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AIM: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 Notch2small 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 PI3KAkt 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).CONCLUSION:Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway
AIM: 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 Notch2small 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 PI3KAkt and MMP9, the PI3K inhibitor LY294002 was used to treat MKN-45 cells.RESULTS: Notch2 expression was changed decreased 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) indicating that the inhibition of Notch signaling was also confirmed by marked reduction of Notch2 intracellular domain at the down-regulation of Notch2 by siRNA enhanced tumor cell invasion (100.00% ± 9.74% vs 162.62% ± 16.84%, P <0.05) 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 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 < Notch2 may negatively regulate c ell invasion byinhibiting the PI3K-Akt signaling pathway