人乳腺癌MDR1基因稳定沉默细胞的筛选及其生物学特性

Screening of stabilizing silencing MDR1 gene cell line of human breast cancer and its biological property

目的筛选MDR1沉默的人乳腺癌细胞并比较其生物学特性。方法采用BLOCK-iT Lentiviral RNAi Expres-sion System生产表达MDR1基因shRNA的慢病毒载体,转导敏感人乳腺癌细胞MCF-7和耐阿霉素人乳腺癌细胞MCF-7/ADM,杀稻瘟菌素(blasticidin)筛选获得MDR1稳定沉默细胞MCF-7/RNAi和MCF-7/ADM/RNAi。定量RT-PCR检测MDR1mRNA表达,流式细胞术检测P-GP蛋白表达和功能,MTT法检测药物敏感性。结果经10 mg/L blasticidin筛选12 d获得MCF-7/RNAi和MCF-7/ADM/RNAi细胞。MCF-7、MCF-7/RNAi、MCF-7/ADM和MCF-7/ADM/RNAi细胞的MDR1mRNA相对表达水平分别为1、0.13、17.14和2.01;P-GP表达分别为(1.5±0.3)%、(1.2±0.2)%、(89.4±3.6)%和(16.3±1.9)%;细胞内Rh123的潴留分别为(92.4±3.1)%、(90.6±4.0)%、(13.6±1.6)%、(72.4±2.8)%;IC50值分别为0.90、0.92、19.61和4.04 mg/L。结论应用慢病毒载体稳定沉默MDR1基因可有效逆转人乳腺癌细胞耐药性。

Objective To screen human breast cancer cell line of MDR1 gene silenced and to compare its biological properties. Methods The lentiviral vector expressing MDR1 gene short hairpin RNA （shRNA） was produced by BLOCK-iT Lentiviral RNAi Expression System and then transducted into human breast cancer cell line MCF-7. Corresponding doxorubicin-resistant human breast cancer cell line MCF-7/ADM, which were selected subsequently with Blasticidin and MDR1 gene silenced cells including MCF-7/RNAi and MCF-7/ADM/RNAi were obtained. MDR1 mRNA level was measured by quantitative RT-PCR. The expression and function of P-glycoprotein（P-GP） were measured by flow cytometry. The drug sensitivity was evaluated by MTr assay. Results Two stably transducted selected cell lines, MCF-7/RNAi and MCF-7/ADM/RNAi, were obtained after being selected with 10 g/mL Blasticidin for 12 days. The relative expression levels of MDR1 mRNA in four cell lines,including MCF-7,MCF-7/ RNAi,MCF-7/ADM and MCF-7/ADM/RNAi were 1,0. 13,17.14 and 2. 01 respectively; P-GP expression levels were （ 1.5 ±0. 3 ） %, （ 1.2 ± 0. 2） %, （89.4±3.6 ） % and （ 16.3 ±1.9） %, respectively ; the retention of cellular rhodamine 123 were （92. 4 ± 3.1 ） %, （90. 6 ±4. 0 ） %, （ 13.6 ±1.6 ） %, and （ 72. 4 ± 2. 8 ） %, respectively. IC50 of four cell lines for ADM were 0. 90,0. 92,19. 61 and 4. 04 μg/mL,respectively. Conclusion The data indicate that MDR1 gene stably silenced by lentivirals vector can effectively reverse muhidrug resistance of human breast cancer cells.