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中华乳腺病杂志(电子版)

中华乳腺病杂志(电子版) ›› 2011, Vol. 05 ›› Issue (03) : 313 -322. doi: 10.3877/cma.j.issn.1674-0807.2011.03.007

实验研究

短发夹RNA 沉默促肝细胞再生磷酸酶-3 基因表达对乳腺癌MCF-7 细胞生长和侵袭能力的影响
钟琰1, 吴爱国1,†(), 纪术峰1, 沈三弟1   
  1. 1.510282 广州,南方医科大学珠江医院普外科
  • 收稿日期:2011-03-02 出版日期:2011-06-01
  • 通信作者: 吴爱国
  • 基金资助:
    广东省科技计划资助项目(2008B030301345)

Effect of shRNA silencing PRL-3 gene expression on the growth and invasiveness of breast cancer MCF-7 cells

Yan ZHONG1, Ai-guo WU1,†(), Shu-feng JI1, San-di SHEN1   

  1. 1.Department of General Surgery, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, China
  • Received:2011-03-02 Published:2011-06-01
  • Corresponding author: Ai-guo WU
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钟琰, 吴爱国, 纪术峰, 沈三弟. 短发夹RNA 沉默促肝细胞再生磷酸酶-3 基因表达对乳腺癌MCF-7 细胞生长和侵袭能力的影响[J/OL]. 中华乳腺病杂志(电子版), 2011, 05(03): 313-322.

Yan ZHONG, Ai-guo WU, Shu-feng JI, San-di SHEN. Effect of shRNA silencing PRL-3 gene expression on the growth and invasiveness of breast cancer MCF-7 cells[J/OL]. Chinese Journal of Breast Disease(Electronic Edition), 2011, 05(03): 313-322.

目的

构建以促肝细胞再生磷酸酶-3(PRL-3)为靶基因的短发夹状小干扰RNA(short hairpin RNA,shRNA)表达载体,并探讨PRL-3- shRNA 表达载体对人乳腺癌MCF-7 细胞增殖、凋亡和侵袭能力的影响。

方法

构建PRL-3 基因特异性shRNA 表达载体,使用脂质体法将PRL-3-shRNA 表达载体转染入MCF-7 细胞。 采用Real-time PCR 和Western blot 分别检测转染后MCF-7 细胞中PRL-3 基因mRNA 和蛋白的表达;运用四甲基偶氮唑蓝(methyl thiazolyl tetrazolium, MTT)比色法检测转染后MCF-7 细胞的增殖水平,用流式细胞仪检测细胞凋亡的情况;采用Transwell 小室法检测细胞侵袭力变化。 计量资料采用单因素方差分析或重复测量方差分析。

结果

酶切鉴定和测序分析证实PRL-3-shRNA 表达载体成功构建。 转染成功后,shRNA-1 ～3 组PRL-3 基因mRNA 表达分别为(0.31±0.27)、(0.15±0.14)和(0.31±0.03),与空白组和阴性组(1.00±0.00、0.98±0.18)相比,差异有统计学意义( P＜0.05)。 PRL-3-shRNA-2 组PRL-3 蛋白的表达量明显低于阴性组和空白组(0.50±0.02 比0.91±0.03 和0.89±0.02,P＜0.05);PRL-3-shRNA-2转染入MCF-7 细胞后能明显降低其增殖水平(P＜0.05);PRL-3-shRNA-2 组凋亡率明显高于空白组和阴性组[(8. 37±1. 85)%比(1. 60±1. 58)%和(0.16±0. 05)%,P ＜0. 05];Transwell 小室侵袭实验显示,PRL-3-shRNA-2 组穿膜细胞数明显低于阴性组和空白组(P＜0.05)。

结论

沉默PRL-3 基因表达可抑制MCF-7 细胞的增殖,促进凋亡,抑制其侵袭能力。

关键词: 乳腺肿瘤, 促肝细胞再生磷酸酶-3, 短发夹状小干扰RNA, 基因治疗

Objective

To study the effect of short hairpin RNA(shRNA) targeting phosphatase of regenerating liver-3(PRL-3) gene on the proliferation, apoptosis, and invasiveness of breast cancer MCF-7 cells.

Methods

The PRL-3 specific shRNA expression vector was constructed and confirmed by sequencing analysis. PRL-3-shRNA expression vector was transfected into MCF-7 cells via lipofectamineTM 2000. The expression level of mRNA and protein after transfection were determined by Real-time PCR and Western bolt respectively. Flow cytometry and MTT assay were performed to assess the effects of the PRL-3-shRNA on the proliferation and apoptosis of MCF-7 cells. Invasion capability of stably transfected MCF-7 cells was evaluated by transwell chamber model assay in vitro. Comparison between quantitative data was performed using one-way ANOVA or repeated measures ANOVA.

Results

PRL-3-shRNA expression vector was successfully constructed and transfected into MCF-7 cells. The PRL-3 mRNA levels in the groups of shRNA-1 ～3 were respectively reduced to (0.31±0.27),(0.15±0.14)and(0.31±0.03)after transfection,and were significantly lower than both the blank group (1.00±0.00) and the negative group(0.98±0.18); the difference was statistically significant (P＜0.05). The PRL-3 protein level in the group of shRNA-2(0.50±0.02)was also significantly lower than both the blank group(0. 89±0. 02) and the negative group(0. 91±0. 03),with statistically significant difference (P＜0. 05). MTT results showed that the growth of MCF-7 cells after PRL-3-shRNA-2 transfection was decreased markedly (P＜0.05). The apoptotic rate in the PRL-3-shRNA-2 group (8.37±1.85% ) was increased significantly compared with the blank group(1.60±1.58%) and the negative group (0.16±0.05% ) (P＜0.05). Transwell chamber model assay showed that the trans-membrane cell numbers in the PRL-3-shRNA-2 group were greatly decreased compared with the blank group and the negative group (P ＜0. 05).

Conclusions

Silencing the expression of PRL-3 gene can effectively suppress the proliferation and invasion capability, and promote the apoptosis of MCF-7 cells.

Key words: breast neoplasms, phosphatase of regenerating liver-3, shRNA, gene therapy
图1 重组载体的酶切鉴定图 M:DL2000 标记条带;1:PRL-3-shRNA-1;2:PRL-3-shRNA-2;3:PRL-3-shRNA-3
表1 转染后72 h 各组PRL-3mRNA 相对表达量
组别 n PRL-3mRNA
空白组 5 1.00±0.00
阴性组 5 0.98±0.18
PRL-3-shRNA-1组 5 0.30±0.27a
PRL-3-shRNA-2组 5 0.15±0.14a
PRL-3-shRNA-3组 5 0.31±0.03a
F值 1339.99
P值 0.00
图2 Real-time PCR 检测PRL-3mRNA 的表达 M:标记条带;1: PRL-3-shRNA-3 组;2: PRL-3-shRNA-2 组;3: PRL-3-shRNA-1 组;4:空白组;5:阴性组
表2 各组MCF-7 细胞PRL-3 蛋白的相对灰度值比较
组别 n PRL-3蛋白相对灰度值
阴性组 5 0.91±0.03
空白组 5 0.89±0.02
PRL-3-shRNA-2组 5 0.50±0.02a
F值 608.26
P值 0.00
图3 各组PRL-3 蛋白的表达 1:空白组; 2:阴性组; 3:PRL-3-shRNA-2 组
表3 转染后各组细胞的吸光度值比较
组别 1 d 2 d 3 d 4 d
阴性组 0.19±0.03 0.35±0.01 0.61±0.02 0.91±0.10
空白组 0.20±0.02 0.36±0.02 0.60±0.03 0.90±0.12
PRL-3-shRNA-2组 0.19±0.02 0.31±0.02a 0.45±0.02a 0.70±0.05a
图4 各组细胞的增殖能力比较 a: P＜0.05,与空白组比较
表4 细胞周期分布情况 (%)
组别 G1 S G2 凋亡率
阴性组 38.58±2.41 34.84±4.56 26.49±4.80 0.16±0.05
空白组 53.50±5.46 35.18±1.67 23.82±0.63 1.60±1.58b
PRL-3-shRNA-2组 40.67±1.16a 20.79±7.75a 25.70±9.08 8.37±1.85a
F值 15.89 7.25 0.16 29.06
P值 0.00 0.03 0.86 0.00
图5 各组细胞的凋亡率比较 a:P＜0.05,分别与阴性组和空白组比较
表5 各组穿膜细胞数比较
组别 穿膜细胞数(个)
阴性组 8.00±1.41a
空白组 7.25±0.96
PRL-3-shRNA-2组 1.25±0.96b
F值 42.85
P值 0.00
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