Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45

Abstract

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 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).

Conclusion: Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway in gastric cancer.

Keywords: Cancer; Epithelial mesenchymal transition; Invasion; Matrix metallopeptidase 9; Notch2; RNA interference; Stomach.

Figure 1

Verification of successful transfection and knockdown of Notch2. ^aP < 0.05, ^bP < 0.01 vs the Mock or Scra groups. Scra: Scrambled small interfering RNA (siRNA); qPCR: Quantitative reverse transcription polymerase chain reaction.

Figure 2

Knockdown of Notch2 led to an increased migration and invasion of MKN-45 cells. Cells were transfected with scrambled small interfering RNA (siRNA) (Scra, A, C) or Notch2 siRNA (B, D) for 48 h, and the effect of the migration (A, B, E) and invasion (C, D, F) were assayed as described in “Materials and Methods”. The number of migrated cells or invaded cells were quantitated (E, F). ^aP < 0.05 vs the Scra groups. A, B, ×200; C, D, ×400.

Figure 3

Knockdown of Notch2 enhanced the expression and activity of matrix metallopeptidase 9. The small interfering RNA (siRNA) mediated knockdown of Notch2 (A) and Notch2 intracellular domain (N2ICD) (B, C) was associated with a marked increase in the expression (A, B, C) and activity (D) of matrix metallopeptidase 9 (MMP9). ^aP < 0.05, ^bP < 0.01 vs the blank control groups. qPCR: Quantitative reverse transcription polymerase chain reaction.

Figure 4

Knockdown of Notch2 enhanced the expression of matrix metallopeptidase 9 via increased phosphorylation of p-Akt in MKN-45 cells. A, B: Knockdown of Notch2 led to an increased phosphorylation of Akt (p-Akt); C, D: Blockade of PI3K/Akt pathway by LY294002 (20 μmol/L) abolished the effect of Notch small interfering RNA (siRNA) in Akt phosphorylation and matrix metallopeptidase 9 (MMP9). ^aP < 0.05 vs the all control groups. DMSO: Dimethyl sulfoxide.