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

doi:10.3877/cma.j.issn.1674-1366.2016.03.001

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

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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. 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 Huang^,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: huanghongzhang@tom.com

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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′	?

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

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