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

doi:10.7518/hxkq.2021.01.011

Abstract

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

CLC Number:

R 739.81

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.

Figures/Tables 8

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References 23

1	Malik UU, Zarina S, Pennington SR. Oral squamous cell carcinoma: key clinical questions, biomarker discovery, and the role of proteomics[J]. Arch Oral Biol, 2016, 63: 53-65.
2	Singhvi HR, Malik A, Chaturvedi P. The role of chronic mucosal trauma in oral cancer: a review of literature[J]. Indian J Med Paediatr Oncol, 2017, 38(1): 44-50.
3	Naruse T, Yanamoto S, Matsushita Y, et al. Cetuximab for the treatment of locally advanced and recurrent/metastatic oral cancer: an investigation of distant metastasis[J]. Mol Clin Oncol, 2016, 5(2): 246-252.
4	Liu S, Liu D, Li J, et al. Regulatory T cells in oral squamous cell carcinoma[J]. J Oral Pathol Med, 2016, 45(9): 635-639.
5	Jo A, Denduluri S, Zhang B, et al. The versatile functions of sox9 in development, stem cells, and human diseases[J]. Genes Dis, 2014, 1(2): 149-161.
6	Francis JC, Capper A, Ning J, et al. Sox9 is a driver of aggressive prostate cancer by promoting invasion, cell fate and cytoskeleton alterations and epithelial to mesenchymal transition[J]. Oncotarget, 2018, 9(7): 7604-7615.
7	Chakravarty G, Moroz K, Makridakis NM, et al. Prognostic significance of cytoplasmic sox9 in invasive ductal carcinoma and metastatic breast cancer[J]. Exp Biol Med (Maywood), 2011, 236(2): 145-155.
8	楚广民, 张建波, 孙淼淼. RNA干扰沉默SOX9对肾细胞癌786-O细胞体外增殖、凋亡及裸鼠成瘤能力的影响[J]. 分子诊断与治疗杂志, 2018, 10(3): 174-195.
	Chu GM, Zhang JB, Sun MM. Effect of silencing SOX9 by RNA interference on proliferation and apoptosis of renal cancer 786-O cells in vitro and tumorigenicity in nude mice[J]. J Mol Diagnosis Ther, 2018, 10(3): 174-195.
9	Kim AL, Back JH, Chaudhary SC, et al. Sox9 transcriptionally regulates mTOR-induced proliferation of basal cell carcinomas[J]. J Invest Dermatol, 2018, 138(8): 1716- 1725.
10	Hong Y, Chen W, Du X, et al. Upregulation of sex-determining region Y-box 9 (sox9) promotes cell proliferation and tumorigenicity in esophageal squamous cell carcinoma[J]. Oncotarget, 2015, 6(31): 31241-31254.
11	Thomson PJ. Perspectives on oral squamous cell carcinoma prevention-proliferation, position, progression and prediction[J]. J Oral Pathol Med, 2018, 47(9): 803-807.
12	Cree IA, Charlton P. Molecular chess? Hallmarks of anti-cancer drug resistance[J]. BMC Cancer, 2017, 17(1): 10.
13	Zou ZT, Hu WT, Cao W, et al. relationship between expression level of sex-determining region y box 9 and metastasis of oral squamous cell carcinoma[J]. Chin J Stomatol, 2018, 53(10): 688-693.
14	Sumita Y, Yamazaki M, Maruyama S, et al. Cytoplasmic expression of sox9 as a poor prognostic factor for oral squamous cell carcinoma[J]. Oncol Rep, 2018, 40(5): 2487-2496.
15	Ren D, Yang C, Liu N, et al. Gene expression profile analysis of u251 glioma cells with shrna-mediated SOX9 knockdown[J]. J BUON, 2018, 23(4): 1136-1148.
16	Yan J, Huang W, Huang X, et al. A negative feedback loop between long noncoding RNA NBAT1 and Sox9 inhibits the malignant progression of gastric cancer cells[J]. 2018, 38(6): BSR20180882.
17	冯蓓, 王媛, 刘宁宁. HPV-16E6/E7通过调控上皮间质转化对宫颈癌模型小鼠存活、肿瘤生长及转移的影响[J]. 医学分子生物学杂志, 2019, 16(1): 46-51.
	Feng B, Wang Y, Liu NN. Effects of HPV-16E6/E7 on survival, tumor growth and metastasis of cervical cancer model mice by regulating epithelial mesenchymal transformation[J]. J Med Mol Biol, 2019, 16(1): 46-51.
18	Huang J, Guo L. Knockdown of sox9 inhibits the proliferation, invasion, and emt in thyroid cancer cells[J]. Oncol Res, 2017, 25(2): 167-176.
19	Huang JQ, Wei FK, Xu XL, et al. SOX9 drives the epithelial-mesenchymal transition in non-small-cell lung cancer through the wnt/beta-catenin pathway[J]. J Transl Med, 2019, 17(1): 143.
20	Duchartre Y, Kim YM, Kahn M. The wnt signaling pathway in cancer[J]. Crit Rev Oncol Hematol, 2016, 99: 141-149.
21	Park HW, Kim YC, Yu B, et al. Alternative wnt signaling activates YAP/TAZ[J]. Cell, 2015, 162(4): 780-794.
22	Nusse R, Clevers H. Wnt/beta-catenin signaling, disease, and emerging therapeutic modalities[J]. Cell, 2017, 169(6): 985-999.
23	Lyu Q, Jin L, Yang X, et al. Lncrna mincr activates wnt/beta-catenin signals to promote cell proliferation and migration in oral squamous cell carcinoma[J]. Pathol Res Pract, 2019, 215(5): 924-930.

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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