
对照组	1.02±0.14	1.00±0.13	0.87±0.11	0.85±0.09
茶黄素低剂量组	0.57±0.05^a	0.63±0.07^a	0.47±0.08^a	0.46±0.07^a
茶黄素高剂量组	0.31±0.05^ab	0.29±0.04^ab	0.11±0.02^ab	0.09±0.01^ab
茶黄素高剂量+空载组	0.33±0.07	0.30±0.06	0.13±0.03	0.10±0.03
茶黄素高剂量+Snail过表达组	0.98±0.16^c	0.94±0.18^c	0.82±0.10^c	0.80±0.13^c
F	63.958^＊＊	57.763^＊＊	132.685^＊＊	129.660^＊＊

图3 流式细胞术检测各组SCC-25细胞凋亡率

Fig.3 Apoptosis rate of SCC-25 cells in each group detected by flow cytometry

表3 各组SCC-25细胞存活率、凋亡率比较（n=6，%，$\bar{x}±s$）

Tab.3 Comparison of survival rate and apoptosis rate of SCC-25 cells between the five groups

组别	存活率	凋亡率
对照组	100.00±14.26	2.71±0.64
茶黄素低剂量组	61.75±6.20^a	39.26±5.17^a
茶黄素高剂量组	34.02±5.32^ab	64.98±7.28^ab
茶黄素高剂量+空载组	31.46±4.93	65.17±8.13
茶黄素高剂量+Snail过表达组	93.13±15.41^c	5.05±1.28^c
F	57.918^＊＊	191.004^＊＊

表4 各组SCC-25细胞迁移率、侵袭数比较（n=6，$\bar{x}±s$）

Tab.4 Comparison of migration rate and invasion number of SCC-25 cells between the five groups

组别	迁移率/%	侵袭数/（个/视野）
对照组	83.70±12.35	342.13±45.63
茶黄素低剂量组	52.53±6.37^a	219.25±24.18^a
茶黄素高剂量组	24.64±3.96^ab	94.82±9.27^ab
茶黄素高剂量+空载组	26.01±4.49	105.12±11.34
茶黄素高剂量+Snail过表达组	72.98±13.40^c	320.56±50.26^c
F	52.681^＊＊	74.663^＊＊

图4 细胞划痕实验检测各组SCC-25细胞迁移率（×200）

Fig.4 Migration rate of SCC-25 cells in each group detected by cell scratch test (×200)

图5 Transwell侵袭实验检测各组SCC-25细胞侵袭数（结晶紫染色，×200）

Fig.5 Invasion number of SCC-25 cells in each group detected by Transwell invasion test (crystal violet staining, ×200)

表5 各组SCC-25细胞凋亡及上皮间质转化相关蛋白相对表达水平比较（n=6，$\bar{x}±s$）

Tab.5 Comparison of relative expression levels of SCC-25 cell apoptosis and epithelial mesenchymal transformation related proteins between the five groups

组别	Bax	Bcl-2	ZO-1	E-cadherin	N-cadherin
对照组	0.08±0.01	0.89±0.13	0.09±0.02	0.15±0.03	1.19±0.22
茶黄素低剂量组	0.46±0.08^a	0.51±0.09^a	0.50±0.11^a	0.58±0.12^a	0.72±0.08^a
茶黄素高剂量组	0.83±0.13^ab	0.14±0.04^ab	0.95±0.14^ab	1.06±0.20^ab	0.21±0.05^ab
茶黄素高剂量+空载组	0.81±0.15	0.13±0.03	0.96±0.13	1.03±0.23	0.22±0.06
茶黄素高剂量+Snail过表达组	0.11±0.03^c	0.84±0.17^c	0.12±0.01^c	0.18±0.04^c	1.14±0.18^c
F	84.340^＊＊	71.048^＊＊	110.548^＊＊	52.992^＊＊	72.711^＊＊

图6 免疫印迹检测各组SCC-25细胞凋亡及上皮间质转化相关蛋白表达 A：对照组；B：茶黄素低剂量组；C：茶黄素高剂量组；D：茶黄素高剂量+空载组；E：茶黄素高剂量+Snail过表达组。

Fig.6 Immunoblotting results of expression of SCC-25 cell apoptosis and epithelial mesenchymal transformation related proteins in each group

表6 各组SCC-25移植瘤裸鼠肿瘤质量、肿瘤体积比较（n=10，$\bar{x}±s$）

Tab.6 Comparison of tumor mass and tumor volume of SCC-25 transplanted tumor in nude mice between the five groups

组别	肿瘤质量/g	肿瘤体积/mm³
对照组	0.85±0.11	940.32±53.76
茶黄素低剂量组	0.52±0.07^a	620.43±48.07^a
茶黄素高剂量组	0.20±0.05^ab	314.85±28.15^ab
茶黄素高剂量+空载组	0.22±0.04	320.94±30.36
茶黄素高剂量+Snail过表达组	0.78±0.10^c	908.89±45.12^c
F	148.199^＊＊	514.771^＊＊

参考文献 20

[1]	NIU Q, SUN Q, BAI R, et al. Progress of nanomaterials-based photothermal therapy for oral squamous cell carcinoma[J]. Int J Mol Sci, 2022, 23(18):10428. doi:10.3390/ijms231810428.
[2]	FERREIRA E COSTA R, LEÃO M, SANT'ANA M, et al. Oral squamous cell carcinoma frequency in young patients from referral centers around the world[J]. Head Neck Pathol, 2022, 16(3):755-762. doi:10.1007/s12105-022-01441-w.
[3]	O'NEILL E J, TERMINI D, ALBANO A, et al. Anti-cancer properties of theaflavins[J]. Molecules, 2021, 26(4):987-1011. doi:10.3390/molecules26040987.
[4]	XU J, WANG S J, BU S S, et al. Theaflavin promoted apoptosis in nasopharyngeal carcinoma unexpectedly via inducing autophagy in vitro[J]. Iran J Basic Med Sci, 2022, 25(1):68-74. doi:10.22038/IJBMS.2021.59190.13143.
[5]	GHOSH A, LAHIRI A, MUKHERJEE S, et al. Prevention of inorganic arsenic induced squamous cell carcinoma of the skin in Swiss albino mice by black tea through epigenetic modulation[J]. Heliyon, 2022, 8(8):e10341. doi:10.1016/j.heliyon.2022.e10341.
[6]	WANG H, LI Q F, CHOW H Y, et al. Arginine deprivation inhibits pancreatic cancer cell migration,invasion and EMT via the down regulation of Snail,Slug,Twist,and MMP1/9[J]. J Physiol Biochem, 2020, 76(1):73-83. doi:10.1007/s13105-019-00716-1.
[7]	CHEN D D, CHENG J T, CHANDOO A, et al. microRNA-33a prevents epithelial-mesenchymal transition,invasion,and metastasis of gastric cancer cells through the Snail/Slug pathway[J]. Am J Physiol Gastrointest Liver Physiol, 2019, 317(2):G147-G160. doi:10.1152/ajpgi.00284.2018.
[8]	CHO Y A, KIM E K, CHO B C, et al. Twist and Snail/Slug expression in oropharyngeal squamous cell carcinoma in correlation with lymph node metastasis[J]. Anticancer Res, 2019, 39(11):6307-6316. doi:10.21873/anticanres.13841.
[9]	DANTAS R, GUIMARÃES V, DE SOUZA R O, et al. Immunodetection of epithelial-mesenchymal transition and tumor proliferation markers in GLi-1-positive oral squamous cell carcinoma[J]. Appl Immunohistochem Mol Morphol, 2021, 29(5):335-344. doi:10.1097/PAI.0000000000000866.
[10]	BHATTACHARYA R, CHATTERJEE R, MANDAL A, et al. Theaflavin-containing black tea extract:A potential DNA methyltransferase inhibitor in human colon cancer cells and ehrlich ascites carcinoma-induced solid tumors in mice[J]. Nutr Cancer, 2021, 73(11/12):2447-2459. doi:10.1080/01635581.2020.1828943.
[11]	邓力, 蔡婷, 王静雷, 等. 黄芪多糖通过JAK/STAT3通路抑制口腔鳞癌SCC-25裸鼠移植瘤[J]. 中国临床解剖学杂志, 2019, 37(2):169-173.
	DENG L, CAI T, WANG J L, et al. Astragalus polysaccharide inhibits oral squamous cell carcinoma cell line SCC-25 xenograft tumor by suppressing JAK/STAT3 signaling pathway[J]. Chinese Journal of Clinical Anatomy, 2019, 37(2):169-173. doi:10.13418/j.issn.1001-165x.2019.02.011.
[12]	BRENNAN P A, DYLGJERI F, COLETTA R D, et al. Surgical tumour margins and their significance in oral squamous cell carcinoma[J]. J Oral Pathol Med, 2022, 51(4):311-314. doi:10.1111/jop.13276.
[13]	LI H, ZHANG Y, XU M, et al. Current trends of targeted therapy for oral squamous cell carcinoma[J]. J Cancer Res Clin Oncol, 2022, 148(9):2169-2186. doi:10.1007/s00432-022-04028-8.
[14]	JANIAK-KISZKA J, NOWACZEWSKA M, KAŹMIERCZAK W. Oral squamous cell carcinoma - clinical characteristics, treatment, and outcomes in a single institution retrospective cohort study[J]. Otolaryngol Pol, 2022, 76(3):12-17. doi:10.5604/01.3001.0015.7567.
[15]	MAITY R, CHATTERJEE M, BANERJEE A, et al. Gold nanoparticle-assisted enhancement in the anti-cancer properties of theaflavin against human ovarian cancer cells[J]. Mater Sci Eng C Mater Biol Appl, 2019, 104:109909. doi:10.1016/j.msec.2019.109909.
[16]	JUNEJA T, PANDYA M D, SHAH S. Molecular Landscape and computational screening of the natural inhibitors against HPV16 E6 oncoprotein[J]. Asian Pac J Cancer Prev, 2021, 22(8):2461-2469. doi:10.31557/APJCP.2021.22.8.2461.
[17]	SHI S, MA B, SUN F, et al. Theaflavin binds to a druggable pocket of TMEM16A channel and inhibits lung adenocarcinoma cell viability[J]. J Biol Chem, 2021, 297(3):101016. doi:10.1016/j.jbc.2021.101016.
[18]	BUYUK B, JIN S, YE K. Epithelial-to-mesenchymal transition signaling pathways responsible for breast cancer metastasis[J]. Cell Mol Bioeng, 2022, 15(1):1-13. doi:10.1007/s12195-021-00694-9.
[19]	PENG J, WU H J, ZHANG H F, et al. miR-143-3p inhibits proliferation and invasion of hepatocellular carcinoma cells by regulating its target gene FGF1[J]. Clin Transl Oncol, 2021, 23(3):468-480. doi:10.1007/s12094-020-02440-5.
[20]	NOGUCHI S, HIRANO K, TANIMOTO N, et al. SLUG is upregulated and induces epithelial mesenchymal transition in canine oral squamous cell carcinoma[J]. Vet Comp Oncol, 2022, 20(1):134-141. doi:10.1111/vco.12755.

茶黄素调节Snail/Slug信号通路对口腔鳞癌细胞生物学行为的影响

Impacts of theaflavin on biological behavior of oral squamous cell carcinoma cells by regulating Snail/Slug signaling pathway

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