title: Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45.
abstract: OBJECTIVE
  To clarify the role of activated Notch2 in the invasiveness of gastric cancer.
  
  
  METHODS
  To investigate the invasiveness of silencing Notch2 gene expression, we established a Notch2 small interfering RNA (siRNA) transfected cell line using the MKN-45 gastric cancer cell line. After the successful transfection confirmed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, migration and invasion assays were employed to evaluate the aggressiveness of the gastric cancer. RT-PCR and Western blottings were employed to confirm the down-regulation of Notch2 and to evaluate the expression of epithelial mesenchymal transition-related gene matrix metallopeptidase 9 (MMP9), Akt, p-Akt. To confirm the relationship between PI3K-Akt and MMP9, the PI3K inhibitor LY294002 was used to treat MKN-45 cells.
  
  
  RESULTS
  Notch2 expression was dramatically decreased after Notch2 siRNA transfection (100.00% ± 9.74% vs 11.61% ± 3.85%, P < 0.01 by qRT-PCR). There was also a marked reduction of Notch target gene Hes1 (100.00% ± 4.74% vs 61.61% ± 3.58%, P < 0.05) at the mRNA, indicating an inhibition of Notch signaling. Inhibition of Notch signaling was also confirmed by the marked reduction of Notch2 intracellular domain at the protein levels (100.00% ± 9.74% vs 65.61% ± 7.58%, P < 0.05). Down-regulation of Notch2 by siRNA enhanced tumor cell invasion (100.00% ± 21.64% vs 162.22% ± 16.84%, P < 0.05) and expression of MMP9 (1.56 fold, P < 0.05), and activated the pro-MMP9 protein to its active form (1.48 fold, P < 0.05). There was no significant difference in the protein levels of Akt between the two groups (100.00% ± 10.87% vs 96.61% ± 7.33%, P > 0.05), while down-regulation of Notch2 elevated p-Akt expression (100.00% ± 9.87% vs 154.61% ± 13.10%, P < 0.05). Furthermore, p-Akt and MMP9 was down-regulated in response to the inhibitor LY294002 (p-Akt 100.00% ± 8.87% vs 58.27% ± 5.01%, P < 0.05; MMP9 100.00% ± 9.17% vs 50.03% ± 4.88%, P < 0.05).
  
  
  CONCLUSIONS
  Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway in gastric cancer.
authors: The Second Clinical Medical School of Lanzhou University, The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China.
authors: Ling-Yun
authors: LY
authors: Guo
authors: Yu-Min
authors: YM
authors: Li
authors: Liang
authors: L
authors: Qiao
authors: Tao
authors: T
authors: Liu
authors: Yuan-Yuan
authors: YY
authors: Du
authors: Jun-Qiang
authors: JQ
authors: Zhang
authors: Wen-Ting
authors: WT
authors: He
authors: Yong-Xun
authors: YX
authors: Zhao
authors: Dong-Qiang
authors: DQ
authors: He
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement; D002867:Chromones / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D009361:Neoplasm Invasiveness; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051883:Receptor, Notch2 / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013274:Stomach Neoplasms / Q000201:enzymology* / Q000235:genetics*
chemical_list: D002867:Chromones; D009025:Morpholines; C497764:NOTCH2 protein, human; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D051883:Receptor, Notch2; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; C579270:MMP9 protein, human; D020780:Matrix Metalloproteinase 9
keywords: Cancer; Epithelial mesenchymal transition; Invasion; Matrix metallopeptidase 9; Notch2; RNA interference; Stomach