Sun Yan-lin, Zhou Geng-yin, Li Kai-nan, Li Wen-tong, Song Xian-rang, Gao Peng
Department of Pathology, Qilu Hospital, University of Shandong, Jinan 250012, China.
Zhonghua Yi Xue Za Zhi. 2005 Mar 2;85(8):518-21.
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.
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.
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).
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.
构建葡糖神经酰胺合酶（GCS）特异性小干扰RNA（siRNA）表达载体，并研究该siRNA对乳腺癌细胞中GCS表达及耐药性的抑制作用。
设计两条GCS基因特异性siRNA，并克隆至表达载体pSUPER中，构建质粒pSUPER-GCS1和pSUPER-GCS2。培养人阿霉素（ADM）耐药的MCF-7/ADR乳腺癌细胞系和人阿霉素敏感的MCF-7乳腺癌细胞系，分别用pSUPER-GCS1、pSUPER-GCS2及空载体pSUPER作为对照进行转染。采用RT-PCR检测GCS mRNA的表达，通过流式细胞术观察GCS蛋白的表达。采用MTT法评估ADM对MCF-7/ADR细胞的半数抑制浓度。通过流式细胞术检测细胞凋亡率。
双酶切分析及DNA测序证实pSUPER-GCS1和pSUPER-GCS2构建成功。pSUPER-GCS1和pSUPER-GCS2转染MCF-7/ADR细胞48小时后，GCS蛋白阳性率分别为8.3%±1.0%和9.2%±0.8%，显著低于转染前（68.3%±0.6%），抑制率分别为89.4%和88.5%（均P<0.01）。pSUPER-GCS1和pSUPER-GCS2转染MCF-7/ADR细胞48小时后，对ADM的相对敏感性逆转率分别为93.7%和91.6%。流式细胞术显示，MCF-7/ADR细胞转染前凋亡率为0.80±0.06，pSUPER-GCS1转染后为15.38±1.16，pSUPER-GCS2转染后为13.92±1.73（均P<0.05），空载体转染细胞凋亡率为0.87±0.12（P>0.05）。
成功构建了GCS特异性小干扰RNA表达载体，该载体可抑制GCS表达，并通过提高耐药细胞的凋亡率逆转乳腺癌细胞的多药耐药性。
Zotero 插件