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中华乳腺病杂志(电子版) ›› 2011, Vol. 05 ›› Issue (05) : 565 -575. doi: 10.3877/cma.j.issn.1674-0807.2011.05.008

实验研究

DNA 聚合酶θ 表达对MCF-7 乳腺癌细胞系体外增殖和早期凋亡的影响
李学孝1, 吴爱国1,(), 胡斌1, 王梦川1, 纪术峰1, 邵国利1, 吴凯1   
  1. 1.510282 广州,南方医科大学珠江医院普外科
  • 收稿日期:2011-04-07 出版日期:2011-10-01
  • 通信作者: 吴爱国
  • 基金资助:
    广东省科技计划基金资助项目(2008B030301345)

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

Xue-xiao LI1, Ai-guo WU,1(), Bin HU1, Meng-chuan WANG1, Shu-feng JI1, Guo-li SHAO1, Kai WU1   

  1. 1.Department of General Surgery, Zhujiang Hospital,Southern Medical University, Guangzhou 510282, China.
  • Received:2011-04-07 Published:2011-10-01
  • Corresponding author: Ai-guo WU
引用本文:

李学孝, 吴爱国, 胡斌, 王梦川, 纪术峰, 邵国利, 吴凯. DNA 聚合酶θ 表达对MCF-7 乳腺癌细胞系体外增殖和早期凋亡的影响[J/OL]. 中华乳腺病杂志(电子版), 2011, 05(05): 565-575.

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/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2011, 05(05): 565-575.

目的

研究DNA 聚合酶θ(POLQ)在MCF-7 乳腺癌细胞系中的表达及其对该细胞系体外增殖和早期凋亡的影响。

方法

经脂质体LipofectamineTM2000 介导将POLQ-siRNA 转染入MCF-7 乳腺癌细胞系中,MCF-7 细胞被分为POLQ-siRNA 组、Control-siRNA 组和空白对照组;用实时荧光定量逆转录-多聚酶链反应(qRT-PCR)和Western blot 分别从基因和蛋白水平检测POLQ 表达率的变化;利用四甲基偶氮唑盐(MTT)法和平板克隆形成实验检测细胞增殖能力的变化;用流式细胞术判断转染前后细胞周期和凋亡率的改变。数据采用单因素方差分析和重复测量数据的方差分析进行统计。

结果

POLQ 在MCF-7 乳腺癌细胞系中高表达,其相对分子质量(Mr)>250 000,同时在Mr 约170 000 和100 000 处容易检测到明显的条带。POLQ-siRNA 组细胞POLQ mRNA 平均表达量是空白对照组的(0.12±0.01)倍(P=0.00),POLQ 蛋白表达率为(32.0±0.7)%。POLQ-siRNA 组细胞增殖能力显著低于空白对照组(P=0.00);POLQ-siRNA 组细胞克隆形成率明显低于空白对照组(P=0.00)。转染后48 h,POLQsiRNA 组G0/G1 期细胞比例显著高于空白对照组(P=0.00),S 期细胞比例显著低于空白对照组(P=0.00),细胞早期凋亡率高于空白对照组(P=0.00),阴性对照组与空白对照组相比差异均无统计学意义(P>0.05)。

结论

POLQ 在MCF-7 乳腺癌细胞系中高表达,阻断该基因表达可以抑制细胞增殖,同时提高乳腺癌细胞的早期凋亡率。

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 LipofectamineTM2000, 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 (Mr)was >250 000.Prominent fragments with Mr =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.

图1 Western blot 证实MCF-7 乳腺癌细胞系中DNA 聚合酶θ 蛋白高表达 1:MCF-7 细胞;2:Hela 细胞
图2 荧光显微镜检测细胞转染效率(×100) a:日常光照射下; b:蓝色激发光照射下
表1 qTR-PCR 测定POLQ mRNA 表达水平
图3 实时荧光定量逆转录-多聚酶链反应溶解曲线
图4 Western blot 检测结果 1:POLQ-siRNA 组; 2:Control-siRNA 组;3:空白对照组
表2 3 组细胞的增殖能力(吸光度值)比较
表3 转染48 h 后MCF-7 细胞周期的变化(%)
图5 流式细胞仪检测转染后MCF-7 细胞周期的变化
图6 流式细胞仪检测转染后MCF-7 细胞早期凋亡率的变化 a:POLQ-siRNA 组;b:Control- siRNA 组;c:空白对照组
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