颗粒蛋白前体通过促进细胞增殖和抗凋亡增强乳腺癌细胞对他莫昔芬的耐药性  

Progranulin promotes tamoxifen-resistance of breast cancer cells by enhancing proliferation and anti-apoptosis

作　　者：叶相森 蒋玉林[1] 刘一锋[1] 张志乾[1] 杨俊鸿[1] 靳倩妮 甘德露 岳姝君 钱胡孙 陈婷梅[1] YE Xiangsen;JIANG Yulin;LIU Yifeng;ZHANG Zhiqian;YANG Junhong;JIN Qianni;GAN Delu;YUE Shujun;QIAN Husun;CHEN Tingmei(Key Laboratory of Laboratory Medical Diagnostics of Ministry of Education, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China)

机构地区：[1]重庆医科大学检验医学院临床检验诊断学教育部重点实验室,重庆400016

出　　处：《肿瘤》2018年第12期1097-1105,共9页Tumor

基　　金：国家自然科学基金资助项目(编号:81272544)~~

摘　　要：目的 :探讨颗粒蛋白前体(progranulin,PGRN)在乳腺癌细胞对他莫昔芬耐药性产生过程中的作用机制。方法 :用4-羟基他莫昔芬(4-hydroxytamoxifen,OHT)处理乳腺癌细胞MCF-7和T47D以及他莫昔芬耐药性细胞MCF-7R和T47DR后,CCK-8法检测OHT对各细胞的半数抑制浓度(50%inhibitory concentration,IC50)。蛋白质印迹法检测各细胞中C-myc、Cyclin D1和Bcl-2蛋白的表达水平,CCK-8法检测各细胞的增殖能力,FCM法检测各细胞的凋亡率,实时荧光定量PCR和蛋白质印迹法分别检测各细胞中PGRN的mRNA和蛋白表达水平。采用脂质体转染法将特异性靶向PGRN基因的siRNA转染至他莫昔芬耐药性细胞MCF-7R和T47DR后,采用蛋白质印迹法检测PGRN、C-myc、Cyclin D1和Bcl-2蛋白表达水平的变化。进一步使用OHT处理干扰PGRN表达后的MCF-7R和T47DR细胞,再采用FCM法检测细胞凋亡的变化。结果 :诱导获得的他莫昔芬耐药性人乳腺癌细胞株MCF-7R和T47DR的IC50值明显高于敏感细胞MCF-7和T47D(P值均<0.001)。耐药细胞MCF-7R和T47DR的增殖速度明显高于MCF-7和T47D细胞(P值均<0.01),凋亡率明显低于MCF-7和T47D细胞(P值均<0.01),增殖相关蛋白C-myc和Cyclin D1以及凋亡相关蛋白Bcl-2的表达水平明显升高(P值均<0.01),PGRNmRNA和蛋白水平也明显升高(P值均<0.05)。干扰PGRN基因表达后,耐药细胞MCF-7R和T47DR中C-myc、Cyclin D1和Bcl-2的表达水平明显降低(P值均<0.01),而OHT处理后细胞凋亡数量明显增多(P值均<0.01)。结论 :PGRN在人乳腺癌他莫昔芬耐药细胞中明显高表达,干扰PGRN基因表达可以增强乳腺癌细胞对他莫昔芬的敏感性。Objective: To investigate the mechanism of progranulin (PGRN) in the development of tamoxifen resistance of human breast cancer cells. Methods: The breast cancer MCF-7 and T47D cells as well as tamoxifen-resistant MCF-7R and T47DR cells were treated by 4-hydroxytamoxifen (OHT). The 50% inhibitory concentration (IC 50 ) of OHT was detected by CCK-8. The expressions of C-myc, Cyclin D1 and Bcl-2 were determined by Western blotting. The cell proliferation was detected by CCK-8, the apoptosis was detected by FCM. The expression levels of PGRN mRNA and protein were detected by real-time fluorescent quantitative PCR and Western blotting, respectively. The expression of PGRN in MCF-7R and T47DR cells was interfered by transfection of PGRN-siRNA. Then the changes of PGRN, C-myc, Cyclin D1 and Bcl-2 expressions were detected by Western blotting. Furthermore, the PGRN-interfered MCF-7R and T47DR cells were stimulated by OHT, then the apoptosis was tested by FCM again. Results: The IC 50 of OHT in tamoxifen-induced MCF-7R or T47DR cells was higher than that of MCF-7 or T47D cells (both P < 0.001). As compared with MCF-7 or T47D cells, the proliferation of MCF-7R or T47DR cells was enhanced (both P < 0.01), the apoptotic rate was decreased (both P < 0.01), the expressions of proliferation-related proteins C-myc and Cyclin D1 as well as apoptosis-related protein Bcl-2 were significantly increased (all P < 0.01), and the mRNA and protein levels of PGRN were significantly increased in MCF-7R and T47DR cells (both P < 0.05). After the transfection of PGRN-siRNA into MCF-7R and T47DR cells, the protein levels of PGRN, C-myc, Cyclin D1 and Bcl-2 were significantly decreased (all P < 0.01). After stimulation with OHT, the apoptosis rate of PGRN-downregulated MCF-7R and T47DR cells was increased (both P < 0.01). Conclusion: PGRN is overexpressed in human breast cancer MCF-7R and T47DR cells with tamoxifen-resistance. Interference of PGRN gene expression can enhance the sensibility of breast cancer cells to tamoxifen.

关 键 词：乳腺肿瘤 RNA干扰 抗药性 肿瘤 他莫昔芬 颗粒蛋白前体 

分 类 号：R737.9[医药卫生—肿瘤]