目的 探讨信号传导及转录激活因子3 (signal transducers and activators of transcription,STAT-3)调控微RNA-21影响人舌鳞状细胞癌侵袭能力的效果和机制.方法 实验共分3大组:空白对照组、二甲基亚砜组(DMSO组)、小分子抑制剂组(WP1066组).采用STAT-3小分子抑制剂WP1066下调STAT-3表达；甲基噻唑基四唑(methyl thiazolyl tetrazolium,MTT)法测定Tscca和Tca8113P160舌癌细胞WP1066的半数抑制浓度(inhibitory concentration,IC5o)；蛋白质印迹法检测WP1066处理后舌癌细胞STAT-3及pSTAT-3表达；实时定量PCR法检测微RNA-21表达水平；用Matrigel基质生长实验和Transwell体外侵袭实验检测肿瘤细胞生长形成球形克隆、侵袭能力；蛋白质印迹法检测肿瘤细胞侵袭相关蛋白表达.荧光素酶报告基因实验验证STAT-3与微RNA-21调控关系.结果 MTT法结果:Tscca、Tca8113P160细胞WP1066的IC50分别为3.1和3.5 μmol/L;WP1066组STAT-3及pSTAT-3表达水平低于空白对照组；WP1066组微RNA-21表达水平低于空白对照组.WP1066组细胞生长形成球形克隆能力减弱,直径减小(Tscca:F=15.751,P=0.004；Tca8113P160:F=12.964,P=0.007)；通过Transwell小室聚碳酸酯膜的细胞数少于空白对照组(Tscca:F=1688.926,P=0.000；Tca8113 P160:F=327.528,P=0.000);基质金属蛋白酶2/9蛋白表达水平下调；金属蛋白酶抑制剂蛋白表达水平上调.荧光素酶报告基因实验证明微RNA-21是STAT-3的调控靶点.结论 抑制舌癌细胞中STAT-3活性可以下调微RNA-21表达并降低舌癌细胞的侵袭能力；为进一步探究STAT-3参与调控舌癌细胞侵袭转移能力的分子机制提供实验依据.%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 (ICs0 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 downregulated(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.
展开▼
