摘要
目的利用RNA干扰技术抑制乳腺癌耐药细胞株MCF-7／ADR乳腺癌耐药蛋白（BCRP）基因的表达并提高对阿霉素（ADR）的敏感性。方法针对BCRP的mRNA序列，设计两条RNA干扰链（BCRP-A和BCRP-B），构建出小干扰RNA（siRNA）表达载体pG—BCRP—AsiRNA-增强型绿色荧光蛋白（EGFP）和pG—BCRP-BsiRNA—EGFP。实验分为4组：A、B、C3组分别转染BCRP-A、BCRP-B、HK-siRNA（对照组）；D组转染过程中未加入任何质粒（空白组）。通过反转录-聚合酶链反应（RT-PCR）和Westernblot检测siRNA阻断BCRP基因表达的效率，应用噻唑蓝（MTT）法分析siRNA干扰后MCF-7／ADR的耐药性变化。结果RT—PCR和Westernblot法检测结果表明BCRP—A和BCRP-B两条干扰链干扰效率不一，前者较后者对BCRPmRNA和蛋白质表达抑制率均明显优于后（P〈0．05）；MTr法分析结果说明BCRP-A组对ADR的药物敏感性明显提高，其IC50为（184．6±25．3）ng／ml，与BCRP—B干扰组和对照组比较差异有统计学意义（P〈0．05）。结论RNA干扰技术可用于抑制BCRP基因的表达，并提高对ADR的敏感性，但不同干扰链的干扰效率差异较大。
Objective To inhibit the expression of breast cancer resistance protein （BCRP） gene in breast cancer MCF -7/ADR cell line and to improve the sensitivity to adriamycin. Methods Two small interfering RNAs （ siRNAs ： BCRP - A and BCRP - B） were designed according to the mRNA sequence of BCRP, and the siRNA expression vectors pG - BCRP - A siRNA - enhanced green fluorescent protein （EGFP） and pG - BCRP - B siRNA - EGFP were constructed. Four groups were set up. The MCF - 7/ ADR cells in groups A, B, and C were treated by pG - BCRP - A siRNA - EGFP, pG - BCRP - B siRNA - EGFP and HK - siRNA, respectively. The cells in group D were untreated. Reverse transcriptase - polymer- ase chain reaction （ RT - PCR） and Western blotting were used to verify the efficiency of siRNA in inhibi- ting the expression of BCRP in MCF-7/ADR. Methyl thiazol tetrazolium （MTT） assay was used for ana- lysing the change of adriamycin resistance. Results The RT - PCR and Western blotting assay showed that the interference efficiency of the two interference chains was different, and BCRP - A was significantly better than BCRP- B （P〈0.05）. The results of MTr showed that the ICso of BCRP -A group was （184.6 ± 25. 3 ） ng/ml, and the drug sensitivity of BCRP - A group was significantly higher than the other two groups （P 〈 0.05）. Conclusion RNA interference technology can be used to inhibit the expression of BCRP gene, and improve the sensitivity to adriamycin, but the interference efficiency of different interference chains is different.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2016年第4期933-935,共3页
Chinese Journal of Experimental Surgery
关键词
RNA干扰
乳腺癌
乳腺癌耐药蛋白基因
阿霉素耐药性
RNA interference
Breast cancer
Breast cancer resistance protein gene
Adria- mycin resistance
