反义寡核苷酸阻断乳腺癌细胞中葡萄糖神经酰胺合成酶的表达和意义

Antisense oligonucleotides block glucosylceramide synthase expression in breast cancer cells and its significance

目的探讨靶向葡萄糖神经酰胺合成酶(glucosylceramide synthase,GCS)的基因试剂MBO-asGCS逆转乳腺癌多药耐药的作用机制及其意义。方法MBO-asGCS处理体外培养的人乳腺癌细胞MCF-7及其耐药型MCF-7/AdrR,采用细胞毒性分析测定MBO-asGCS对MCF-7-AdrR存活率的影响,RT-PCR和Western印迹分析对耐药型细胞GCS的表达变化,检测对caspase 3/7的活性影响及流式细胞仪测定MBO-asGCS对耐药型细胞的凋亡情况。结果MBO-asGCS转染人耐药型乳腺癌细胞MCF-7/AdrR后,与未转染组细胞的IC50分别是0.18μmol/L和12.50μmol/L,药物敏感性提高了69倍;MBO-asGCS抑制GCS的mRNA表达70%,减少GCS蛋白表达39%(P<0.01);转染MBO-asGCS后耐药型细胞的caspase 3/7活性上升53%(P<0.05),细胞凋亡率上升5.6倍(P<0.01)。结论GCS过表达是耐药型乳腺癌细胞的特征,靶向人GCS的反义寡核苷酸可直接抑制GCS的过表达,诱导耐药细胞凋亡,有效逆转其耐药性,GCS可能是治疗耐药型乳腺癌的一个潜在靶点。

Objective To investigate the effect of antisense oligonucleotides targeting human glucosylceramide synthase and study its significance on reversing multidrug-resistance in breast adenocarcenoma cells. Methods MBO-asGCS （ antisense GCS, mixed-backbone oligonucleotides） were transfected to human breast cacer cell MCF-7 and its drug-resistant ceil MCF-7/AdrR. Cell viability assay was performed to assess the cytotoxic influence on MBO-asGCS, while RT-PCR and Western blot were adapted to evaluate the expression of GCS mRNA and protein levels respectively. The activity of caspase 3/7 was measured by caspase kit, and flow cytometry analysis was performed to detect the ratio of apoptosis. Results The ICs0 of MCF-7/AdrR treated with MBO-asGCS and vehicle was 0.18 μmol/L and 12.5μmol/L, respectively. MBO-asGCS specifically suppressed GCS mRNA 70% and protein expression 39 % in MCF-7-AdrR cell ; whereas the activity of caspase 3/7 dramatically increased 53 % （ P 〈 0.05 ） and the ratio of apoptosis for MCF-7-AdrR cell treated with MBO-asGCS was up to 5.6-fold, compared with Doxorubicin control. Conclusion GCS overexpression is a character of MDR breast cancer cell, antisense oligonucleotides targeting human GCS can markedly restore cellular sensitivity to Doxorubicin, significantly suppress GCS mRNA and protein expression, and increase apoptosis of drug-resistant cells. Reversal of drug-resistance demonstrates GCS is a potential target for the treatment of chemotherapy-refractory breast cancer.