Effect of down-regulation of sex determining region Y-box 9 on epithelial mesenchymal transition and cloning of oral squamous carcinoma cells

doi:10.7518/hxkq.2019.01.003

Abstract

Abstract:

Objective To investigate the effect of sex determining region Y-box 9 (SOX9) on epithelial mesenchymal transition (EMT) and cloning of oral squamous cell carcinoma (OSCC). Methods siRNA control, SOX9 siRNA were trans-fected into BcaCD885 cells in OSCC. Simultaneously, cells that did not undergo transfection were used as the control. Quanti-tative real time polymerase chain reaction (qRT-PCR) and Western blot were used to select SOX9 siRNA1 with enhanced interference effect. A cell cloning assay was used to determine the cell’s clone formation ability. E-cadherin and Vimentin expressions were detected by immunofluorescence. The expressions of E-cadherin, matrix metalloprotease 2 (MMP-2), Vi-mentin and matrix metalloprotease 9 (MMP-9) were detected by Western blot. Cell invasion and migration were detected in the Transwell compartment. Results The levels of SOX9 mRNA and protein in SOX9 siRNA cells were significantly lower than those of the control (P<0.05). An increase in the number of SOX9 siRNA1 cell clonesled to the considerable decrease of the number of cell invasion and migration. In addition, levels of MMP-2 and MMP-9 proteins in cells decreased significantly compared with the control (P<0.05). The level of Vimentin expression in SOX9 siRNA1 cells decreased, and expression level of E-cadherin was elevated. Cell EMT was inhibited com-pared with the control, and the difference was statistically significant (P<0.05). Conclusion Down-regulation of SOX9 inhibited EMT, clonogenic formation, cell invasion and OSCC migration.

Key words: sex determining region Y-box 9, oral squa-mous cell carcinoma, epithelial mesenchymal transition, cell clones

CLC Number:

Q785

Wenli Yang,Minglei Sun,Peng Zhang,Weiwei Yu,Haixia Zhou,Qiang Sun. Effect of down-regulation of sex determining region Y-box 9 on epithelial mesenchymal transition and cloning of oral squamous carcinoma cells[J]. West China Journal of Stomatology, 2019, 37(1): 13-18.

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References 21

[1]	Sahu A, Nandakumar N, Sawant S , et al. Recurrence pre-diction in oral cancers: a serum Raman spectroscopy study[J]. Analyst, 2015,140(7):2294-2301. doi: 10.1039/C4AN01860E URL
[2]	Zhang H, Cai K, Wang J , et al. MiR-7, inhibited indirectly by lincRNA HOTAIR, directly inhibits SETDB1 and reverses the EMT of breast cancer stem cells by downregulating the STAT3 pathway[J]. Stem Cells, 2015,32(11):2858-2868.
[3]	Sun L, Mathews LA, Cabarcas SM , et al. Epigenetic regu-lation of SOX9 by the NF-κB signaling pathway in pancreatic cancer stem cells[J]. Stem Cells, 2013,31(8):1454-1466. doi: 10.1002/stem.1394 URL
[4]	Chen W, Zhao W, Zhang L , et al. MALAT1-miR-101-SOX9 feedback loop modulates the chemo-resistance of lung can-cer cell to DDP via Wnt signaling pathway[J]. Oncotarget, 2017,8(55):94317-94329.
[5]	Wang H, Leav I, Ibaragi S , et al. SOX9 is expressed in human fetal prostate epithelium and enhances prostate cancer inva-sion[J]. Cancer Res, 2008,68(6):1625-1630. doi: 10.1158/0008-5472.CAN-07-5915 URL
[6]	Nantajit D, Lin D, Li JJ . The network of epithelial-mesenc-hymal transition: potential new targets for tumor resistance[J]. J Cancer Res Clin Oncol, 2015,141(10):1697-1713. doi: 10.1007/s00432-014-1840-y URL
[7]	Kim GJ, Sock E, Buchberger A , et al. Copy number varia-tion of two separate regulatory regions upstream of SOX9 causes isolated 46,XY or 46,XX disorder of sex development[J]. J Med Genet, 2015,52(4):240-247. doi: 10.1136/jmedgenet-2014-102864 URL
[8]	Xia S, Feng Z, Qi X , et al. Clinical implication of Sox9 and activated Akt expression in pancreatic ductal adenocarci-noma[J]. Med Oncol, 2015,32(1):358. doi: 10.1007/s12032-014-0358-0 URL
[9]	Liu N, Zhang L, Wang Z , et al. MicroRNA-101 inhibits pro-liferation, migration and invasion of human glioblastoma by targeting SOX9[J]. Oncotarget, 2017,8(12):19244-19254.
[10]	Vetro A, Dehghani MR, Kraoua L , et al. Testis development in the absence of SRY: chromosomal rearrangements at SOX9 and SOX3[J]. Eur J Hum Genet, 2015,23(8):1025-1032. doi: 10.1038/ejhg.2014.237
[11]	Li Z, Li B, Niu L , et al. miR-592 functions as a tumor sup-pressor in human non-small cell lung cancer by targeting SOX9[J]. Oncol Rep, 2017,37(1):297-304. doi: 10.3892/or.2016.5275 URL
[12]	Wu JH, Liang XA, Wu YM , et al. Identification of DNA methylation of SOX9 in cervical cancer using methylated-CpG island recovery array[J]. Oncol Rep, 2013,19(1):125-132.
[13]	吴姗 . SOX9基因沉默对大肠癌细胞系生物学行为的影响[D]. 杭州: 浙江大学, 2011.
	Wu S . Effects of SOX9 gene silencing on biological beha-vior of colorectal cancer cell lines[D]. Hangzhou: Zhejiang University, 2011.
[14]	Res C . Correction: miR145 targets the SOX9/ADAM17 axis to inhibit tumor-initiating cells and IL6-mediated paracrine effects in head and neck cancer[J]. Cancer Res, 2015,75(13):2761. doi: 10.1158/0008-5472.CAN-15-1351 URL
[15]	Attramadal CG, Kumar S, Boysen ME , et al. Tumor budding, EMT and cancer stem cells in T1-2/N0 oral squamous cell carcinomas[J]. Anticancer Res, 2015,35(11):6111-6120.
[16]	Natsuizaka M, Whelan KA, Kagawa S , et al. Interplay be-tween Notch1 and Notch3 promotes EMT and tumor initia-tion in squamous cell carcinoma[J]. Nat Commun, 2017,8(1):1758. doi: 10.1038/s41467-017-01500-9 URL
[17]	Lu MH, Huang CC, Pan MR , et al. Prospero homeobox 1 promotes epithelial-mesenchymal transition in colon cancer cells by inhibiting E-cadherin via miR-9[J]. Clin Cancer Res, 2012,18(23):6416-6425. doi: 10.1158/1078-0432.CCR-12-0832 URL
[18]	Palen K, Weber J, Dwinell MB , et al. E-cadherin re-expression shows in vivo evidence for mesenchymal to epithelial tran-sition in clonal metastatic breast tumor cells[J]. Oncotarget, 2016,7(28):43363-43375.
[19]	Liu H, Liu Z, Jiang B , et al. SOX9 overexpression promotes glioma metastasis via Wnt/β-Catenin signaling[J]. Cell Bio-chem Biophys, 2015,73(1):205-212. doi: 10.1007/s12013-015-0647-z URL
[20]	Wu SH, Hsiao YT, Kuo CL , et al. Bufalin inhibits NCI-H460 human lung cancer cell metastasis in vitro by inhibiting MAPKs, MMPs, and NF-κB pathways[J]. Am J Chin Med, 2015,43(6):1247-1264. doi: 10.1142/S0192415X15500718 URL
[21]	Li W, Li S, Deng L , et al. Decreased MT1-MMP in gastric cancer suppressed cell migration and invasion via regulating MMPs and EMT[J]. Tumor Biol, 2015,36(9):6883-6889. doi: 10.1007/s13277-015-3381-7 URL

组别	SOX9 mRNA	SOX9蛋白
control	1.00	0.84±0.06
siRNA control	1.02±0.09^#	0.86±0.07^#
SOX9 siRNA1	0.21±0.05*	0.15±0.02*
SOX9 siRNA2	0.36±0.02*^&	0.29±0.05*^&

组别	E-cadherin	Vimentin
control	0.25±0.06	0.82±0.09
siRNA control	0.22±0.04^#	0.84±0.10^#
SOX9 siRNA1	0.79±0.05*	0.28±0.06*

组别	MMP-2	MMP-9
control	0.81±0.09	0.48±0.04
siRNA control	0.80±0.11^#	0.49±0.06^#
SOX9 siRNA1	0.35±0.04*	0.30±0.05*