摘要
目的:研究RNA干扰人抗原R(human antigen R,HuR)基因的表达对人乳腺癌耐药细胞株MCF-7/Adr对多柔比星(doxorubicin)敏感性的影响。方法:构建靶向HuR基因的shRNA表达质粒(pGenesil-siHuR),稳定转染至MCF-7/Adr细胞,real-time PCR检测细胞中MDR1 mRNA的表达,Western blotting检测MCF-7/Adr细胞中由MDR1基因编码的P糖蛋白(P-glycoprotein,P-gp)的表达,MTT法检测pGenesil-siHuR转染后MCF-7/Adr细胞在多柔比星作用后的存活率和IC50,流式细胞术检测MCF-7/Adr细胞的凋亡率。结果:与未转染的MCF-7/Adr细胞比较,pGenesil-siHuR质粒转染MCF-7/Adr细胞中MDR1 mRNA的表达水平明显减低[(0.184±0.029)vs(1.203±0.026),P<0.01],P-gp表达水平明显降低。pGenesil-siHuR质粒转染MCF-7/Adr细胞后,MCF-7/Adr细胞对多柔比星的IC50从未转染的(148.2±2.3)nmol/L降至(42.9±0.4)nmol/L;经多柔比星处理后,pGenesil-siHuR质粒转染组MCF-7/Adr细胞的凋亡率明显上升[(34.6±1.1)%vs(1.1±0.2)%,P<0.01]。结论:RNA干扰HuR的表达能抑制MDR1基因的表达,增加耐药乳腺癌MCF-7/Adr细胞对多柔比星的敏感性。
Objective： To investigate the effect of interference of human antigen R （HuR） expression on sensitivity of human multidrug-resistant human breast cancer MCF-7/Adr cell line to Doxorubicn. Methods： The shRNA expression vector targeting HuR gene （pGenesil-siHuR） has been constructed and stably transfected into human breast cancer MCF-7/Adr cell line. The expression level of MDR1 mRNA in MCF-7/Adr cells was assayed by real-time PCR. The P-gp protein （encoded by the MDR1 gene） expression were determined by Western blotting. The survival rate and ICs0 of MCF-7/Adr cells to doxorubicin after pGenesil-siHuR transfection were evaluated by MTT method. The apoptosis rate of MCF-7/Adr cells was detected by flow cytometry. Results： Compared with untransfected MCF-7/Adr cells, the MDR1 mRNA （ [ 0.184 ± 0. 029 ] vs [ 1. 203 ± 0. 026 ], P 〈 0.01 ） and P-gp protein expressions （ [ 0.314 ± O. 011 ] vs [ 0. 796 ± 0. 007 ], P 〈 0.01 ） were significantly reduced in pGenesil-siHuR transfected MCF-7/Adr cells （ P 〈 O. 01 ）. The IC50 of MCF-7/Adr cells to doxorubicin decreased from （148.2 ± 2.3 ） nmol/L to （42.9 ±O. 4） nmol/L after pGenesil-siHuR transfection. Compared with untransfected MCF-7/Adr ceils, the ratio of cell apoptosis was significantly increased in pGenesil-siHuR transfected MCF-7/Adr cells （ [ 34.6 ＋ 1.1 ] % vs [ 1.1 ± 0.2 ] %, P 〈 0.01 ） after the treatment with doxorubicin. Conclusion ： RNA interference of HuR can inhibit the expression of MDR1 gene and increase the sensitivity of multidrug-resistant breast cancer cells to doxorubicin.
出处
《中国肿瘤生物治疗杂志》
CAS
CSCD
北大核心
2013年第2期182-186,共5页
Chinese Journal of Cancer Biotherapy
基金
国家自然科学基金资助项目(No.30901788
No.81272619)
山东省自然科学基金资助项目(No.ZR2010HQ038
No.ZR2010HM059)~~
