小干扰RNA抑制LRP16基因表达限制了MCF-7乳腺癌细胞增殖

Inhibition of Cell Proliferation by Small Interference RNA Against LRP16 Gene in Human Breast Cancer MCF-7 Cells

雌激素雌二醇上调人乳腺癌细胞MCF 7中LRP16基因表达 ,该基因过表达促进MCF 7细胞增殖 .为进一步探讨LRP16基因不同表达水平对MCF 7细胞增殖的影响以及对雌激素的反应性增殖能力 ,采用针对LRP16基因特异的小干扰RNA策略 ,通过逆转录病毒介导及抗性筛选构建了LRP16基因被稳定抑制的 2个MCF 7细胞系 ,针对绿色荧光蛋白的干扰序列作为阴性对照 .Northern印迹实验检测了LRP16基因在各个细胞株中mNRA的水平 ,与对照组细胞比较 ,针对LRP16基因不同位置的 2个小干扰RNA可分别将该基因抑制 90 %和 6 0 % .细胞增殖试验结果显示 ,MCF 7细胞中LRP16基因表达抑制率越高 ,细胞增殖速率减慢越显著 (P <0 0 5 ) ;软琼脂集落形成试验结果显示 ,抑制LRP16基因在MCF 7细胞中表达 ,限制了细胞锚定非依赖性生长 ;细胞周期分析结果表明 ,LRP16基因抑表达使MCF 7细胞G1 S周期转换受抑 ;Western印迹结果表明 ,LRP16基因表达抑制的细胞中细胞周期蛋白E及细胞周期蛋白D1蛋白水平显著下调 ,但未检测到P5 3及Rb蛋白表达水平的影响 .雌二醇刺激的增殖实验结果显示 ,抑制LRP16基因表达没有消除MCF 7细胞的反应性增殖特征 .上述结果表明 ,LRP16基因表达量与MCF 7细胞增殖能力密切相关 ,抑制其表达可有效限制MCF 7细胞的增殖能力 ,提?

Estradiol(β E2) up regulate LRP16 gene expression in human breast cancer MCF 7 cells,and ectopic expression of the LRP16 gene promote MCF 7 cells proliferation.The effects of LRP16 gene expression on growth and response to 17β E2 of MCF 7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16 inhibitory MCF 7 cell lines.Hairpin small interference RNA(siRNA) strategy,by which hairpin siRNA was released by U6 promoter and was mediated by pLPC based retroviral vector,was adopted to knockdown endogenous LRP16 level in MCF 7 cells.And the hairpin siRNA against green fluorescence protein(GFP) was used as the negative control.Ninety and sixty perecnt suppressant efficiency of the LRP16 gene expression were confirmed by Nothern blot.The results from cell proliferation assays suggested that down regulation of LRP16 gene expression is capable of inhibiting MCF 7 breast cancer cell growth and down regulation of the LRP16 gene expression is able to inhibit anchorage independent growth of breast cancer cells in soft agar.Cell cycle analysis showed that suppression of the LRP16 gene expression in MCF 7 cells effectively arrested the G 1/S transition.Western blot analysis demonstrated that cyclin E and cyclin D1 protein were much lower in the LRP16 inhibitory cells than in the control cells,while the expression levels of P53 and Rb protein were not different between these two groups.Nevertheless,MCF 7 cells with the LRP16 gene deficiency maintained the proliferation response to β E2.These data suggest that LRP16 gene play an important role in MCF 7 cells proliferation by regulating the pathway of the G 1/S transition and it may function as an important modulator in regulating the process of tumorigenesis in human breast.