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

doi:10.3877/cma. j. issn.1674-0807.2013.01.004

Abstract

Abstract:

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-C1-beclin 1 by Lipofectamine^TM 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 cells were cultured in 1640 medium with and without 10% fetal bovine serum (FBS), respectively. The growth rate and cell survival were detected by MTT 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(χ² =7.7, P=0.00).

Conclusion

Transfecting beclin 1 to induce autophagy in triple-negative breast cancer BT-549 cells can inhibit cell proliferation in normal condition and improve cell survival in starvation.

Key words: breast neoplasms, beclin 1, autophagy

Meng-chuan WANG, Ai-guo WU, Ri-wei WANG, Hong-jun YUAN, Xue-xiao LI, Bin HU, Guo-li SHAO, Shu-feng JI, Kai WU, Xu-long FAN, Lin-hai ZHENG. Influence on cell survival by transfecting beclin 1 to induce autophagy in triple-negative breast cancer BT-549 cells[J]. Chinese Journal of Breast Disease(Electronic Edition), 2013, 07(01): 12-19.

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Figures/Tables 11

References 14

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组别	增殖率(%)					F值	P值
组别	24h	48h	72h	96h	合计	F值	P值
空白组	120.3±0.8	149.3±0.9	180.6±3.6	264.7±5.6	178.7±55.3	2065.1	0.00
脂质体组	115.2±3.7	135.2±2.3	179.6±5.3	259.5±1.3	172.4±56.7	2021.1	0.00
空载体组	123.1±3.9	142.7±7.3	176.7±1.7	261.7±1.8	176.1±54.4	1219.1	0.00
目的基因组	122.0±1.4	144.4±1.3	163.3±2.3	203.3±6.6	158.2±30.7	541.1	0.00
合计	120.2±4.0	142.9±6.3	175.0±7.8	247.0±26.4	171.4±50.3	5936.1^a	0.00^a
F值	9.2	13.4	31.5	260.7	112.1^a	103.6^b
P值	0.00	0.00	0.00	0.00	0.00^a	0.00^b

组别	增殖率(%)					F值	P值
组别	24h	48h	72h	96h	合计	F值	P值
空白组	120.3±0.8	149.3±0.9	180.6±3.6	264.7±5.6	178.7±55.3	2065.1	0.00
脂质体组	115.2±3.7	135.2±2.3	179.6±5.3	259.5±1.3	172.4±56.7	2021.1	0.00
空载体组	123.1±3.9	142.7±7.3	176.7±1.7	261.7±1.8	176.1±54.4	1219.1	0.00
目的基因组	122.0±1.4	144.4±1.3	163.3±2.3	203.3±6.6	158.2±30.7	541.1	0.00
合计	120.2±4.0	142.9±6.3	175.0±7.8	247.0±26.4	171.4±50.3	5936.1^a	0.00^a
F值	9.2	13.4	31.5	260.7	112.1^a	103.6^b
P值	0.00	0.00	0.00	0.00	0.00^a	0.00^b

组别	存活率(%)					F值	P值
组别	12h	24h	36h	48h	合计	F值	P值
空白组	99.2±1.8	93.2±6.5	85.0±2.2	75.7±3.2	88.3±9.7	41.6	0.00
脂质体组	96.0±2.9	91.5±3.9	83.2±1.5	69.9±0.9	85.1±10.4	120.7	0.00
空载体组	97.8±1.3	93.2±3.5	81.6±1.8	71.5±2.5	86.0±10.7	144.2	0.00
目的基因组	98.5±2.3	96.9±2.2	91.5±2.0	85.7±3.3	93.2±5.6	32.7	0.00
合计	97.9±2.3	93.7±4.5	85.3±4.3	75.7±6.8	88.2±9.7	273.5^a	0.00^a
F值	2.6	1.7	31.4	43.4	37.5^a	5.9^b
P值	0.08	0.20	0.00	0.00	0.00^a	0.00^b

组别	存活率(%)					F值	P值
组别	12h	24h	36h	48h	合计	F值	P值
空白组	99.2±1.8	93.2±6.5	85.0±2.2	75.7±3.2	88.3±9.7	41.6	0.00
脂质体组	96.0±2.9	91.5±3.9	83.2±1.5	69.9±0.9	85.1±10.4	120.7	0.00
空载体组	97.8±1.3	93.2±3.5	81.6±1.8	71.5±2.5	86.0±10.7	144.2	0.00
目的基因组	98.5±2.3	96.9±2.2	91.5±2.0	85.7±3.3	93.2±5.6	32.7	0.00
合计	97.9±2.3	93.7±4.5	85.3±4.3	75.7±6.8	88.2±9.7	273.5^a	0.00^a
F值	2.6	1.7	31.4	43.4	37.5^a	5.9^b
P值	0.08	0.20	0.00	0.00	0.00^a	0.00^b

组别	凋亡率	坏死率	G₀/G₁期	G₂/M期	S期
空白组	2.8±0.0	10.0±0.4	57.6±0.1	26.0±0.2	16.5±0.3
脂质体组	2.8±0.0	11.7±0.2^a	55.4±0.3^a	28.1±0.5^a	16.5±0.2
空载体组	2.8±0.1	10.1±0.2	58.2±0.3	25.8±0.2	16.0±0.1
目的基因组	8.7±0.1^a	5.8±0.2^a	62.5±0.4^a	21.7±0.6^a	15.8±0.2^a
阳性对照组	5.2±0.1^a	3.3±0.0^a	63.6±0.6^a	20.0±0.5^a	14.1±0.3^a
F值	3914.4	607.2	258.8	195.4	55.6
P值	0.00	0.00	0.00	0.00	0.00

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