Beclin 1蛋白增强三阴性乳腺癌细胞对5-Fu敏感度的研究

doi:10.3971/j.issn.1000-8578.2015.05.003

Beclin 1蛋白增强三阴性乳腺癌细胞对5-Fu敏感度的研究

Beclin 1 Protein Enhances Sensitivity of Triple-negative Breast Cancer Cell Line BT-549 to 5-Fu by Promoting Cell Apoptosis

摘要

摘要: 目的探讨Beclin 1蛋白与三阴性乳腺癌细胞对5-Fu敏感度的关系。方法应用慢病毒介导pLenO-GTP-BECN1表达载体稳定转染至BT-549细胞，嘌呤霉素筛选阳性细胞，设立对照组。使用不同浓度5-Fu处理各组细胞，MTT法比较细胞对5-Fu的敏感度；流式细胞术和Hoechst33342实验检测细胞凋亡情况；Western blot检测细胞凋亡相关蛋白Caspase-3和Caspase-8表达情况。结果成功构建稳定表达Beclin 1蛋白的BT-549细胞株。pLenO-GTP-BECN1组对5-Fu敏感度显著高于其他两组；5-Fu作用48 h后，pLenO-GTP-BECN1组的IC₅₀为（3.54±0.20）μg/ml，显著低于空白载体pLenO-GTP组（14.45±1.81）μg/ml和空白组（79.40±8.34）μg/ml（F=207.902；P<0.00）。5-Fu作用前，pLenO-GTP-BECN1组凋亡率为（12.14±0.76）%，同pLenO-GTP组（13.57±1.04）%和空白组（13.75±1.29）%（F=2.096；P=0.204）之间差异并无统计学意义；5-Fu作用48 h后，pLenO-GTP-BECN1组凋亡率为（51.01±3.26）%，显著高于pLenO-GTP组（42.17±4.6）%和空白组（38.42±2.93）%（F=9.32；P<0.01）；接受5-Fu处理后，pLenO-GTP-BECN1组细胞核凋亡特征改变较其他两组更明显。5-Fu作用后，pLenO-GTP-BECN1组细胞Caspase-3相对表达量为（0.57±0.0028），显著高于pLenO-GTP组（0.42±0.010）和空白组（0.30±0.046）（F=46.134；P<0.01）；pLenO-GTP-BECN1组细胞Caspase-8相对表达量为（0.44±0.0038），显著高于pLenO-GTP组（0.18±0.0095）和空白组（0.27±0.00060）（F=1006.757；P<0.00）。结论 Beclin 1显著提高三阴性乳腺癌细胞对5-Fu的敏感度，其中促进凋亡发生可能是重要机制之一。

Abstract: Objective To investigate the effect of Beclin 1 protein on the sensitivity of triple-negative breast cancer cell line BT-549 to 5-Fu. Methods pLenO-GTP-BECN1 vectors were transfected into BT-549 cells respectively via lentiviral method. Puromycin selection was used to select positive cells. The negative and the blank control groups were established. BT-549 cells of each group were treated with different concentrations of 5-Fu. MTT assay was performed to compare the sensitivity of BT-549 cells to 5-Fu. FCM and Hoechst33342 dying method were employed to investigate cell apoptosis. Western blot was performed to detect the expression of apoptosis-related protein Caspase-3 and Caspase-8. Results BT-549 cells with stable expression of Beclin 1 were successfully established. The pLenO-GTP-BECN1 group was more sensitive to 5-Fu than other two groups. After treated by 5-Fu for 48h, IC50 of 5-Fu in pLenO-GTP-BECN1 group was(3.54±0.20) μg/ml, which was significantly lower than those in pLenO-GTP group(14.45±1.81) μg/ml and blank group (79.40±8.34)μg/ml(F=207.902;P<0.00). Before 5-Fu treatment, the apoptosis rate of pLenO-GTP-BECN1 group was (12.14±0.76)%, which had no significant difference with those of pLenOGTP group(13.57±1.04)% and blank group(13.75±1.29)% (F=2.096; P=0.204);After treated by 5-Fu for 48h, the apoptosis rate of pLenO-GTP-BECN1 group was (51.01±3.26)%, which was significantly higher than those of pLenO-GTP group(42.17±4.6)% and blank group (38.42±2.93)%(F=9.32;P<0.01).The characteristic changes of nucleus apoptosis in pLenO-GTP-BECN1 group were more apparent than those in the other two groups. After 5-Fu treatment, the relative expression of Caspase-3 in pLenO-GTP-BECN1 group was(0.57±0.0028),which was significantly higher than those in pLenO-GTP group(0.42±0.010) and blank group(0.30±0.046)(F=46.134; P<0.01);and the relative expression of Caspase-8 in pLenO-GTP-BECN1 group was(0.44±0.0038),which was significantly higher than those in pLenO-GTP group (0.18±0.0095) and blank group(0.27 ±0.00060)(F=1006.757; P<0.00). Conclusion Beclin 1 greatly enhances the sensitivity of BT-549 cells to 5-Fu, in which the promotion of apoptosis may be an important mechanism.

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