siRNA抑制人乳腺癌KLK6基因表达的实验研究

Research of silencing kallikrein 6 gene with siRNA in human breast cancer

目的探讨siRNA沉默KLK6基因表达对乳腺癌细胞生长的抑制作用及其机制,为乳腺癌的基因诊断和治疗提供理论依据。方法针对KLK6 mRNA序列设计合成siRNA,构建2个重组质粒PGCsilencerTMH1/GFP/Neo/KLK6和PGCsilen-cerTMH1/GFP/Neo/Non;将重组质粒导入乳腺癌MCF-7细胞株,G418筛选获得稳定转染的细胞株;实验分为脂质体对照、阴性质粒转染对照及KLK6 siRNA重组质粒转染组,实时荧光定量PCR法检测KLK6 mRNA的表达的变化,用四甲基偶氮唑盐(MTT)法测量细胞生长情况。结果测序证实表达载体构建成功,在稳定转染重组质粒PGCsilencerTMH1/GFP/Neo/KLK6的乳腺癌MCF-7细胞株中,KLK6 mRNA抑制率(76%)明显高于阴性质粒对照组(2.7%),MCF-7细胞增殖活性显著低于阴性对照组和脂质体对照组。结论重组质粒PGCsilencerTMH1/GFP/Neo/KLK6可抑制乳腺癌细胞中KLK6基因的表达,并抑制乳腺癌细胞的生长。

Objective To investigate the inhibitory effect of silencing KLK6 gene with siRNA on the growth of breast cancer cells and its mechanism,and to provide evidence in diagnosis and treatment for breast cancer.Methods Specific siRNA for KLK6 mRNA was designed and synthesized.Two recombinant plasmids PGCsilencerTMH1/GFP/Neo/KLK6 and PGCsilencerTMH1/GFP/Neo/Non were constructed.The two plasmids were transfected into MCF-7 cells and the positive cell clones were obtained by G418 selection.The breast cancer cel1 line MCF-7 were divided into three groups：liposome-treated control group,negative plasmid-transfected control group and KLK6-siRNA transfected group.The expression of KLK6 was determined by real time quantitative PCR,and the proliferation was also observed by MTT assay.Results The recombinant plasmid was successfully constructed.The results of real time quantitative PCR indicated that the inhibitory rates of mRNA in PGCsilencerTMH1/GFP/Neo/KLK6 transfected group（76%）was higher than that in negative plasmid transfected control group（2.7 %）.The MTT assay results showed that the cell proliferation in KLK6-siRNA transfected group was lower than that in negative plasmid transfected control group and liposome-treated control group（P0.01）.Conclusion The recombinant plasmid PGCsilencerTMH1/GFP/Neo/KLK6 can suppress the expression of KLK6 in breast cancer cells and proliferation of MCF-7 cells.