摘要
目的探讨let-7a介导的基因表达调控在肺癌形成中的作用。方法依据miRBase数据库中hsa-let-7a序列,设计两条寡核苷酸片段,退火处理后克隆至pGenesil-1质粒,构建重组质粒pGenesil-let-7a,同时设计并构建一个阴性对照质粒pGenesil-control。将pGenesil-let-7a及pGenesil-control转染肺癌A549细胞,经G418筛选,建立稳定表达let-7a-GFP及control-GFP的细胞株。采用MTT比色法检测活细胞数并绘制生长曲线;半定量RT-PCR、Western blotting及免疫细胞化学检测k-Ras表达的差异。结果经测序证实重组质粒构建成功,与未处理的A549细胞和稳定表达pGenesil-control的细胞相比,在稳定表达pGenesil-let-7a的A549细胞中,细胞生长明显减慢,k-Ras的蛋白表达明显降低,而k-Ras的mRNA水平无明显变化。结论 let-7a在翻译水平抑制了k-Ras的表达,并对细胞生长具有显著的抑制作用。
Objective To elucidate the role of let-7a-mediated gene regulation in the pathogenesis of lung cancer.Methods Two template DNA sequences were designed based on hsa-let-7a sequence in miRBase database.The let-7a expression construct and a control plasmid,namelypGeneil-let-7a and pGeneil-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.Results The recombinant vectors were verified by sequencing.The cell growth curves indicated that the proliferation of the cells transfected with pGenesillet-7a were inhibited significantly compared with that of cells transfected with pGenesil-control and A549 cells.Semiquantitative 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.Conclusion let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.
出处
《南方医科大学学报》
CAS
CSCD
北大核心
2010年第11期2427-2431,共5页
Journal of Southern Medical University
基金
国家自然科学基金落选预研资助[重医大(2010)2号文件]
重庆医科大学校级重点科研课题(XBZD201001)
