title: Activator protein-1 involvement in proliferation inhibition by gene silencing of Twist in gastric cancer cells.
abstract: OBJECTIVE
  To study the role of the Twist gene in growth of gastric cancer cell line MKN45 and the possible mechanisms involved.
  
  
  METHODS
  Human gastric carcinoma MKN45 cells were stably transfected with Twist antisense plasmid using the lipofectamine transfection technique. Expression of Twist in Twist antisense plasmid transfected cells (TwistAS), non-transfected cells (NT) and non-specific Twist antisense plasmid transfected cells (CON) were examined by Western blotting. Cell growth ability in vitro was evaluated by MTT and clone formation assays. Xenograft cancer models were established by nude mouse transfer method. Activator protein-1 (AP-1) DNA binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of c-Jun and c-Fos were examined by Western blotting. The mRNA level of cyclin D1 was detected by RT-PCR.
  
  
  RESULTS
  TwistAS inhibited cell growth and proliferation. In vitro, the cloning efficiency of TwistAS cells (8.0  ±  0.6%) was significantly lower compared to that in NT (26.5  ±  1.1%) and CON (22.7  ±  1.2%). In vivo, the average tumour weight was lighter in the TwistAS group (425.3  ±  20.8  mg) compared with the CON group (1217.0  ±  50.2  mg) and the NT group (1120.6  ±  75  mg). TwistAS inhibited AP-1 activity in MKN45 cells (15.3  ±  3.2% versus 50.2  ±  3.6% and 52.4  ±  3.8%). TwistAS inhibited the expression of c-Fos in MKN45 cells (20.4  ±  3.8% versus 72.5  ±  3.6% and 75.3  ±  4.0%) but not c-Jun (p < 0.05). cyclin D1 mRNA level was significantly lower in TwistAS cells (40.5  ±  3.8%) than that in CON (132  ±  5.4%) and NT cells (130  ±  5.2%).
  
  
  CONCLUSIONS
  This study demonstrated that down-regulation of the Twist gene suppressed the proliferation of MKN45 gastric cancer cells by negatively regulating the AP-1 activity resulting in the cyclin D1 mRNA level decreasing.
authors: Department of Pathology, Xiangya Medical School, Central South University, China.
authors: Geng-Qiu
authors: GQ
authors: Luo
authors: Jing-He
authors: JH
authors: Li
authors: Lan
authors: L
authors: Cao
authors: Yan-Hong
authors: YH
authors: Zhou
authors: Ji-Fang
authors: JF
authors: Wen
mesh_terms: D000818:Animals; D015153:Blotting, Western; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000096:biosynthesis; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000235:genetics*; D020868:Gene Silencing; D006801:Humans; D051379:Mice; D008819:Mice, Nude; D009687:Nuclear Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013274:Stomach Neoplasms / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D018808:Transcription Factor AP-1 / Q000235:genetics* / Q000378:metabolism; D014162:Transfection; D014183:Transplantation, Heterologous; D051879:Twist-Related Protein 1 / Q000235:genetics* / Q000378:metabolism
chemical_list: D009687:Nuclear Proteins; D012333:RNA, Messenger; C103173:TWIST1 protein, human; D018808:Transcription Factor AP-1; D051879:Twist-Related Protein 1; D019938:Cyclin D1