miR-363对顺铂耐药乳腺癌细胞的作用及机制

Effect and Mechanism of miR-363 on Cisplatin-treated Breast Cancer Cells

关键词:  miR-363  Mcl-1  MCF-7-R  顺铂

Key words:  miR-363  Mcl-1  MCF-7-R  cisplatin

摘要:

目的 研究miR-363对顺铂耐药乳腺癌细胞的作用及机制。方法 采集以顺铂为主要化疗药物的中晚期乳腺癌患者血清，用定量PCR法检测化疗前和化疗后血清标本miR-363的表达水平。构建顺铂抵抗MCF-7细胞系(MCF-7-R)，MTT法检测不同浓度顺铂对MCF-7及MCF-7-R细胞活力的影响。在MCF-7-R细胞中转染miR-363，MTT法检测miR-363是否能提高顺铂对MCF-7-R的杀伤活性。利用生物信息学、定量PCR及western blot方法验证miR-363是否调节MCF-7-R细胞中Mcl-1的表达。构建Mcl-1真核表达载体，MTT法检测Mcl-1表达载体转染对miR-363联合顺铂治疗MCF-7-R疗效的影响。结果 中晚期乳腺癌患者顺铂治疗后血清miR-363水平相较化疗前显著下降。MTT结果表明相同浓度顺铂对MCF-7-R的杀伤活性显著低于MCF-7细胞，且miR-363转染能显著提高顺铂对MCF-7-R的杀伤活性。生物信息学、定量PCR及western blot结果表明miR-363转染可显著降低MCF-7-R细胞中Mcl-1的表达。miR-363联合顺铂在Mcl-1表达载体转染后对MCF-7-R细胞的杀伤活性显著低于未转染Mcl-1表达载体的miR-363联合顺铂组。结论 MiR-363能增强耐顺铂乳腺癌细胞对顺铂杀伤作用的敏感性。

Abstract:

OBJECTIVE to investigate the role and mechanism of miR-363 on cisplatin-resistant breast cancer cells. METHODS collected the serum from the advanced breast cancer patients who were treated with cisplatin-based chemotherapy. Then the expression of miR-363 in breast cancer patients’ serum was detected before or after chemotherapy using qPCR analysis. Constructed the cisplatin-resistant MCF-7 cell line, the cytotoxicity of cisplatin to MCF-7 cell line and MCF-7-R cell line was measured by MTT assay. Transfected the MCF-7-R cells with miR-363 to determine whether the transfection of miR-363 enhanced cytotoxicity of cisplatin to MCF-7-R cells. Confirmed whether the expression of Mcl-1 was regulated by miR-363 using bioinformatics, qPCR and western blot. Constructed the Mcl-1 expression vector, and detected the cytotoxicity of cisplatin combing with the cotransfection of miR-363 and Mcl-1 expression vector by MTT assay. RESULTS miR-363 levels were significantly decreased in advanced breast cancer patients treated with cisplatin-based chemotherapy. MiR-363 levels were also lower in MCF-7-R cells than in MCF-7 cells. Exogenous miR-363 significantly overcame cisplatin resistance in MCF-7-R cells, whereas miR-363 knockdown increased the cell viability during cisplatin treatment. It was demonstrated that miR-363 directly targeted to Mcl-1, and the downregulation of miR-363 resulted in upregulation of Mcl-1. miR-363 decreased cisplatin resistance of MCF-7-R cells, partly by targeting Mcl-1. CONCLUSION downregulation of miR-363 increases drug resistance in cisplatin-treated MCF-7 by downregulating Mcl-1.