title: [Construction of let-7a expression plasmid and its inhibitory effect on k-Ras protein in A549 lung cancer cells].
abstract: 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, 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  let-7a over-expression represses the expression of k-Ras protein and significantly inhibits the growth of lung cancer cells.
authors.affiliation: Department of Cell Biology and Genetics, Chongqing Medical University, Chongqing 400016, China. hexiaoyanaa@163.com
authors.forename: Xiao-yan
authors.initials: XY
authors.lastname: He
authors.forename: Jun-xia
authors.initials: JX
authors.lastname: Chen
authors.forename: Xi
authors.initials: X
authors.lastname: Ou-Yang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhang
authors.forename: Hui-min
authors.initials: HM
authors.lastname: Peng
mesh_terms: D045744:Cell Line, Tumor; D015972:Gene Expression Regulation, Neoplastic; D005822:Genetic Vectors; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D016283:Proto-Oncogene Proteins p21(ras) / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D014162:Transfection
chemical_list: D035683:MicroRNAs; D012333:RNA, Messenger; C487931:mirnlet7 microRNA, human; D016283:Proto-Oncogene Proteins p21(ras)