切换至 "中华医学电子期刊资源库"
高级检索
中华口腔医学研究杂志(电子版)

中华口腔医学研究杂志(电子版) ›› 2016, Vol. 10 ›› Issue (03) : 155 -160. doi: 10.3877/cma.j.issn.1674-1366.2016.03.001

所属专题： 文献；

基础研究

5-Aza对CDH1启动子甲基化及舌鳞状细胞癌细胞迁移能力的影响
蔡伟鑫1, 王成1, 马辉彬1, 侯劲松1, 刘习强1, 陈小华1, 黄洪章1,†()   
  1. 1. 510055　广州，中山大学光华口腔医学院·附属口腔医院，广东省口腔医学重点实验室
  • 收稿日期:2016-02-14 出版日期:2016-06-01
  • 通信作者: 黄洪章
  • 基金资助:
    国家自然科学基金(81272949、81202136、81572661); 高等学校博士学科点专项科研基金(20120171120068); 中央高校基本科研业务费专项资金(11ykpy47); 广州市珠江科技新星专项(2014J2200045)

5-Aza inhibits cell migration and methylation of CDH1 gene in tongue squamous cell carcinomacell lines

Weixin Cai1, Cheng Wang1, Huibin Ma1, Jinsong Hou1, Xiqiang Liu1, Xiaohua Chen1, Hongzhang Huang1,†()   

  1. 1. Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangdong Provincial Key Laboratory of Stomatology, Guangzhou 510055, China
  • Received:2016-02-14 Published:2016-06-01
  • Corresponding author: Hongzhang Huang
  • About author:
    Corresponding author: Huang Hongzhang, Email:
全文 ( ) 下载PDF ( ) 导出引用
引用本文：

蔡伟鑫, 王成, 马辉彬, 侯劲松, 刘习强, 陈小华, 黄洪章. 5-Aza对CDH1启动子甲基化及舌鳞状细胞癌细胞迁移能力的影响[J/OL]. 中华口腔医学研究杂志(电子版), 2016, 10(03): 155-160.

Weixin Cai, Cheng Wang, Huibin Ma, Jinsong Hou, Xiqiang Liu, Xiaohua Chen, Hongzhang Huang. 5-Aza inhibits cell migration and methylation of CDH1 gene in tongue squamous cell carcinomacell lines[J/OL]. Chinese Journal of Stomatological Research(Electronic Edition), 2016, 10(03): 155-160.

目的

探讨甲基化抑制剂5-氮杂胞苷（5-Aza）对CDH1启动子甲基化状态及舌鳞状细胞癌（TSCC）细胞侵袭迁移的影响。

方法

5-Aza处理TSCC细胞株UM1和UM2，倒置相差显微镜下观察处理前后细胞形态及排列情况；划痕试验和Transwell试验检测处理前后细胞迁移和侵袭能力；Western blot法和免疫荧光检测E-cadherin表达；甲基化特异PCR（MSP）检测处理前后UM1、UM2细胞E-cadherin编码基因CDH1的甲基化状态。细胞相对侵袭率的比较采用χ2检验，P<0.05为差异有统计学意义。

结果

UM1细胞呈小圆形、多边形或梭形，细胞排列松散，未见明显的细胞间的紧密连接；UM2细胞则表现为多边形，细胞与细胞之间紧密接触，呈典型的"铺路石"样排列。UM1细胞E-cadherin表达较UM2低，细胞划痕经过48 h后完全铺满，而UM2细胞划痕48 h后不足75%。加入去甲基化药物5-Aza后，UM1细胞形态稍不规则，以多边、多角形细胞为主，且出现类似UM2细胞的铺路石样排列的细胞团，可见细胞间的紧密连接，细胞划痕经过48 h后仍不足70%，细胞相对侵袭率为加药前的102% (χ2= 0.651，P>0.05)，E-cadherin表达上调。MSP结果显示，UM1细胞的E-cadherin编码基因CDH1的呈现过甲基化，去甲基化药物5-Aza作用后其甲基化程度降低。

结论

5-Aza可诱导E-cadherin编码基因CDH1去甲基化，导致E-cadherin上调抑制其迁移能力。

关键词: 舌, 癌，鳞状细胞, 迁移, 甲基化, E钙粘着糖蛋白, 上皮细胞-间质细胞转换
Objective

To evaluate the effect of 5-Aza on methylation of CDH1 and the migration and mobility of tongue squamous cell carcinoma (TSCC) cell lines.

Methods

Two TSCC cell lines UM1, UM2 were treated with or without 5-Aza. Cell morphology and growth patterns were observed under the light microscope. Wound healing and transwell assay were performed to determine the ability of cell migration. Western blot and immunofluorescence were used to detect the expression of E-cadherin. Methylation specific polymerase chain reaction (MSP) was used to determine the methylated profile of CDH1. Chi-square test was performed to compare relative invasion rate and the significance level was set at P<0.05.

Results

UM1 cells appeared to be dispersed, spindle-shaped with migratory protrusions, whereas, UM2 cells presented with cobblestone-like pattern and an apical-basal polarity. A higher expression of E-cadherin with lower mobility was demonstrated in UM2 than in UM1. The wound area was completely covered in UM1 after 48 h, while less than 75% in UM2. UM1 displayed a higher methylation profile in the MSP detection. After treated with 5-Aza in UM1 cells, the cell morphology became irregular and UM2-like with firm cell connection. The 48 h wound cover area of 5-Aza treated UM1 was less than 70%, the relative invasion rate was 102% (χ2 = 0.651, P>0.05) compared to the untreated cells and E-cadherin expression was increased, while a demethylation of CDH1 gene was observed.

Conclusion

Demethylation of CDH1 was involved in 5-Aza mediated inhibition of cell migration and motility in TSCC cells.

Key words: Tongue, Carcinoma, squamous cell, Migration, Methylation, E-cadherin, Epithelial-mesenchymal transition
表1 CDH1甲基化和非甲基化引物序列
基因 引物序列 扩增片段长度 (bp)
CDH1?M F:5′?TTAGGTTAGAGGGTTATCGCGT?3′ 97
? R:5′?TAACTAAAAATTCACCTACCGAC?3′ ?
CDH1?U F:5′?TAATTTTAGGTTAGAGGGTTATTGT?3′ 237
? R:5′?CACAACCAATCAACAACACA?3′ ?
图1 加药前后UM1、UM2细胞形态学观察（× 200）
图2 细胞迁移和侵袭能力检测（× 20）
图3 E-cadherin表达情况分析
图4 MSP分析CDH1甲基化状态
[1]
Hugo H, Ackland ML, Blick T，et al. Epithelial-mesenchymal and mesenchymal-epithelial transitions in carcinoma progression[J]. J Cell Physiol，2007，213(2):374-383.
[2]
Yang J, Weinberg RA. Epithelial-mesenchymal transition：at the crossroads of development and tumor metastasis[J]. Dev Cell，2008，14(6):818-829.
[3]
Rodriguez FJ, Lewis-Tuffin LJ, Anastasiadis PZ. E-cadherin′s dark side：possible role in tumor progression[J]. Biochim Biophys Acta，2012，1826(1):23-31.
[4]
Kudo Y, Kitajima S, Ogawa I，et al. Invasion and metastasis of oral cancer cells require methylation of E-cadherin and/or degradation of membraneous beta-catenin[J]. Clin Cancer Res，2004，10(16):5455-5463.
[5]
Wang C, Liu X, Chen Z，et al. Polycomb group protein EZH2-mediated E-cadherin repression promotes metastasis of oral tongue squamous cell carcinoma[J]. Mol Carcinog，2013，52(3):229-236.
[6]
Wang C, Liu X, Huang HZ，et al. Deregulation of Snai2 is associated with metastasis and poor prognosis in tongue squmous cell carcinoma[J]. Int J Cancer，2012，130(10):2249-2258.
[7]
Acloque H, Adams MS, Fishwick K，et al. Epithelial-mesenchymal transitions：theimportance of changing cell state in development and disease[J]. J Clin Invest，2009，119(6):1438-1449.
[8]
Yan C, Grimm WA, Garner WL，et al. Epithelial to mesenchymal transition in human skin wound healing is induced by tumor necrosis factor-alpha through bone morphogenic protein-2[J]. Am J Pathol，2010，176(5):2247-2258.
[9]
López-Novoa JM, Nieto MA. Inflammation and EMT：an alliance towards organ fibrosis and cancer progression[J]. EMBO Mol Med，2009，1(6-7):303-314.
[10]
Chaffer CL, Brennan JP, Slavin JL，et al. Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis：role of fibroblast growth factor receptor-2[J]. Cancer Res，2006，66(23):11271-11278.
[11]
Lee TK, Man K, Poon RT，et al. Signal transducers and activators of transcription 5b activation enhances hepatocellular carcinoma aggressiveness through induction of epithelial-mesenchymal transition[J]. Cancer Res，2006，66(20):9948-9956.
[12]
Alonso SR, Tracey L, Ortiz P，et al. A high-throughput study in melanoma identifies epithelial-mesenchymal transition as a major determinant of metastasis[J]. Cancer Res，2007，67(7):3450-3460.
[13]
Zhong LP, Li J, Zhang CP，et al. Expression of E-cadhefin in cervical lymph nodes from primary oral squamous cell carcinoma patients[J]. Arch Oral Biol，2007，52(8):740-747.
[14]
Wang C, Huang H, Huang Z，et al. Tumor budding correlates with poor prognosis and epithelial-mesenchymal transition in tongue squamous cell carcinoma[J]. J Oral Pathol Med，2011，40(7):545-551.
[15]
Nakayama S, Sasaki A, Mese H，et al. Establishment of high and low metastasis cell lines derived from a human tongue squamous cell carcinoma[J]. Invasion Metastasis，1998-1999,18(5-6):219-228.
[16]
Park YJ, Claus R, Weichenhan D，et al. Genome-wide epigenetic modifications in cancer[J]. Prog Drug Res，2011(67):25-49.
[1] 陈金业, 凌潜龙, 朱冰, 骆杰. 补体B因子在结直肠癌中的表达及临床意义[J/OL]. 中华普通外科学文献(电子版), 2024, 18(03): 192-198.
[2] 谭智勇, 付什, 李宁, 王海峰, 王剑松. 膀胱小细胞癌发病机制及其诊疗研究进展[J/OL]. 中华腔镜泌尿外科杂志(电子版), 2024, 18(02): 183-187.
[3] 刘锦程, 王斌, 张雯, 张明周, 刘禹, 叶东樊, 黄赞胜, 邱凌霄, 卿斌, 王创业, 王南博, 王苹, 郭宇航, 周培花, 程秋霞, 徐智. 肺泡灌洗液RASSF1A及SHOX2甲基化联合径向超声特征对肺结节性质鉴别诊断的意义[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(04): 505-511.
[4] 赵蒙蒙, 黄洁, 余荣环, 王葆青. 过表达小GTP酶Rab32抑制非小细胞肺癌细胞侵袭性生长[J/OL]. 中华肺部疾病杂志(电子版), 2024, 17(04): 512-518.
[5] 赵旭鹏, 王集琛, 田硕, 李宏召, 李修彬, 张旭. EP300 通过上调FKBP10 促进膀胱肿瘤细胞迁移和侵袭[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(05): 264-274.
[6] 曾聿理, 雷发容, 肖慧, 邱德亮, 谢静, 吴寻. 氯普鲁卡因通过调控circRNA-ZKSCAN1表达抑制肝癌Huh-7细胞体外生长和转移的研究[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(04): 220-228.
[7] 李晶, 潘侠, 周芳, 汪晶, 洪佳. 普鲁卡因通过上调lncRNA DGCR5抑制胃癌细胞增殖、迁移和侵袭[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 151-158.
[8] 刘杜先, 张杰东, 付鲁渝, 熊志强, 龚程, 张小雅, 高明悦, 孟俊宏, 刘兰侠. 沉默circXPO1抑制肝癌细胞恶性生物学行为[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 159-166.
[9] 李博, 马秀岩, 孙杰. lncRNA TINCR对滋养层HTR-8/SVneo细胞生物学行为的影响及其机制[J/OL]. 中华细胞与干细胞杂志(电子版), 2024, 14(03): 167-172.
[10] 夏辉, 戴斌, 冉君, 王威, 龚昭, 周程. DEP结构域蛋白1B在肝细胞癌中的表达及功能[J/OL]. 中华肝脏外科手术学电子杂志, 2024, 13(02): 205-213.
[11] 崔精, 鲍一帆, 沈晓明, 杨增辉, 高森, 鲍传庆. 结直肠癌中circMFSD12对肿瘤细胞功能及5-FU敏感性的调控[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(04): 294-302.
[12] 邵文哲, 孙倩男, 王道荣. Galectin-9在结直肠癌中的表达及其临床意义[J/OL]. 中华结直肠疾病电子杂志, 2024, 13(02): 112-120.
[13] 王国强, 张纲, 唐建坡, 张玉国, 杨永江. LINC00839 调节miR-17-5p/WEE1 轴对结直肠癌细胞增殖、凋亡和迁移的影响[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 491-499.
[14] 孙晗, 于冰, 武侠, 周熙朗. 基于循环肿瘤DNA 甲基化的结直肠癌筛查预测模型的构建与验证[J/OL]. 中华消化病与影像杂志(电子版), 2024, 14(06): 500-506.
[15] 陈学敬, 周立娟, 赵宏, 张莉, 孙组钰, 车南颖. SHOX2和RASSF1A基因甲基化检测联合细胞学检查在支气管灌洗液标本诊断肺癌中的应用价值[J/OL]. 中华临床医师杂志(电子版), 2024, 18(02): 139-143.
阅读次数
全文


摘要

版权所有 中华医学会 中华医学电子音像出版社有限责任公司  京ICP备14006079号-1  网络出版服务许可证：(署)网出证(京)字第075号