性别决定区Y框蛋白9诱导人口腔鳞状细胞癌CAL27微管形成和上皮间质转化的机制初探

doi:10.7518/hxkq.2021.01.011

华西口腔医学杂志 ›› 2021, Vol. 39 ›› Issue (1): 74-80.doi: 10.7518/hxkq.2021.01.011

性别决定区Y框蛋白9诱导人口腔鳞状细胞癌CAL27微管形成和上皮间质转化的机制初探

黄盛¹(), 张七援², 何爱娥³, 李洪波⁴, 张智星⁵

^1.武汉理工大学马房山校区东院医院口腔科，武汉 430000
^2.武汉大学口腔医院牙体牙髓二科，武汉 430000
^3.武汉市第五医院口腔科，武汉 430000
^4.武汉大学口腔医院修复科，武汉 430000
^5.同济医科大学附属医院口腔科，武汉 430000

收稿日期:2020-01-16 修回日期:2020-09-17 出版日期:2021-02-01 发布日期:2021-03-02
通讯作者: 黄盛 E-mail:yao200708@163.com
作者简介:黄盛，主治医师，硕士，E-mail：yao200708@163.com
基金资助:
湖北省自然科学基金(ZRMS2017000033)

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells

Huang Sheng¹(), Zhang Qiyuan², He Aie³, Li Hongbo⁴, Zhang Zhixing⁵

^1.Dept. of Stomatology, East Hospital, Mafangshan Campus, Wuhan University of Science and Technology, Wuhan 430000, China
^2.Dept. of Cariology and Endodontics, School of Stomatology, Wuhan University, Wuhan 430000, China
^3.Dept. of Stomatology, The Fifth Hospital of Wuhan, Wuhan 430000, China
^4.Dept. of Prosthodontics, School of Stomatology, Wuhan University, Wuhan 430000, China
^5.Dept. of Stomatology, Affiliated Hospital of Tongji Medical University, Wuhan 430000, China

Received:2020-01-16 Revised:2020-09-17 Online:2021-02-01 Published:2021-03-02
Contact: Huang Sheng E-mail:yao200708@163.com
Supported by:
Natural Science Foundation of Hubei Province(ZRMS2017000033)

摘要/Abstract

摘要： 目的

探究性别决定区Y框蛋白9（SOX9）对人口腔鳞状细胞癌（OSCC）CAL27微管形成和上皮间质转化的影响及其作用机制。

方法

设计合成SOX9-shRNA1和SOX9-shRNA2，将SOX9-shRNA1和SOX9-shRNA2转染到CAL27细胞中，实时荧光定量聚合酶链反应检测SOX9的表达水平；微管形成实验检测微管结节数目的变化；免疫荧光检测波形蛋白（Vimentin）的含量；免疫印迹法检测钙黏蛋白（E-cadherin）、神经钙黏素（N-cadherin）、纤连蛋白（Fibronectin）、Wnt、β-连环蛋白（β-catenin）和T细胞4（TCF-4）蛋白的相对表达量。

结果

SOX9-shRNA1和SOX9-shRNA2转染CAL27细胞后，SOX9的表达水平显著降低（F=578.000，P=0.000；F=96.850，P=0.000）。干扰SOX9后可抑制OSCC细胞上皮间质转化。干扰SOX9后微管结节数目和Vimentin阳性细胞数显著减少（F=169.700，P=0.000）；E-cadherin蛋白表达水平显著升高（F=181.400，P=0.000）；N-cadherin、Fibronectin、Wnt、β-catenin、TCF-4蛋白表达水平显著降低（N-cadherin：F=101.400，P=0.000；Fibronectin：F=122.300，P=0.000； Wnt：F=70.290，P=0.000；β-catenin：F=81.740，P=0.000；TCF-4：F=37.020，P=0.000）。

结论

干扰SOX9降低Vimentin的含量，抑制微管形成，影响上皮间质转化标记分子的蛋白表达以及抑制Wnt/β-catenin通路的激活，结果表明SOX9可诱导人OSCC细胞CAL27微管形成和上皮间质转化，这一作用与Wnt/β-catenin通路激活抑制存在关联。

关键词: 性别决定区Y框蛋白9, Wnt/β-catenin通路, 口腔鳞状细胞癌, 微管形成, 上皮间质转化

Abstract: Objective

This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.

Methods

SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).

Results

The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (F=578.000, P=0.000; F=96.850, P=0.000). Interference with SOX9 inhibited the EMT of OSCC. After interference with SOX9, the number of tubules and Vimentin positive cells decreased significantly (F=169.700, P=0.000). The expression level of E-cadherin significantly increased (F=181.400, P=0.000). The expression levels of N-cadherin, Fibronectin, Wnt, β-catenin, and TCF-4 proteins significantly decreased (N-cadherin: F=101.400, P=0.000; Fibronectin: F=122.300, P=0.000; Wnt: F=70.290, P=0.000; β-catenin: F=81.740, P=0.000; TCF-4: F=37.020, P=0.000).

Conclusion

Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.

Key words: sex determining region Y-box 9, Wnt/β-catenin pathway, oral squamous cell carcinoma, microtubule formation, epithelial-mesenchymal transition

中图分类号:

R 739.81

黄盛, 张七援, 何爱娥, 李洪波, 张智星. 性别决定区Y框蛋白9诱导人口腔鳞状细胞癌CAL27微管形成和上皮间质转化的机制初探[J]. 华西口腔医学杂志, 2021, 39(1): 74-80.

Huang Sheng, Zhang Qiyuan, He Aie, Li Hongbo, Zhang Zhixing. Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells[J]. West China Journal of Stomatology, 2021, 39(1): 74-80.

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组别	E-cadherin	N-cadherin	Fibronectin
对照组	2.34±0.15	41.23±5.42	32.76±4.22
Scramble组	3.13±0.23	44.46±8.41	34.39±5.74
SOX9-shRNA1组	19.16±2.04*	6.78±1.03*	4.06±0.98*
SOX9-shRNA2组	35.33±4.76*	1.01±0.25*	1.52±0.43*

组别	Wnt	β-catenin	TCF-4
对照组	0.78±0.15	0.93±0.18	0.33±0.08
Scramble组	0.81±0.14	1.02±0.17	0.42±0.11
SOX9-shRNA1组	0.21±0.03*	0.15±0.03*	0.10±0.02*
SOX9-shRNA2组	0.06±0.01*	0.04±0.01*	0.03±0.01*

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性别决定区Y框蛋白9诱导人口腔鳞状细胞癌CAL27微管形成和上皮间质转化的机制初探

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells

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