He Xiao-yan, Chen Jun-xia, Ou-Yang Xi, Zhang Zheng, Peng Hui-min
Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2010 Nov;30(11):2427-31.
To elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer.
Two template DNA sequences were designed based on hsa-let-7a sequence in miRBase database. The let-7a expression construct and a control plasmid, namely pGenesil-let-7a and pGenesil-control, respectively, were generated by cloning the annealed oligonucleotides into pGenesil-1 and then transfected into A549 cells, which were selected by G418 to establish the lung cancer cell lines stably expressing let-7a-GFP and control-GFP. The living cells were counted by MTT assay and cell growth curves were drawn to analyze the cell proliferation. The k-Ras mRNA level was assessed by semi-quantitative RT-PCR, and the expression of k-Ras protein was determined by Western blotting and immunocytochemistry.
The recombinant vectors were verified by sequencing. The cell growth curves indicated that the proliferation of the cells transfected with pGenesil- let-7a were inhibited significantly compared with that of cells transfected with pGenesil-control and A549 cells. Semi- quantitative RT-PCR analysis showed that the levels of k-Ras mRNA almost remained unchanged in cells with or without the treatments. Western blotting and immunocytochemistry demonstrated a significant decrease of k-Ras protein levels in cells transfected with pGenesil-let-7a, but not in cells transfected with pGenesil-control, when compared to A549 cells.
let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.
阐明let-7a介导的基因调控在肺癌发病机制中的作用。
基于miRBase数据库中的hsa-let-7a序列设计两条模板DNA序列。通过将退火的寡核苷酸克隆到pGenesil-1中，分别构建let-7a表达载体和对照质粒，即pGenesil-let-7a和pGenesil-control，然后转染到A549细胞中，用G418筛选以建立稳定表达let-7a-GFP和对照-GFP的肺癌细胞系。采用MTT法计数活细胞并绘制细胞生长曲线以分析细胞增殖。通过半定量RT-PCR评估k-Ras mRNA水平，通过蛋白质免疫印迹法和免疫细胞化学法测定k-Ras蛋白的表达。
重组载体经测序验证。细胞生长曲线表明，与转染pGenesil-control的细胞和A549细胞相比，转染pGenesil-let-7a的细胞增殖受到显著抑制。半定量RT-PCR分析显示，无论有无处理，细胞中k-Ras mRNA水平几乎保持不变。蛋白质免疫印迹法和免疫细胞化学法表明，与A549细胞相比，转染pGenesil-let-7a的细胞中k-Ras蛋白水平显著降低，而转染pGenesil-control的细胞中则未降低。
let-7a过表达可抑制k-Ras蛋白的表达并显著抑制肺癌细胞的生长。
