沉默Bmi-1基因表达对MCF-7细胞阿霉素敏感性的影响

Silencing of Bmi-1 Gene Can Effect Doxorubicin Sensitive of MCF-7 Cells

目的研究Bmi-1对MCF-7细胞阿霉素敏感性的影响及机制。方法阿霉素处理MCF-7／Bmilsi、MCF-7／GFPsi和MCF一7细胞株，M1Tr法检测阿霉素的IC50；DAPI检测阿霉素处理后细胞的凋亡，计算凋亡指数（apoptosisindex，AI）；Western印迹检测相关蛋白P53，phospho—Akt（Ser473）（pAkt），totle—Akt（tAkt），Bcl-2，Bax的表达。结果阿霉素处理72h的MCF-7／Bmi．1si组生长抑制率明显高于MCF-7和MCF-7／GFPsi组，MCF-7／Bmilsi组的IC50为（0．15±0．02）μg／ml，而MCF-7组和MCF-7／GFPsi组的IC50分别为（0．87±0．06）μg/ml和（0．81±0．02）μg/ml（P〈0．05）。阿霉素处理48h后用DAPI检测凋亡发现，MCF-7／Bmi．lsi＋doxorubiein组可见大量凋亡细胞，而MCF-7＋doxorubicin和MCF-7／GFPsi＋doxo—rubicin组出现较少的凋亡细胞，MCF-7／Bmi-1si＋doxorubicin组凋亡指数明显高于对照组（P〈0．05）。进一步研究发现：MCF-7／Bmi．1si＋doxorubicin组与MCF-7＋doxorubiein及MCF-7／GFPsi＋doxorubiein组相比，P53表达量增加，tAkt表达未发生改变，而pAkt的表达明显减少，另外，Bcl-2表达量减少而Bax表达量增加，差异具有显著性（P〈0．05）。结论沉默Bmi—l基因表达能增加MCF-7细胞对阿霉素的敏感性，增加阿霉素引起的凋亡。

Objective This study aim to research effect and mechanism of silencing Bmi-1 gene on MCF-7 doxorubicin sensitivity. Methods The three cell lines MCF-7, MCF-7/GFPsi and MCF-7/Bmisi were treated with doxorubicin. MTY assay was performed to evaluate the IC50 values of doxorubicin. Apoptosis was detected by DAPI staining and AI was caculated. Western blot was performed to evaluate the expression of related genes （ p53, phospho-Akt （ ser473 ） （ pAkt）, totle- Akt （ tAkt）, Bcl-2 and Bax） were studied by western blot. Results After treatment with doxorubicin for 72 h, cells in MCF-7/Bmi-lsi showed higher IR than that in MCF-7 and MCF-7/GFPsi. The IC50 value of doxorubicin in MCF-7/Bmi-lsi was 0. 15 ±0. 02 μg/ml, while the IC50 value of doxorubi- cin in MCF-7 and MCF-7/GFPsi were 0. 87 ±0. 06 μg/ml and 0. 81 ±0. 02 μg/ml. There were signifi- cant differences between the IC50 of MCF-7/Bmi-lsi and that of controls. Apoptosis was examined after treating with doxorubicin for 48 h. AI in MCF-7/Bmi-lsi cells treated with doxorubicin was great than the controls, and apoptosis cells were frequently observed. Compared with MCF-7 and MCF-7/GFPsi, there were increased expression of p53 and decreased Akt phosphorylation without affecting tAkt expression. There were decreased expression of Bcl-2 and increasion of Bax, and Bcl-2/Bax ratio was down-regulated （P 〈 0. 05 ） . Conehlsion The silencing of Bmi-l can render MCF-7 cells more sensitive to doxombicin and induce a significantly higher percentage of apoptosis cells.