| 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 |
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| 引用本文： | 孙妍琳,周庚寅,孙建国,林晓燕,李红,白艳花,张翠娟. 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[J]. 基础医学与临床, 2006, 26(7): 704-709 |
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| 作者姓名： | 孙妍琳 周庚寅 孙建国 林晓燕 李红 白艳花 张翠娟 |
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| 作者单位： | 山东大学,齐鲁医院,病理科,山东,济南,250012;莱阳市山前店医院,山东,莱阳,265210;山东省立医院病理科,山东,济南,250012 |
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| 基金项目： | 中国科学院资助项目;山东省科技厅资助项目;高等学校博士学科点专项科研项目 |
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| 摘 要： | 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。
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| 关 键 词： | 短发夹状RNA 乳腺癌 葡萄糖神经酰胺合成酶 凋亡 |
| 文章编号： | 1001-6325(2006)07-0704-06 |
| 收稿时间： | 2005-06-16 |
| 修稿时间： | 2006-02-07 |
Inhibition of glucosylceramide synthase expression and reversal of multidrug resistance in human breast cancer cells by shRNA |
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| SUN Yan-lin,ZHOU Geng-yin,SUN Jian-guo,LIN Xiao-yan,LI Hong,BAI Yan-hua,ZHANG Cui-juan. Inhibition of glucosylceramide synthase expression and reversal of multidrug resistance in human breast cancer cells by shRNA[J]. Basic Medical Sciences and Clinics, 2006, 26(7): 704-709 |
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| Authors: | SUN Yan-lin ZHOU Geng-yin SUN Jian-guo LIN Xiao-yan LI Hong BAI Yan-hua ZHANG Cui-juan |
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| Affiliation: | 1. Department of Pathology, Qilu Hospital, Shandong University, Jinan 250012; 2. Shangian Dian Hospital of Laiyang, Laiyang 265210; 3. Department of Pathology, Shendeng Procincial Hospital, Jinan 250021, China |
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| Abstract: | 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 MTT 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 higherand 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. |
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| Keywords: | small hairpin RNA carcinoma breast cells glucosylceramide synthase apoptosis |
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