葡萄糖糖基神经酰胺合成酶特异性小干扰RNA表达载体的构建及其逆转乳腺癌细胞耐药的研究

目的　构建葡萄糖糖基神经酰胺合成酶(GCS)小干扰RNA表达载体,观察其对乳腺癌耐药细胞(MCF- 7 /ADR)GCS基因表达和耐药性的影响。方法　设计合成2对GCS基因的特异性siRNA,分别定向克隆入真核表达载体pSUPER,构建pSUPER -GCS1和pSUPER -GCS2重组质粒,脂质体LipofectAMINE2000介导转染MCF -7 /ADR细胞,空载体pSUPER转染作为对照,逆转录聚合酶链反应(RT- PCR)分析GCSmRNA的表达,流式细胞仪观察GCS蛋白的表达,四氮唑盐法检测阿霉素对MCF -7 /ADR细胞的半数抑制浓度(IC50 ),流式细胞技术检测细胞凋亡率的改变。结果　酶切分析和测序证实成功构建了针对GCS的siRNA表达载体pSUPER -GCS1和pSUPER GCS2。两对重组质粒均可特异性抑制GCS基因表达,转染后48hGCSmRNA抑制率分别为89 .4%、88. 5%,GCS蛋白含量分别下降为8 .3%±1 .0%, 9. 2%±0 .8%,四氮唑盐法显示重组质粒对阿霉素耐药性相对逆转率分别为93. 7%、91. 6%,明显提高了乳腺癌细胞对于阿霉素的药物敏感性,流式细胞仪结果表明细胞凋亡率增加为15. 38±1 .16, 13. 92±1 .73,而空载体对照组无以上作用。结论　GCS特异性siRNA表达载体构建成功,且有效抑制了GCS基因表达,并通过诱导耐药细胞凋亡率增加,逆转乳腺癌细胞多药耐药。

Construction of glucosylceramide synthase-specific siRNA expression vector and its efficiency in reversal of drug resistance in breast carcinoma cells

OBJECTIVE: To construct a glucosylceramide synthase (GCS)-specific small interfering RNA (siRNA) expression vector and to investigate the inhibitory effect of this siRNA on GCS expression and drug resistance in breast carcinoma cells. METHODS: Two GCS gene-specific siRNAs were designed and cloned into the expression vector pSUPER to generate the plasmids pSUPER-GCS1 and pSUPER-GCS2. Human adriamycin (ADM)-resistant breast carcinoma cells of the line MCF-7/ADR and human adriamycin-sensitive breast carcinoma cells of the line MCF-7 were cultured and transfected with pSUPER-GCS1, pSUPER-GCS2, and blank vector pSUPER as controls. The expression of GCS mRNA was assayed by RT-PCR and the expression of GCS protein was observed by flow cytometry. The 50% inhibition concentration of ADM on MCF-7/ADR cells was evaluated by MTT method. Flow cytometry was performed to determine the ratio of apoptosis. RESULTS: Double enzyme digestion analysis and DNA sequencing confirmed that pSUPER-GCS1 and pSUPER-GCS2 were successfully constructed. The GCS protein positive rate of the MCF-7/ADR cells 48 hours after transfection with pSUPER-GCS1 and pSUPER-GCS2 were 8.3% +/- 1.0% and 9.2% +/- 0.8% respectively, significantly lower than that before transfection (68.3% +/- 0.6%), with a inhibition rate of 89.4% and 88.5% respectively (both P < 0.01). Forty-eight hours after transfection with pSUPER-GCS1 and pSUPER-GCS2, the relative reversal rates of sensitivity to ADM of the MCF-7/ADR cells were 93.7% and 91.6%. Flow cytometry showed that the apoptotic rate of the MCF-7/ADR cells was 0.80 +/- 0.06 before transfection, 15.38 +/- 1.16 after transfection with pSUPER-GCS1 and 13.92 +/- 1.73 after transfection with pSUPER-GCS2 (both P < 0.05), and was 0.87 +/- 0.12 in the cells transfected with blank vector (P > 0.05). CONCLUSION: A GCS-specific small interfering RNA expression vector has been constructed successfully that suppresses the GCS expression and reverses the multidrug resistance in breast carcinoma cells by increasing the ratio of apoptosis in drug-resistant cells.