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

• Published in: World journal of gastroenterology : WJG Vol. 18; no. 48; pp. 7262 - 7270
• Main Authors: Guo, Ling-Yun, Li, Yu-Min, Qiao, Liang, Liu, Tao, Du, Yuan-Yuan, Zhang, Jun-Qiang, He, Wen-Ting, Zhao, Yong-Xun, He, Dong-Qiang
• Format: Journal Article
• Language: English
• Published: United States Baishideng Publishing Group Co., Limited 28.12.2012
• Subjects: Cell Line, Tumor; Cell Movement; Chromones - pharmacology; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Neoplastic; Humans; Matrix Metalloproteinase 9 - metabolism; Morpholines - pharmacology; Neoplasm Invasiveness; Original; Phosphatidylinositol 3-Kinases - metabolism; Protein Kinase Inhibitors - pharmacology; Proto-Oncogene Proteins c-akt - metabolism; Receptor, Notch2 - metabolism; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering - metabolism; Signal Transduction; siRNA; Stomach Neoplasms - enzymology; Stomach Neoplasms - genetics; Western印迹法; 信号通路; 定量RT-PCR; 矩阵; 胃癌细胞; 蛋白质水平; 逆转录聚合酶链反应; Stomach; RNA interference; Notch2; Epithelial mesenchymal transition; Matrix metallopeptidase 9; Invasion; Cancer
• ISSN: 1007-9327; 2219-2840
• DOI: 10.3748/wjg.v18.i48.7262

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