[信号转导与转录激活因子3通过靶向微小RNA-21在体外调节人鳞状细胞癌侵袭]

[Signal transducers and activators of transcription-3 modulates human squamous cell carcinoma invasion via targeting mircoRNA-21 in vitro].

作者信息

Liu Ai-qin, Li Sha-sha, Zhou Xuan, Wang Xu-dong, Zhang Lun

机构信息

Department of Maxillofacial & E.N. T (ear, nose, and throat) Oncology, Tianjin Medical University Cancer Institute and Hospital, Key Laboratory of Cancer Prevention and Therapy (Tianjin), Tianjin 300060, China.

出版信息

Zhonghua Kou Qiang Yi Xue Za Zhi. 2013 Sep;48(9):539-44.

PMID:24314280

Abstract

OBJECTIVE

To investigate the effect and mechanism of signal transducers and activators of transcription 3 (STAT-3) modulates human tongue squamous cell carcinoma invasion ability via targeting mircoRNA-21.

METHODS

Tscca and Tca8113P160 human tongue squamous cell carcinoma cell lines were used.WP1066 (STAT-3 inhibitor) , the small molecule inhibitor of STAT-3 was used to suppress the STAT-3 expression. The half maximal inhibitory concentration (IC50 value) of WP1066 in the two cell lines was determined by methyl thiazolyl tetrazolium (MTT) assay. The expression level of STAT-3 and phosphorylation of STAT-3 (pSTAT-3) was examined by Western blotting. Real-time PCR was used to detect the mircoRNA-21 expression after treated with WP1066. Matrigel matrix and transwell assay were used to determine cancer cell colony formation and invasion ability after treated with WP1066. Tumor invasion related proteins in Tscca and Tca8113P160 cell lines were measured by Western blotting. Luciferase reporter gene assay was conducted to detect the relationship between STAT-3 and mircoRNA-21.

RESULTS

The IC50 to WP1066 in Tscca cell was 3.1 and 3.5 µmol/L for Tca8113P160 cell respectively. STAT-3/pSTAT-3 protein level was suppressed significantly (Tscca: STAT-3: F = 887.154, P = 0.000; pSTAT-3: F = 332.212, P = 0.000; Tca8113P160: STAT-3: F = 322.895, P = 0.000; pSTAT-3:F = 788.357, P = 0.000). mircoRNA-21 expression was down-regulated (Tscca:F = 32.157, P = 0.000; Tca8113P160: F = 11.349, P = 0.007). The diameters of culture clone in cell treated with WP1066 were less than control groups (Tscca:F = 15.751, P = 0.004; Tca8113P160: F = 12.964, P = 0.007). The number of tongue cancer cell migrating through the transwell membrane in WP1066 treated group was less than in control groups (Tscca: F = 1688.926, P = 0.000; Tca8113P160: F = 327.528, P = 0.000). In addition, MMP-2/9 protein expression was decreased in both of the cell lines treated with WP1066, while TIMP-3 was up regulated dramatically. STAT-3 could modulate mircoRNA-21 directly.

CONCLUSIONS

Reduction of STAT-3 can inhibit tongue cancer cell invasion ability via targeting mircoRNA-21.

摘要

目的

探讨信号转导与转录激活因子3（STAT-3）通过靶向微小RNA-21调节人舌鳞状细胞癌侵袭能力的作用及机制。

方法

采用Tscca和Tca8113P160人舌鳞状细胞癌细胞系。使用STAT-3小分子抑制剂WP1066抑制STAT-3表达。通过甲基噻唑基四氮唑（MTT）法测定WP1066在两种细胞系中的半数抑制浓度（IC50值）。采用蛋白质免疫印迹法检测STAT-3的表达水平及磷酸化的STAT-3（pSTAT-3）。用实时荧光定量PCR检测WP1066处理后微小RNA-21的表达。采用基质胶基质和Transwell小室实验检测WP1066处理后癌细胞集落形成及侵袭能力。通过蛋白质免疫印迹法检测Tscca和Tca8113P160细胞系中肿瘤侵袭相关蛋白。进行荧光素酶报告基因实验以检测STAT-3与微小RNA-21之间的关系。

结果

WP1066对Tscca细胞的IC50为3.1 μmol/L，对Tca8113P160细胞的IC50为3.5 μmol/L。STAT-3/pSTAT-3蛋白水平显著降低（Tscca：STAT-3：F = 887.154，P = 0.000；pSTAT-3：F = 332.212，P = 0.000；Tca8113P160：STAT-3：F = 322.895，P = 0.000；pSTAT-3：F = 788.357，P = 0.000）。微小RNA-21表达下调（Tscca：F = 32.157，P = 0.000；Tca8113P160：F = 11.349，P = 0.007）。WP1066处理的细胞中培养克隆的直径小于对照组（Tscca：F = 15.751，P = 0.004；Tca8113P160：F = 12.964，P = 0.007）。WP1066处理组中穿过Transwell小室膜的舌癌细胞数量少于对照组（Tscca：F = 1688.926，P = 0.000；Tca8113P160：F = 327.528，P = 0.000）。此外，WP1066处理的两种细胞系中MMP-2/9蛋白表达均降低，而TIMP-3显著上调。STAT-3可直接调节微小RNA-21。