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

doi:10.3877/cma.j.issn.1674-0807.2011.03.007

Abstract

Abstract:

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 lipofectamine^TM 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

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]. Chinese Journal of Breast Disease(Electronic Edition), 2011, 05(03): 313-322.

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URL: https://zhrxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.1674-0807.2011.03.007

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Figures/Tables 10

References 14

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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±0.27^a
PRL-3-shRNA-2组	5	0.15±0.14^a
PRL-3-shRNA-3组	5	0.31±0.03^a
F值		1339.99
P值		0.00

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±0.27^a
PRL-3-shRNA-2组	5	0.15±0.14^a
PRL-3-shRNA-3组	5	0.31±0.03^a
F值		1339.99
P值		0.00

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±0.02^a
F值		608.26
P值		0.00

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±0.02^a
F值		608.26
P值		0.00

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±0.02^a	0.45±0.02^a	0.70±0.05^a

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