RANKL/RANK通路介导人乳腺癌MCF-7细胞株对阿霉素耐药机制的研究  

Mechanism of RANKL/RANK Signaling Pathway-mediated the Drug-resistance for Adriamycin in Human Breast Cancer Cell Line MCF-7

作　　者：王柚熙 唐振宁[2] 刘奇伦[2] WANG Youxi TANG Zhenning LIU Qilun(Ningxia Medical University, Yinchuan 750004 Department of Medical Oncology, the General Hospital of Ningxia Medical University, Yinchuan 750004)

机构地区：[1]宁夏医科大学,银川750004 [2]宁夏医科大学总医院肿瘤医院,银川750004

出　　处：《宁夏医科大学学报》2017年第2期144-148,158,F0003,共7页Journal of Ningxia Medical University

基　　金：国家自然科学基金(81360390);宁夏自然科学基金(NZ13161);宁夏医科大学特殊人才科研启动项目(XT201327)

摘　　要：目的观察核因子κB活化因子受体(RANK)/核因子κB活化因子受体配体(RANKL)通路对乳腺癌细胞耐药性的影响,并研究其作用机制。方法培养人乳腺癌细胞株MCF-7和人乳腺癌阿霉素耐药细胞株MCF7/ADR,用免疫荧光显微技术及Western blot检测MCF-7及MCF7/ADR中RANK蛋白表达情况;分别用RANKL和RANKL抑制剂人骨保护素(OPG)处理两种细胞,用Western blot检测乳腺癌干细胞相关乙醛脱氢酶1(ALDH1)蛋白表达情况,CCK-8法检测不同浓度的RANKL与OPG处理后MCF-7细胞在阿霉素作用下的细胞存活率。结果免疫荧光显微技术及Western blot结果显示,RANK蛋白在MCF7/ADR中的表达较MCF-7均明显增强(P<0.05)。相同RANKL浓度预处理时,MCF7/ADR中ALDH1蛋白表达均高于MCF-7(P<0.05)。随着RANKL浓度的增高(0、50、100ng·mL^(-1)),两种细胞中的ALDH1蛋白表达均升高;RANKL100ng·mL^(-1)及OPG 500ng·mL^(-1)的共同预处理MCF-7细胞,其ALDH1蛋白的表达明显低于RANKL 100ng·mL^(-1)组(P<0.05)。CCK-8法检测MCF-7细胞在RANKL及OPG预处理后的细胞存活率实验中,在阿霉素浓度为2.5~5μmol·L-1范围内,随着RANKL浓度升高,细胞存活率也随之增高,而RANKL抑制剂OPG可以逆转RANKL的作用(P>0.05)。结论 RANKL/RANK通路参与了乳腺癌耐药过程,其可能通过维持肿瘤干细胞群介导乳腺癌细胞耐药。Objective To investigate the effect of RANKL/RANK （the receptor activator of nuclear factor KB ligand / receptor activator of nuclear factor KB, RANKL/RANK） signaling breast cancer cell line, and to analyze its potential mechanism. Methods pathway on the drug resistance in Human breast cancer cell lines MCF-7 and adriamycin-resistant human breast cancer cell line MCF7/ADR were cultured. Expression levels of RANK in MCF-7 and MCF7/ADR cells were detected by Immunofluorescence and Western blot. Two kinds of cells were pre-treated with RANKL and RANKL inhibitor osteoprotegerin （OPG） respectively. Protein levels of RANK and breast cancer stem cell associated ALDH1 were detected by Western blot. The drug resistance to adriamycin of MCF-7, pre-treated with RANKL and OPG, were detected by cell counting kit 8（ CCK-8 ） and cell survival rate were calculated. Results Immunofluorescence and Western blot showed that the expression of RANK protein in MCF7/ADR were significantly increased （P〈0.05）. In the same concentration of RANKL, the protein level of ALDH1 in MCF7/ADR was significantly higher than that of MCF-7 （P〈0.05）. With the increase of the concentration of RANKL （0,50, 100ng· mL-1 ）, the expression of two kinds of cell were gradually increased（P〈0.05 ）. The protein levels of ALDH1 in MCF-7 which pre-treated with RANKL 100ng·mL-1 and OPG 500ng ·mL-1 were significantly lower than thoseof the RANKL 100ng ·mL-1 group （P〈0.05）. In CCK-8 trials, when Adriamyein were from 2.5 μmol· L-1 to 5 μmol· L-1, with the increase of the concentration of RANKL, the eell survival rate of MCF-7 was significantly dose-dependent and there was a gradual increasing trend. However OPG could reverse the phenomenon （P 〉0.05）. Conclusion RANKL/RANK signaling pathway may have participated in drug resistance in breast cancer. It may have promoting effect on the process of drug resistance by maintaining stem cells population.

关 键 词：RANKL/RANK 乳腺癌 阿霉素 癌症干细胞 

分 类 号：R737.9[医药卫生—肿瘤]