摘要
目的:观察靶向S100A4基因的短发夹RNA(short hairpin RNA,shRNA)对乳腺癌MCF-7细胞生长和侵袭的抑制作用。方法:构建S100A4基因特异性shRNA表达载体,经过脂质体介导将S100A4-shRNA表达载体转染入MCF-7细胞。应用实时荧光定量PCR(real-time fluorogentic quantitative PCR,RFQ-PCR)和Western印迹法检测转染后MCF-7细胞中S100A4的表达水平,运用MTT法和FCM法检测转染后MCF-7细胞的增殖水平和凋亡率变化。转染细胞经G418筛选及克隆化培养后获得稳定转染株,运用Transwell小室法和划痕实验检测其侵袭力和迁移力的变化,裸鼠体内成瘤实验检测体内细胞增殖情况。结果:经测序鉴定证实成功构建了S100A4-shRNA表达载体。所构建的S100A4-shRNA表达载体转染MCF-7细胞48 h后,能有效抑制S100A4的表达,并且显著降低MCF-7细胞增殖水平,以及诱导细胞进入晚期凋亡。稳定转染S100A4-shRNA表达载体的MCF-7细胞形态无显著变化,但其侵袭力和迁移力明显下降;裸鼠体内成瘤实验也显示实验组裸鼠移植瘤的体积和质量明显小于空白对照组和阴性对照组。结论:S100A4-shRNA表达载体能有效地抑制乳腺癌MCF-7细胞中S100A4的表达,从而降低细胞增殖水平以及细胞侵袭力和迁移力。
Objective：To study the inhibitory effects of short hairpin RNA（shRNA） targeting S100A4 gene on the growth and invasiveness of breast cancer MCF-7 cells.Methods：The S100A4 specific shRNA expression vector was constructed and confirmed by sequencing analysis.S100A4-shRNA expression vector was transfected into MCF-7cells via lipofectamineTM 2000,and the expression levels of S100A4 mRNA and protein were determined by real-time fluorogentic quantitative PCR and Western blotting at 48 h after transfection.Flow cytometry and MTT assay were performed to assess the effect of the S100A4-shRNA on the apoptosis and proliferation of MCF-7 cells.The cell line with stable expression of S100A4 was obtained by G418 screening and colony culture.Invasion and migration capability of stably transfected MCF-7 cells was evaluated by using Transwell chamber model and wound healing assay in vitro.The proliferation of stably transfected MCF-7 cells was evaluated by tumor formation test in nude mice.Results： S100A4-shRNA expression vector was successfully constructed and transfected into MCF-7 cells.At 48 h after transfection,S100A4 mRNA and protein expression levels were decreased significantly and the proliferation of MCF-7 cells was markedly suppressed.Transfection of S100A4-shRNA expression vector induced significant apoptosis of MCF-7 cells.The morphology of stably transfected MCF-7 cells did not change,but the invasion and migration capability decreased significantly.Tumor formation test indicated that the tumor size and weight were greatly decreased compared with control group and negative group.Conclusion：S100A4-shRNA expression vector effectively suppresses S100A4 expression in breast cancer MCF-7 cells,and thereby decreasing the proliferation,invasion,and migration capability of MCF-7 cells.
出处
《肿瘤》
CAS
CSCD
北大核心
2009年第8期715-720,共6页
Tumor
基金
广东省科技计划资助项目(编号:2008B030301345)
