转染beclin1基因诱导自噬对三阴性乳腺癌BT-549细胞生长的影响

Influence on cell survival by transfecting beclin 1 to induce autophagy in triple-negative breast cancer BT-549 cells

目的探讨转染beclin1基因诱导三阴性乳腺癌BT-549细胞发生自噬及其对细胞生长的影响。方法将BT-549细胞分为4组,包括空白组、空载体组、脂质体组和目的基因组,并以MDA-MB-231细胞作为阳性对照组。采用Lipofectamine^(TM)2000携带含有beclin1基因的质粒转染BT-549细胞,分别于转染后24、36、48、60h用荧光显微镜观察转染效率;于转染后48h利用RT-PCR检测各组细胞beclin1 mRNA表达情况;转染后各组细胞分别在含10%胎牛血清1640培养基和无胎牛血清1640培养基内培养,并分别于24、48、72、96h和12、24、36、48h采用MTT法检测细胞的增殖率和存活率;各组细胞在无胎牛血清1640培养基内培养24h后,采用流式细胞仪检测其细胞凋亡和细胞周期情况;各组细胞在无胎牛血清1640培养基内生长36h后,经吖啶橙染色观察并比较发生自噬的细胞数。MTT数据按重复测量的方差分析进行统计,其余计量资料按单因素方差分析进行统计,计数资料按照R×C表资料的χ~2检验进行统计分析。结果 48h时携带目的基因的质粒经脂质体转染BT-549细胞的效率最高;目的基因组细胞beclin1 mRNR水平显著高于空白组、脂质体组、空载体组和阳性对照组(P<0.01);在含10%胎牛血清1640培养基内,目的基因组细胞的增殖率显著低于空白组(F=112.1,P=0.00),而在无胎牛血清1640培养基内,目的基因组细胞36h和48h的存活率显著高于空白组(F=37.5,P=0.00);目的基因组细胞的凋亡比例增加(F=3914.4,P=0.00),且细胞处于G0/G1期的比例升高(F=258.8,P=0.00);目的基因组细胞发生自噬的数量显著增加(χ~2=7.7,P=0.00)。结论向三阴性乳腺癌BT-549细胞转染beclin1基因,可提高细胞的自噬水平。该作用可抑制细胞在正常培养环境下的增殖,但对低营养环境下的细胞存活具有保护作用。

Objective To investigate the effect of autophagy on cell growth in triple-negative breast cancer BT-549 cells by transfecting beclin 1. Methods BT-549 cells were divided into 4 groups,including the blank group,the empty vector group, the liposome group and the target gene group （beclin 1 group） ;besides, MDA-MB-231 cells served as the positive control group. BT-549 cells were transfected with pDsRed-Cl-beclin 1 by LipofectamineTM 2000 and the efficiency was evaluated by fluorescence at 24, 36, 48, 60 h after transfection ; beclin 1 mRNA levels in each group were examined by RT-PCR at 48 h after transfection. Then the transfected ceils were cultured in 1640 medium with and without 10% fetal bovine serum （ FBS）, respectively. The growth rate and cell survival were detected by MTr at 24, 48, 72, 96 h or 12, 24, 36, 48 h, respectively. The cell apoptosis and distribution of cell cycle were determined by flow cytometry after 24 h of starvation; the numbers of cells containing autophagic vesicles were calculated by acridine orange staining after the culture in serumdeprived 1640 medium for 36 h. Measurement data were calculated by repeated measurement one-way ANOVA; categorical data were calculated by chi-square test. Results The transfection efficiency was the highest at 48 h. RT-PCR showed that the mRNA level in beclin 1 was higher than that in other groups（P〈0. 01 ）. MTT showed that in 1640 medium containing 10% FBS, the cell proliferation rate in beclin 1 group was lower than that in control group （F= 112. 1, P= 0.00） while in serum-deprived 1640 medium, the cell survival at 36, 48 h in beclin 1 group was significantly higher than that in control group （F = 37.5, P = 0. 00）. Flow cytometry showed that the percentage of apoptotic cells in beclin 1 group （F= 3914. 4, P=0. 00） and the cells in G0/G1 phase （F= 258.8, P = 0. 00 ） increased; the number of cells with autophagic vesicles stained by acridine orange obviously increased in beclin 1 group （ χ^2 = 7. 7, P = 0. 00）. Conclusion Transfecting beclin l to induce autophagy in triple-negative breast cancer BT-549 cells can inhibit cell proliferation in normal condition and improve cell survival in starvation.