启动子甲基化状况对唾液腺恶性多形性腺瘤体外细胞系细胞E-cadherin表达的影响

摘要/Abstract

摘要： 目的通过体外改变人唾液腺恶性多形性腺瘤（malignant pleomorphic adenoma,MPA）细胞系细胞E-cadherin基因启动子甲基化情况,探讨启动子甲基化对E-cadherin表达的影响。方法采用合适浓度的5-Aza-CdR和TGF-β1处理MPA细胞系SM-AP4和SM-AP1细胞,分别使用蛋白免疫印迹法、亚硫酸盐测序法、实时荧光定量PCR检测用药前、后E-cadherin蛋白表达、E-cadherin基因启动子甲基化状况、mRNA表达;同时采用划痕实验、Transwell小室检测用药前、后细胞迁移能力的变化。应用 SPSS13.0 软件包对数据进行独立样本t检验。结果经5-Aza-CdR处理后,SM-AP4细胞E-cadherin启动子区甲基化率显著降低（P<0.01）,E-cadherin蛋白和mRNA表达显著升高（P<0.05）,相应的细胞迁移能力显著降低（P<0.05）。而经TGF-β1处理后的SM-AP1细胞表达Vimentin蛋白,E-cadherin启动子区甲基化率显著升高（P<0.01）,E-cadherin蛋白和mRNA表达显著降低（P<0.05）,相应的细胞迁移能力显著提高（P<0.05）。结论 DNA甲基化是调节MPA体外细胞系细胞中E-cadherin表达的重要机制之一,E-cadherin表达升高对MPA细胞转移具有潜在的抑制作用。

关键词: E-cadherin, 5-Aza-CdR, TGF-β, 1, DNA甲基化, 恶性多形性腺瘤, 唾液腺

Abstract: PURPOSE: To investigate the effect of promoter methylation on the expression of E-cadherin by changing the E-cadherin gene promoter methylation level in human salivary malignant pleomorphic adenoma cell lines in vitro. METHODS: After appropriate concentrations of 5-Aza-CdR and TGF-β1 treatment, E-cadherin promoter methylation, protein and mRNA expression were detected by bisulfite sequencing, Western blotting, fluorescence quantitative real-time PCR respectively. Wound-healing test and Transwell test were used to identify the corresponding changes of SM-AP4 and SM-AP1 cells when E-cadherin expression was altered. SPSS 13.0 software package was used for statistical analysis. RESULTS: After treatment with 5-Aza-CdR, E-cadherin promoter methylation rate was decreased significantly in SM-AP4 cell (P<0.001). E-cadherin protein and mRNA expression was significantly elevated (P<0.05) and the corresponding ability of cell migration significantly decreased (P<0.05). After TGF-β1 treatment in SM-AP1, vimentin expression was induced. E-cadherin promoter methylation rate significantly increased (P<0.001) and E-cadherin protein and mRNA expression significantly decreased (P<0.05). The corresponding migration ability of SM-AP1 cell significantly increased (P<0.05). CONCLUSIONS: DNA methylation is an important mechanism to regulate the expression of E-cadherin in MPA, which can improve the level of E-cadherin protein expression and decrease the ability of tumor metastasis by reducing the methylation level of E-cadherin gene promoter.

Key words: E-cadherin, 5-Aza-CdR, TGF-β1, DNA methylation, Malignant pleomorphic adenoma, Salivary gland

中图分类号:

R739.8

夏亮, 张春叶, 胡宇华, 钱佳骏, 李江, 田臻. 启动子甲基化状况对唾液腺恶性多形性腺瘤体外细胞系细胞E-cadherin表达的影响[J]. 中国口腔颌面外科杂志, 2016, 14(4): 308-314.

XIA Liang, ZHANG Chun-ye, HU Yu-hua, QIAN Jia-jun, LI Jiang, TIAN Zhen. Promoter methylation level affects the expression of E-cadherin in salivary malignant pleomorphic adenoma cell lines[J]. China J Oral Maxillofac Surg, 2016, 14(4): 308-314.

参考文献

[1] Tian Z, Li L, Wang L, et al. Salivary gland neoplasms in oral and maxillofacial regions: a 23-year retrospective study of 6982 cases in an eastern Chinese population[J]. Int J Oral Maxillofac Surg,2010,39(3): 235-242.
[2] Guzzo M, Locati LD, Prott FJ, et al. Major and minor salivary gland tumors [J]. Crit Rev Oncol Hemat, 2010, 74(2): 134-148.
[3] Canel M, Serrels A, Frame MC, et al. E-cadherin-integrin crosstalk in cancer invasion and metastasis[J]. J Cell Sci, 2013, 126(Pt 2): 393-401.
[4] Berx G, Van Roy F. The E-cadherin/catenin complex: an important gatekeeper in breast cancer tumorigenesis and malignant progression [J]. Breast Cancer Res, 2001, 3(5): 289-293.
[5] Qu Y, Dang S, Hou P. Gene methylation in gastric cancer [J]. Clin Chim Acta, 2013, 424: 53-65.
[6] Li YX, Lu Y, Li CY, et al. Role of CDH1 promoter methylation in colorectal carcinogenesis: a meta-analysis [J]. DNA Cell Biol, 2014, 33(7): 455-462.
[7] Liu S, Ye D, Guo W, et al. G9a is essential for EMT-mediated metastasis and maintenance of cancer stem cell-like characters in head and neck squamous cell carcinoma[J]. Oncotarget, 2015, 6(9): 6887-6901.
[8] Zhang CY, Mao L, Li L, et al. Promoter methylation as a common mechanism for inactivating E-cadherin in human salivary gland adenoid cystic carcinoma [J]. Cancer, 2007, 110(1): 87-95.
[9] Diniz-Freitas M, Garcia-Caballero T, Antúnez-López J, et al. Reduced E-cadherin expression is an indicator of unfavourable prognosis in oral squamous cell carcinoma [J]. Oral Oncol, 2006, 42(2): 190-200.
[10] Hansford S, Kaurah P, Li-Chang H, et al. Hereditary diffuse gastric cancer syndrome: CDH1 mutations and beyond [J]. JAMA Oncol, 2015, 1(1): 23-32.
[11] Kim JH, Choi KY, Lee DJ, et al. Loss of heterozygosities in five tumor suppressor genes (FHIT Gene, p16, pRb, E-Cadherin and p53) in thyroid tumors [J]. Clin Exp Otorhinolaryngol, 2014, 7(1): 53-58.
[12] 邓亚伟, 夏荣辉, 张春叶, 等. 唾液腺恶性多形性腺瘤细胞系中E-cadherin的表达差异及表观遗传调控机制[J]. 中国口腔颌面外科杂志, 2015, 13(4): 297-302.
[13] 韩一凡, 李江, 王旭, 等. 5-氮杂-2'-脱氧胞苷对涎腺腺样囊性癌细胞系细胞MGMT和hMLH1基因表达的影响[J]. 中华口腔医学杂志, 2012, 47(z1): 222-226.
[14] Dong C, Wu Y, Yao J, et al. G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer [J]. J Clin Invest, 2012, 122(4): 1469-1486.
[15] Koutsimpelas D, Pongsapich W, Heinrich U, et al. Promoter methylation of MGMT, MLH1 and RASSF1A tumor suppressor genes in head and neck squamous cell carcinoma: pharmacological genome demethylation reduces proliferation of head and neck squamous carcinoma cells [J]. Oncol Rep, 2012, 27(4): 1135-1141.
[16] Wozniak RJ, Klimecki WT, Lau SS, et al. 5-Aza-2'-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation [J]. Oncogene, 2007, 26(1): 77-90.
[17] Lombaerts M, van Wezel T, Philippo K, et al. E-cadherin transcriptional downregulation by promoter methylation but not mutation is related to epithelial-to-mesenchymal transition in breast cancer cell lines [J]. Br J Cancer, 2006, 94(5): 661-671.
[18] Sleeman JP, Thiery JP. SnapShot: The epithelial-mesenchymal transition [J]. Cell, 2011, 145(1): 162.e1.