转录信号转导子与激活子3小分子干扰RNA增强乳腺癌对多柔比星的药物敏感性

Small Interfering RNA Targeting STAT3 Enhances the Drug Susceptibility of Adriamycin in Breast Cancer Cells

目的进一步明确转录信号转导子与激活子3(signal transducers and activators of transcription 3,STAT3)信号通路在调节乳腺癌多柔比星耐药中的作用,促进转录信号转导子与激活子3信号通路抑制剂在逆转肿瘤耐药方面的临床应用。方法首先在临床乳腺癌组织标本水平,应用Western blotting检测转录信号转导子与激活子3与活化磷酸化转录信号转导子与激活子3(phosphorylated STAT3,p STAT3)的表达水平,同时应用RT-PCR及Western blotting检测转录信号转导子与激活子3/磷酸化转录信号转导子与激活子3在乳腺癌敏感及耐药细胞中的表达水平;再应用小分子干扰RNA(small interfering RNA,siRNA)技术阻断转录信号转导子与激活子3表达,通过Western blotting方法检测转录信号转导子与激活子3-小分子干扰RNA对转录信号转导子与激活子3表达的影响;最后分别应用细胞免疫荧光法和四甲基偶氮唑蓝法,检测阻断转录信号转导子与激活子3表达后,乳腺癌细胞的增殖活性及IC50值,从而观察转录信号转导子与激活子3-小分子干扰RNA是否能够抑制乳腺癌细胞增殖,以及能否增强乳腺癌细胞对多柔比星的敏感性。结果转录信号转导子与激活子3/磷酸化转录信号转导子与激活子3在肿瘤组织以及在多柔比星敏感及耐药的乳腺癌细胞中均呈现高水平表达,并且在多柔比星耐药乳腺癌细胞中的表达高于在相应敏感细胞中的表达水平。转录信号转导子与激活子3-小分子干扰RNA明显下调转录信号转导子与激活子3蛋白表达,并抑制了敏感乳腺癌细胞的生长。转录信号转导子与激活子3-小分子干扰RNA和多柔比星联合应用后,与单独应用多柔比星比较,使多柔比星耐药乳腺癌细胞的IC50降低了4倍,同时抑制了转录信号转导子与激活子3信号通路介导的抗凋亡蛋白的表达。结论转录信号转导子与激活子3信号通路活化与乳腺癌发生相关,在多柔比星耐药乳腺癌细胞中显著活化,阻断乳腺癌多柔比星耐药细胞中转录信号转导子与激活子3信号通路可提高其对多柔比星的敏感性,并促进细胞凋亡。本试验为探索阻断转录信号转导子与激活子3信号通路逆转肿瘤耐药的可行性,以及临床联合应用转录信号转导子与激活子3抑制剂与化疗药物克服肿瘤细胞多药耐药现象提供实验基础。

ABSTRACT ：OBJECTIVE To further define the modulation effect of signal transducers and activators of transcription 3 （ STAT3 ） in adriamycin-resistant breast cancer and to promote the clinical application of the inhibitors of STAT3 in reversing mnltidrng resistance in cancer. METHODS Firstly, the levels of STAT3 and phosphorylated STAT3 （ pSTAT3 ） expression in clinical breast cancer tissue samples were determined by Western blotting. The expression of STAT3 and pSTAT3 in adriamyein-sensitive and adriamycin-resistant breast cancer cell lines was evaluated by RT-PCR and Western blotting. Secondly, the expression of STAT3 was detected by Western blotting after blocking the STAT3 signal pathway with small interfering RNA targeting STAT3 （STAT3-siRNA）. Finally, after the ex- pression of STAT3 was blocked by STAT3-siRNA, immunofluorescence was performed to study the proliferation activity of breast cancer cells and MTI＂ was used to determine the ICs0of the cells in order to observe whether STAT3-siRNA had any inhibitory effects on the growth of breast cancer cells and whether it could promote the efficacy of adriamycin in breast cancer cells. RESULTS STAT3 and pSTAT3 were highly expressed both in clinical breast cancer tissue samples and in adriamycin-sensitive-and-resistant breast cancer ceils. The expression of pSTAT3 in adriamycin-resistant breast cancer cells was significantly higher than in adriamycin-sensitive breast cancer cells. STAT3-siRNA conspicuously decreased the expression of STAT3 protein and inhibited the growth of adriamycin-sensitive breast cancer cells. Compared with the single application of adriamycin, ICs0of adriamycin-resistant breast cancer cells decreased by 4 fold when adriamycin was used in combinaiton with STAT3-siRNA. Meanwhile, an inhibition of the expression of the anti-apoptotic pro- tein mediated by STA33 was observed in adriamycin-resistant breast cancer cells. CONCLUSION This study reveals that the devel- opment of breast cancer is related to the activation of STA33. The activation of STAT3 in adriamycin-resistant breast cancer cells was more notable than in adriamycin-sensitive cells. The inhibition of the STA33 pathway could improve the adriamycin sensitivity of adria- mycin-resistant breast cancer cells and lead to their apoptosis. The results of this study explores the feasibility of the reversal of drug re- sistance in cancer by blocking STA33 pathway and establishes the experimental basis for promoting the clinical use of the inhibitors of STA33 pathway and the chemotherapeutics to overcome the muhidrug resistance in cancer.