miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究

doi:10.19438/j.cjoms.2018.04.001

中国口腔颌面外科杂志 ›› 2018, Vol. 16 ›› Issue (4): 289-295.doi: 10.19438/j.cjoms.2018.04.001

miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究

刘墨¹, 李劲松¹, 阮毅¹, 黄洪章²

1.中山大学孙逸仙纪念医院口腔科,广东广州 510120;
2.中山大学光华口腔医学院口腔颌面外科,广东广州 510055

收稿日期:2017-09-08 修回日期:2018-01-17 出版日期:2018-07-20 发布日期:2018-08-09
通讯作者: 黄洪章,E-mail:huanghongzhang@tom.com
作者简介:刘墨(1978-),女,博士,副主任医师,E-mail：mmliumo@163.com
基金资助:
国家自然科学基金 (81502350）; 广东省基础与应用基础研究专项（省自然基金）自由申请项目（2016A030313352）

Study on miR-181a inhibiting epithelial-mesenchymal transition and drug-resistance by targeting Twist1 in tongue squamous cell carcinoma

LIU Mo¹, LI Jin-song¹, RUAN Yi¹, HUANG Hong-zhang²

1.Department of Stomatology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University. Guangzhou 510120;
2.Department of Oral and Maxillofacial Surgery, Guanghua School and Research Institute of Stomatology, Sun Yat-sen University. Guangzhou 510055, Guangdong Province, China

Received:2017-09-08 Revised:2018-01-17 Online:2018-07-20 Published:2018-08-09

摘要/Abstract

摘要： 目的:探讨miR-181a调控上皮-间充质转化(EMT)促进舌鳞状细胞癌细胞顺铂耐药的机制。方法:分别在CAL27/CDDP、CAL27中转染miR-181a mimics、miR-181a LNA,免疫印迹及免疫荧光检测E-cadherin、Vimentin的表达;免疫印迹检测Twist1、Slug的表达;Transwell及划痕实验检测细胞的侵袭、迁移能力;MTT法检测细胞对顺铂的半抑制率。利用生物信息学方法:预测Twist1可能为miR-181a的靶基因。构建包含Twist1结合序列片段的荧光素酶报告基因质粒,进行双荧光素酶报告基因实验。采用SPSS17.0软件包对结果进行单因素方差分析。结果:转染miR-181a mimics可逆转CAL27/CDDP的EMT表型,E-cadherin表达增强,Vimentin、Twist1及Slug表达降低,细胞的侵袭、迁移能力显著降低,IC₅₀下降47.57%±6.23%（P<0.05）。转染miR-181a LNA可诱导CAL27细胞发生EMT,E-cadherin表达降低,Vimentin、Twist1及Slug表达增强,细胞侵袭、迁移能力显著增强,IC₅₀升高55.61%±7.20%（P<0.05）。双荧光素酶报告基因实验证实,Twist1是miR-181a的靶基因。结论:miR-181a靶向调控Twist1,从而抑制舌鳞癌细胞上皮-间充质转化与顺铂耐药。

关键词: 舌鳞状细胞癌, 上皮-间充质转化, microRNA, Twist1, 耐药性

Abstract: PURPOSE:The aim of this study was to investigate the mechanism of miR-181a regulating epithelial-mesenchymal transition (EMT) and enhancing the capacity of cisplatin-resistance of tongue squamous cell carcinoma cells. METHODS: TSCC CDDP resistant cell line CAL27/CDDP and its parent cell line CAL27 were transfected with miR-181a mimics and miR-181a LNA respectively. Western blot and immunofluorescence staining were used to detect the expression of E-cadherin and Vimentin; Western blot was used to detect the expression of Twist1 and Slug; Transwell assay and scratch test were used to detect the invasion and migration capabilities; MTT was used to analyze the half maximal inhibitory concentration (IC₅₀) values. Bioinformatics analysis was performed and found that Twist1 was directly targeted by miR-181a. Luciferase reporter gene plasmids were constructed and designed obligos including miR-181a targeting site. Dual luciferase reporter gene array was performed. SPSS17.0 software package was used to analyze the results. RESULTS: miR-181a mimics transfection reversed the EMT phenotype of CAL27/CDDP. The expression of E-cadherin was up-regulated and the expression of Vimentin, Twist1 and Slug was down-regulated. The motility was significantly decreased. IC₅₀ of CAL27/CDDP decreased by 47.57%±6.23% (P<0.05). miR-181a LNA transfection induced EMT in CAL27. The expression of E-cadherin was down-regulated and the expression of Vimentin, Twist1 and Slug was up-regulated. The motility was significantly increased. IC₅₀ of CAL27 increased by 55.61%±7.20%(P<0.05).CONCLUSIONS: MiR-181a inhibits EMT and CDDP drug-resistance by directly targeting Twist1 in TSCC.

Key words: Tongue squamous cell carcinoma, Epithelial-mesenchymal transition, microRNA, Twist1, Drug resistance

中图分类号:

R739.8

刘墨, 李劲松, 阮毅, 黄洪章. miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究[J]. 中国口腔颌面外科杂志, 2018, 16(4): 289-295.

LIU Mo, LI Jin-song, RUAN Yi, HUANG Hong-zhang. Study on miR-181a inhibiting epithelial-mesenchymal transition and drug-resistance by targeting Twist1 in tongue squamous cell carcinoma[J]. China J Oral Maxillofac Surg, 2018, 16(4): 289-295.

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miR-181a抑制舌鳞癌细胞上皮-间充质转化及化疗耐药机制的研究

Study on miR-181a inhibiting epithelial-mesenchymal transition and drug-resistance by targeting Twist1 in tongue squamous cell carcinoma

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