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

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

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-ti me 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。

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（P0.05）.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（P0.05）.MTT results showed that the growth of MCF-7 cells after PRL-3-shRNA-2 transfection was decreased markedly（P0.05）.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%）（P0.05）.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（P0.05）.Conclusions Silencing the expression of PRL-3 gene can effectively suppress the proliferation and invasion capability,and promote the apoptosis of MCF-7 cells.