短发夹状RNA抑制GCS基因在人乳腺癌细胞的表达及逆转其多药耐药

Inhibition of glucosylceramide synthase expression and reversal of multidrug resistance in human breast cancer cells by shRNA

目的构建葡萄糖神经酰胺合成酶(GCS)基因的短发夹状RNA(shRNA),观察其对人耐药乳腺癌细胞GCS表达的抑制及多药耐药的逆转作用。方法设计2对GCS基因编码的反向重复序列,中间间隔6个nt,体外合成并克隆至载体pSUPER。转染乳腺癌细胞,Westernblot和免疫细胞化学方法测定GCS表达。Westernblot、比色法检测细胞内caspase-3的表达,流式细胞仪分析细胞凋亡率。四氮唑盐(MTT)法检测半数细胞抑制浓度。结果酶切和DNA测序证实重组质粒构建成功。转染后GCS蛋白含量分别下降80%和82%,GCS表达阳性率降低为18·1%和16·7%。caspase-3表达和活性均显著增强,细胞凋亡率明显增加,细胞对化疗药物耐药性有明显下降。结论shRNA可有效抑制人耐药乳腺癌细胞中GCS表达,并可提高其对多种化疗药物的敏感性。

Objective To construct the plasmid containing short hairpin RNA （shRNA） of GCS in order to suppress the expression of GCS and to reverse the multidrug resistance in drug-resistant breast cancer cells. Methods Two reverse repeated motifs of sequence targeting GCS with 6 bp spacer were designed and synthesized in vitro, then they were inserted into the plasmid pSUPER. The recombinant plasmids were transfected into human breast cancer cells. GCS expression was detected by Western blot and immunocytochemistry, caspase-3 expression and its activity were assessed using Western blot and colorimetry, flow cytometry was performed to analyze the ratio of apoptosis. Results MTr method was used to evaluate the 50% inhibition concentration. Enzyme digestion analysis and DNA sequencing confirmed that the recombinant plasmids were successfully constructed. GCS protein content decreased 80% , 82% respectively after transfection with recombinant plasmids. The positive rate of GCS expression reduced to 18. 1%, 16.7% respectively, caspase-3 expression and its activity were significantly higher and the apoptosis of cells increased dramatically. The drug resistance of breast cancer cells to antineoplastic drugs declined significantly. Conclusion shRNA can suppress GCS expression in human drug-resistant breast cancer cells effectively and restore the sensitivity to several antineoplastic drugs.