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

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,IC_(50)下降47.57%±6.23%(P<0.05)。转染miR-181a LNA可诱导CAL27细胞发生EMT,Ecadherin表达降低,Vimentin、Twist1及Slug表达增强,细胞侵袭、迁移能力显著增强,IC_(50)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。

PURPOSE： The aim of this study was to investigate the mechanism of miR-181 a 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-181 a mimics and miR-181 a 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-181 a. Luciferase reporter gene plasmids were constructed and designed obligos including miR-181 a targeting site. Dual luciferase reporter gene array was performed. SPSS17.0 software package was used to analyze the results. RESULTS： miR-181 a 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-181 a LNA transfection induced EMT in CAL27. The expression of E-cadherin was down-regulated and the expression of Vimentin, Twist1 and Slug wasup-regulated. The motility was significantly increased. IC（50 of CAL27 increased by 55.61% ±7.20%（P 〈0.05）.CONCLUSIONS： MiR-181 a inhibits EMT and CDDP drug-resistance by directly targeting Twist1 in TSCC.