Effect of DNA polymerase θ expression on proliferation and early apoptosis of breast cancer cell line MCF-7 invitro

doi:10.3877/cma.j.issn.1674-0807.2011.05.008

Abstract

Abstract:

Objective

To investigate the expression of DNA polymerase θ (POLQ)in breast cancer cell line MCF-7 and its effect on the proliferation and early apoptosis of the cells invitro.

Methods

Mediated by Lipofectamine^TM2000, POLQ-siRNA was designed and transfected into the MCF-7 cells.Then the MCF-7 cells were divided into a POLQ-siRNA group, a control-siRNA group and a blank control group.Real-time quantitative reverse transcription PCR (qRT-PCR) and Western blot were used to evaluate the POLQ mRNA gene expression and protein expression respectively.The proliferative ability of the cells was measured by MTT assay and colony-forming test.The cell cycle and apoptosis rate before and after transfection were assayed using flow cytometry.One-way analysis of variance and repeated measure analysis of variance were used for statistical analysis.

Results

POLQ highly expressed in the breast cancer MCF-7 cells, and its relative molecular weight (M_r)was ＞250 000.Prominent fragments with M_r =170 000 or 100 000 were detected easily.qRT-PCR and Western Blot revealed that in the POLQ-siRNA group the mean expression volume of POLQ mRNA was (0.12±0.01) times that of the blank control groups (P=0.00), and the POLQ protein expression rate was (32.0±0.7)%.MTT assay showed the cell proliferation of the POLQ-siRNA group was significantly lower than that of the blank control group (P=0.00).Colony-forming test demonstrated the successful cell clone rate of the POLQ-siRNA group was lower than that of the blank control group (P=0.00).At 48 h after transfection, in the POLQ-siRNA group,the percentage of the cells at G₀/G₁ phase was significantly higher than that in the blank control group (P=0.00), the percentage of the cells at S phase was significantly lower than that in the blank control group (P=0.00), and the early apoptosis rate of cells was higher than that in the blank control group(P=0.00);but there was no significant difference between the negative control group and the blank control group (P＞0.05).

Conclusion

POLQ is highly expressed in human breast cancer MCF-7 cells.Inhibition of the POLQ expression could suppress the cell proliferation ability and increase the early apoptosis rate of breast cancer cells.

Key words: breast neoplasms, small interfering RNA, cell proliferation, apoptosis

Xue-xiao LI, Ai-guo WU, Bin HU, Meng-chuan WANG, Shu-feng JI, Guo-li SHAO, Kai WU. Effect of DNA polymerase θ expression on proliferation and early apoptosis of breast cancer cell line MCF-7 invitro[J]. Chinese Journal of Breast Disease(Electronic Edition), 2011, 05(05): 565-575.

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Figures/Tables 9

References 16

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组别	2^-△△CT	F值	P值
POLQ-siRNA组	0.12±0.01^a	29199.99	0.00
Control-siRNA组	1.01±0.01^b
空白对照组	1.00±0.00

组别	2^-△△CT	F值	P值
POLQ-siRNA组	0.12±0.01^a	29199.99	0.00
Control-siRNA组	1.01±0.01^b
空白对照组	1.00±0.00

组别	24h	48h	72h	合计	F值	P值
POLQ-siRNA组^a	0.30±0.01	0.52±0.01	0.76±0.02	0.53±0.19	1834.77	0.00
Control-siRNA组^b	0.32±0.00	0.53±0.01	0.80±0.01	0.55±0.20	4263.10	0.00
空白对照组	0.32±0.01	0.54±0.01	0.81±0.02	0.56±0.21	2439.78	0.00
合计	0.31±0.01	0.53±0.01	0.79±0.03	0.55±0.20	7252.58	0.00
F值	4.85	12.46	14.85	34.32	3.34	0.04
P值	0.02	0.00	0.00	0.00

组别	24h	48h	72h	合计	F值	P值
POLQ-siRNA组^a	0.30±0.01	0.52±0.01	0.76±0.02	0.53±0.19	1834.77	0.00
Control-siRNA组^b	0.32±0.00	0.53±0.01	0.80±0.01	0.55±0.20	4263.10	0.00
空白对照组	0.32±0.01	0.54±0.01	0.81±0.02	0.56±0.21	2439.78	0.00
合计	0.31±0.01	0.53±0.01	0.79±0.03	0.55±0.20	7252.58	0.00
F值	4.85	12.46	14.85	34.32	3.34	0.04
P值	0.02	0.00	0.00	0.00

组别	G₀/G₁期	G₂/M期	S期
POLQ-siRNA组	79.79±0.88^ag	8.99±0.29^bh	11.55±1.27^ci
Control-siRNA组	72.97±1.25^d	8.64±0.35^e	18.38±1.46^f
空白对照组	73.84±1.84	7.85±0.48	18.31±1.59
F值	21.74	7.08	22.13
P值	0.00	0.03	0.00

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