正常和低氧环境下转染beclin-1对乳腺癌细胞BT-549的抑制作用

Inhibition of beclin-1 on breast cancer cell line BT-549 under normal and hypoxic conditions

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 effect of gene beclin-1 on the proliferation, autophagy and apoptosis of triple-negative breast cancer（TNBC） cell line BT-549 under both normal and hypoxic conditions and its influence on the cell migration ability.Methods Eukaryotic vector pDS-RED-Cl-beclin-1 were transfected into human TNBC cell line BT-549 with lipofectamine 2000,which served as experimental group, with pDS-RED-Cl transfected BT-549 cells as negative control, untransfected BT-549 cells as blank control. beclin-1 mRNA and protein were detected by RT-PCR and western blot respectively. The level of proliferation was evaluated by 3-（4,5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） method, the incidence of autophagy was studied by fluorescence microscopy after staining with acridine orange, flow cytometry was applied to identify the induction of the apoptosis and cell scratch test was performed to evaluate the cell migration ability. The results in RT-PCR and Western blot analysis and the proliferation rate measured by MTT were analyzed by single factor analysis of variance, the autophagy and apoptosis rate were analyzed by chi-square test.Results Gene beclin-1 was successfully transfected into BT-549 cells. The expression of beclin-1 mRNA and protein increased evidently after the transfection. Under both normal and hypoxic conditions, the proliferation was inhibited （48 h： P1=0.002,P2=0.000; 72 h： P1=0.006,P2=0.001; P1： normal condition, P2 ： hypoxic condition） and the interaction between beclin-1 and hypoxic stimulation was significant （P=0.016）, which meant that they enhanced the inhibition effect of each other on the cell proliferation. The incidence of autophagy was higher after the transfection（P1=0.004,P2=0.001）,and the transfection of gene beclin-1 also leaded to more apoptosis（P1=0.045,P2=0.008）,meanwhile the cell migration ability was reduced.Conclusion Transfection of beclin-1 inhibits proliferation and increases the incidence of autophagy and apoptosis in TNBC cell line BT-549 under both normal and hypoxic conditions, and reduces the cell migration ability.