BCRP过表达对乳腺癌细胞增殖和凋亡的影响

Influence of BCRP overexpression on proliferation and apoptosis of human breast cancer cells

目的探讨乳腺癌耐药蛋白(BCRP)基因过表达对人乳腺癌细胞MDA-MB-435S增殖和凋亡的影响。方法取对数生长期的MDA-MB-435S细胞,将其随机分为三组,阳性转染组将真核表达质粒pc DNA3.1/BCRP经双酶切和测序鉴定后转染MDA-MB-435S细胞,阴性对照组同步转染2μg pc DNA3.1的空质粒,空白对照组用2 m L原培养液正常培养。采用RT-PCR法和Western blotting法检测BCRP mRNA和蛋白表达。转染后48 h,每组再分设7个丝裂霉素(MMC)浓度(0.01、0.10、2.50、5.00、10.00、20.00、40.00μmol/L)组,每个浓度组3个复孔,加入相应浓度的MMC 10μL,采用CCK-8法检测细胞增殖情况;流式细胞术分析细胞凋亡情况。结果重组真核表达质粒pc DNA3.1/BCRP经双酶切和测序证实该质粒构建正确;阳性转染组转染24、48、72 h后,MDA-MB-435S细胞中BCRP mRNA和蛋白表达均明显增加(P均<0.05);与阴性对照组比较,阳性转染组转染pc DNA3.1/BCRP 48 h后,细胞凋亡率无统计学差异(P>0.05);CCK-8结果显示,化疗药物MMC作用24 h后,与阴性对照组比较,阳性转染组转染pc DNA3.1/BCRP 48h后细胞存活率显著增加(P<0.05)。结论 BCRP过表达可促进人乳腺癌细胞MDA-MB-435S增殖,但对细胞凋亡无明显影响。

Objective To investigate the effect of breast cancer resistance protein（ BCRP） overexpression on cell proliferation and apoptosis of breast cancer cells MDA-MB-435 S. Methods MDA-MB-435 S cells in the logarithmic phase were randomly divided into three groups： the positive transfection group,in which the eukaryotic expression plasmid pc DNA3. 1/BCRP was identified by restriction analysis and sequencing and then was transfected into MDA-MB-435 S cells,the positive control group,in which the cells were transfected with 2 μg pc DNA3. 1 empty plasmid at the same time,and the blank control group,in which the cells were cultured with 2 m L nutrient solution. The mRNA and protein expression of BCRP was determined by RT-PCR and Western blotting,respectively. After 48-hour transfection,7 mitomycin（ MMC）concentration groups（ 0. 01,0. 10,2. 50,5. 00,10. 00,20. 00,40. 00 μmol/L） were set up in each group,each concentration group had 3 complex holes which were added with 10 μL MMC. Cell counting kit-8（ CCK-8） and flow cytometry were used to determine the influence of BCRP on cell proliferation and apoptosis of MDA-MB-435 S cells,respectively. Results Restriction analysis and sequencing proved that recombinant plasmid pc DNA3. 1/BCRP was constructed correctly.Both the expression of BCRP mRNA and protein was up-regulated in MDA-MB-435 S cells after transfection 24,48 or 72 h in the positive transfection group（ P〈0. 05）. No significant difference was found between the positive transfection group and the negative control group（ P〈0. 05）. After MMC treatment of 24 hours,CCK-8 showed that the cell survival rate in the transfection group was significantly higher than that of the negative control group（ P〈0. 05）. Conclusion BCRP overexpression promotes the proliferation of MDA-MB-435 S cells,but has no significant effect on apoptosis.