摘要
目的用RNAi技术干扰乳腺癌细胞BCRP基因,研究其提高乳腺癌MCF-7/ADR细胞对阿霉素的药物敏感性及分子机制。方法乳腺癌MCF-7/ADR细胞分为空白对照、阴性对照(无效siRNA)、BCRP siRNA、阿霉素、BCRP siRNA+阿霉素组。分别用实时荧光定量PCR和Western blot检测乳腺癌细胞BCRP mRNA及蛋白水平,MTT法检测细胞增殖,FCM检测细胞凋亡,实时荧光定量PCR和Western blot方法检测PKC、p53、Bcl-2 mRNA及蛋白表达水平。结果转染BCRP siRNA后,实时荧光定量PCR和Western blot结果显示MCF-7/ADR细胞中BCRP mRNA和蛋白表达量均显著降低(P<0.01)。MTT检测结果显示,与阴性对照组比较,BCRP siRNA组、阿霉素组、BCRP siRNA+阿霉素组MCF-7/ADR细胞增殖能力受到显著抑制;与阿霉素组比较,BCRP siRNA+阿霉素组MCF-7/ADR细胞增殖能力受到显著抑制(P<0.01)。FCM检测显示,与阴性对照组比较,BCRP siRNA组、阿霉素组、BCRP siRNA+阿霉素组早、晚期凋亡细胞数量均显著增加(P<0.01)。实时荧光定量PCR和Western blot实验结果显示,与阴性对照组比较,BCRP siRNA组、阿霉素组、BCRP siRNA+阿霉素组PKC、Bcl-2 mRNA和蛋白表达均下调,而p53 mRNA和蛋白表达上调(P<0.01);与阿霉素组比较,BCRP siRNA+阿霉素组PKC、Bcl-2 mRNA和蛋白表达均下调,而p53 mRNA和蛋白表达上调(P<0.01)。结论沉默BCRP表达通过调控PKC、Bcl-2、p53表达提高乳腺癌MCF-7/ADR细胞对阿霉素的药物敏感性。
Objective To explore the inhibitory effect of silencing BCRP on doxorubicin resistance in breast cancer cells and its mole-cular mechanism.Methods Breast cancer MCF-7/ADR cells were divided into blank control group,negative control group(invalid siRNA),BCRP siRNA group,doxorubicin group,and BCRP siRNA+doxorubicin group.BCRP mRNA and protein levels were mea-sured by real time-PCR and Western blot,respectively.MTT method was used to detect cell proliferation.Flow cytometry was used to examine cell apoptosis.Real time-PCR and Western blot methods were used to detect the expression of PKC,p53 and Bcl-2.Results After transfected with BCRP siRNA,BCRP mRNA levels and protein expression were significantly reduced in MCF-7/ADR cells by real time-PCR and Western blot(P<0.01).MTT assay showed that the proliferation in BCRP siRNA group,doxorubicin group,and BCRP siRNA+doxorubicin group significantly decreased compared to negative control group,and that the proliferation was decreased in BCRP siRNA+doxorubicin group compared to doxorubicin group(P<0.01).FCM examination revealed that compared with negative control group,the numbers of early and late apoptotic cells were increased significantly in BCRP siRNA group,doxorubicin group,and BCRP siRNA+doxorubicin group(P<0.01).Compared with negative control group,the expression of PKC and Bcl-2 decreased,p53 expression was increased in BCRP siRNA group,doxorubicin group,and BCRP siRNA+doxorubicin group(P<0.01).Compared with doxorubicin group,the expression of PKC and Bcl-2 was decreased,while p53 expression was increased in BCRP siRNA+doxorubicin group(P<0.01).Conclusion Silencing BCRP may significantly improve the chemosensitivity of doxorubicin in breast cancer MCF-7/ADR cells by regulating the expression of PKC,Bcl-2 and p53.
作者
马海琳
车少敏
王晓丽
张晓智
MA Hailin;CHE Shaomin;WANG Xiaoli;ZHANG Xiaozhi(Department of Radiation Oncology,First Affiliated Hospital of Xi’an Jiaotong University,Xi’an 710061,China)
出处
《山西医科大学学报》
CAS
2019年第10期1357-1363,共7页
Journal of Shanxi Medical University
基金
国家自然科学基金资助项目(81772995)
关键词
乳腺癌
BCRP
多药耐药
增殖
凋亡
breast cancer
BCRP
multi-drug resistance
proliferation
apoptosis
作者简介
通讯作者:马海琳,男,1979-06生,博士,主治医师,E-mail:mhl7906@163.com。
