下调三磷酸腺苷结合转运蛋白G超家族成员2基因对人乳腺癌多西他赛耐药细胞株BT549/Docetaxel的作用

摘要

Objective Down people docetaxel-resistant breast cancer cell lines expressing multidrug resistance gene adenosine triphosphate binding cassette transporter G2 (ABCG2) BT549/Docetaxel,testing alter their biological properties.Methods moderate concentrations,to establish the role of law intermittent docetaxel in human breast cancer cell lines resistant to BT549/Docetaxel,ABCG2 expression vector construct pRNAT-U6.2/Lenti small interfering RNA (siRNA) transfection BT549/Docetaxel,using the methyl thiazol tetrazolium (MTT) assay after transfection resistance index of cells,high performance liquid chromatography (HPLC) intracellular concentration of docetaxel.Methods Using moderate concentrations 0.08 mg/L,to establish the role of law intermittent docetaxel in human breast cancer cell lines resistant to BT549/Docetaxel,constructed of ABCG2 pRNAT-U6.2/Lenti siRNA expression vector was transfected BT549/Docetaxel,using the MTT assay after transfection cells the resistance index,HPLC intracellular concentration of docetaxel.Results Infection ABCG2si2,ABCG2si1,BT549/Docetaxel cells ABCG2siC after BT549/Docetaxel compared with uninfected cells and ABCG2 mRNA relative expression of β-actin levels were 0.11 t± 0.02,0.26 ± 0.01,0.88 ± 0.03,0.92± 0.02.Infection ABCG2si1,the ABCG2si2 cells reduce ABCG2 expression and control ABCG2siC,BT549/Docetaxel statistically significant compared (P ＜ 0.05),respectively,a decrease of 71.7％ and 88.0％.Four cells were Western bloting showed different ABCG2 protein levels,ABCG2si1,ABCG2 si2 inhibit the expression of the protein,the latter effect is significant (P ＜ 0.05).By MTT assay,infection ABCG2si2,ABCG2siC cells and uninfected cells BT549/Docetaxel 50％ inhibitory dose (IC50) of Docetaxel respectively 31.12,69.55,76.21 mg/L,the resistance index were 8.13,18.16,19.90.Which significantly increases the sensitivity of the former and the latter two Docetaxel compared (P ＜ 0.05).HPLC intracellular docetaxel,the content of the drug within the cells of 7.9 ng/106 BT549/Docetaxel cells infected in the content of intracellular drug BT549/Docetaxel 66.7 ng/106 cells,normal culture medium after 4 h,the content within BT549/Docetaxel cells after infection drugs for 53.1 ng/106 cells,the difference was statistically significant (P ＜ 0.05).Conclusion By ABCG2 gene regulation and breast cancer resistance can be closely related to the reversal of drug resistance BT549/Docetaxel.%目的 下调人乳腺癌多西他赛耐药细胞株BT549/Docetaxel耐药相关基因三磷酸腺苷结合转运蛋白G超家族成员2(ABCG2)的表达,检测其生物学特性的改变.方法 采用中等浓度0.08 mg/L、间歇作用法建立人乳腺癌多西他赛耐药细胞株BT549/Docetaxel,构建ABCG2的pRNAT-U6.2/Lenti小干扰RNA(siRNA)表达载体转染BT549/Docetaxel,采用噻唑蓝(MTT)法测定转染前后细胞的耐药指数,高效液相色谱测定细胞内多西他赛的浓度.结果 感染ABCG2si2、ABCG2si1、ABCG2siC后的BT549/Docetaxel细胞与未感染的BT549/Docetaxel细胞ABCG2mRNA与β-actin比较其相对表达水平分别为0.11 ±0.02、0.26 ±0.01、0.88±0.03、0.92±0.02.感染ABCG2si1、ABCG2si2的细胞内ABCG2的表达减少,与对照ABCG2siC、BT549/Docetaxel比较差异有统计学意义(P＜0.05),分别下降了71.7％和88.0％.4种细胞经Western blot检测显示,ABCG2蛋白表达水平不同,ABCG2si1、ABCG2si2对该蛋白的表达均有抑制作用,其中后者效果显著(P＜0.05).经MTT法检测,感染有ABCG2si2、ABCG2siC的细胞与未感染的BT549/Docetaxel细胞对Docetaxel的半数抑制剂量(IC50)分别是:31.12、69.55、76.21 mg/L,其耐药指数分别为:8.13、18.16、19.90.其中前者与后两者比较对Docetaxel的敏感性显著增加(P＜0.05).高效液相色谱测定细胞内多西他赛的含量,BT549/Docetaxel细胞内药物的含量为7.9 ng/106个细胞,感染后BT549/Docetaxel细胞内药物的含量为66.7 ng/106个细胞,正常培养基培养4h后,感染后BT549/Docetaxel细胞内药物的含量为53.1 ng/106个细胞,BT549/Docetaxel细胞内药物的含量4.3 ng/106个细胞,差异有统计学意义(P＜0.05).结论 通过调控与乳腺癌耐药密切相关的ABCG2基因可以逆转BT549/Docetaxel的耐药性.