The Second Clinical Medical School of Lanzhou University, The Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China.
World J Gastroenterol. 2012 Dec 28;18(48):7262-70. doi: 10.3748/wjg.v18.i48.7262.
To clarify the role of activated Notch2 in the invasiveness of gastric cancer.
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.
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).
Notch2 may negatively regulate cell invasion by inhibiting the PI3K-Akt signaling pathway in gastric cancer.
阐明 Notch2 激活在胃癌侵袭性中的作用。
为了研究 Notch2 基因表达沉默对侵袭性的影响，我们使用 MKN-45 胃癌细胞系建立了 Notch2 小干扰 RNA(siRNA)转染细胞系。通过实时逆转录聚合酶链反应(RT-PCR)和 Western blot 确认成功转染后，采用迁移和侵袭实验评估胃癌的侵袭性。RT-PCR 和 Western blot 用于确认 Notch2 的下调，并评估上皮间质转化相关基因基质金属蛋白酶 9(MMP9)、Akt、p-Akt 的表达。为了确认 PI3K-Akt 和 MMP9 之间的关系，使用 PI3K 抑制剂 LY294002 处理 MKN-45 细胞。
Notch2 siRNA 转染后 Notch2 表达明显降低(100.00%±9.74%vs11.61%±3.85%，qRT-PCR，P<0.01)。Notch 靶基因 Hes1 的 mRNA 水平也显著降低(100.00%±4.74%vs61.61%±3.58%，P<0.05)，表明 Notch 信号通路受到抑制。Notch2 细胞内结构域蛋白水平的显著降低(100.00%±9.74%vs65.61%±7.58%，P<0.05)也证实了 Notch 信号通路的抑制。Notch2 siRNA 的下调增强了肿瘤细胞的侵袭性(100.00%±21.64%vs162.22%±16.84%，P<0.05)和 MMP9 的表达(1.56 倍，P<0.05)，并将前 MMP9 蛋白激活为其活性形式(1.48 倍，P<0.05)。两组之间 Akt 蛋白水平无显著差异(100.00%±10.87%vs96.61%±7.33%，P>0.05)，而 Notch2 的下调则增加了 p-Akt 的表达(100.00%±9.87%vs154.61%±13.10%，P<0.05)。此外，抑制剂 LY294002 可下调 p-Akt 和 MMP9(p-Akt 100.00%±8.87%vs58.27%±5.01%，P<0.05；MMP9 100.00%±9.17%vs50.03%±4.88%，P<0.05)。
Notch2 可能通过抑制胃癌中的 PI3K-Akt 信号通路负调控细胞侵袭。
