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目的研究转染beclin-1基因对三阴性乳腺癌(TNBC)细胞BT-549在正常及低氧环境下的增殖抑制情况,促自噬和促凋亡作用,以及其对细胞迁移能力的影响。方法利用脂质体将真核载体pDS-RED-C1-beclin-1转染进入BT-549细胞作为实验组,空白组细胞和转染pDS-RED-C1空质粒的细胞分别作为空白对照组和阴性对照组,用RT-PCR检测转染后beclin-1mRNA表达,Western blot检测转染后蛋白表达。在正常及低氧环境下分别培养后用四甲基偶氮唑盐(MTT)法测细胞增殖率,吖啶橙染色后用荧光显微镜观察细胞自噬情况,流式细胞仪检测细胞凋亡百分比。通过细胞划痕试验研究转染beclin-1基因对细胞迁移能力的影响。RT-PCR和Western blot结果及MTT所测增殖率结果用单因素方差分析,自噬和凋亡的差异采用卡方检验分析。结果 beclin-1基因被成功转染进细胞;转染后目的基因组beclin-1mRNA和蛋白表达均高于对照组,72h最明显。MTT测定结果:正常与低氧环境下实验组48h和72h增殖率均低于对照组(48hP_1=0.002,P_2=0.000;72hP_1=0.006,P_2=0.001;P_1和P_2分别为正常与低氧环境下统计结果),beclin-1与低氧刺激有明显交互作用(P=0.016),即二者能增强彼此对细胞增殖率的抑制作用。吖啶橙染色镜下观察正常和低氧环境下实验组自噬发生率明显高于空白对照组(P_1=0.004,P_2=0.001),阴性对照组与空白对照组间差异无统计学意义(P>0.050)。流式细胞仪检测正常和低氧环境下实验组凋亡发生率均高于空白对照组(P_1=0.045,P_2=0.008)。体外划痕试验发现转染beclin-1后细胞迁移能力降低。结论在正常和低氧环境下,转染beclin-1基因可抑制TNBC细胞BT-549增殖,促进细胞发生自噬及凋亡,并且降低其迁移能力。
OBJECTIVE: To investigate the effects of beclin-1 gene transfection on the proliferation of triple negative breast cancer (TNBC) cells BT-549 under normal and hypoxia conditions, the role of autophagy and proapoptosis, and their effects on cell migration. Methods The eukaryotic vector pDS-RED-C1-beclin-1 was transfected into BT-549 cells by lipofectamine. The blank cells and the cells transfected with pDS-RED-C1 empty plasmid were used as blank control group and Negative control group, beclin-1mRNA expression was detected by RT-PCR, Western blot detection of protein expression after transfection. Under normal and hypoxia conditions, the cell proliferation rate was measured by MTT assay, autophagy was observed by fluorescence microscopy after acridine orange staining, the percentage of apoptosis was detected by flow cytometry . The effect of beclin-1 gene transfection on cell migration was investigated by cell scratch assay. RT-PCR and Western blot results and MTT measured proliferation results using one-way ANOVA, differences in autophagy and apoptosis using chi-square test analysis. Results The beclin-1 gene was successfully transfected into the cells. The expression of beclin-1 mRNA and protein in the transfected cells was higher than that in the control group, which was the most obvious at 72h. The results of MTT assay showed that the proliferation rates at 48h and 72h in experimental group were lower than those in control group (48hP_1 = 0.002, P_2 = 0.000; 72hP_1 = 0.006, P_2 = 0.001) in normal and hypoxic conditions; P_1 and P_2 were normal and hypoxic Statistical results), beclin-1 and hypoxia stimulation had a significant interaction (P = 0.016), that both can enhance each other on cell proliferation inhibition. Acridine orange staining under normal and hypoxic conditions in the experimental group was significantly higher than the incidence of autophagy in the control group (P 1 = 0.004, P 2 = 0.001), the negative control group and the blank control group, the difference was not statistically significant (P > 0.050). The apoptosis rate of experimental group was higher than that of blank control group (P 1 = 0.045, P 2 = 0.008) by flow cytometry in normal and hypoxic environment. In vitro scratch test showed that the transfection of beclin-1 cell migration decreased. Conclusion Under normal and hypoxia conditions, the transfection of beclin-1 gene can inhibit the proliferation of TNBC cells BT-549, promote cell autophagy and apoptosis, and reduce its migration ability.