Original Article
Copyright ©2012 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Dec 28, 2012; 18(48): 7262-7270
Published online Dec 28, 2012. doi: 10.3748/wjg.v18.i48.7262
Notch2 regulates matrix metallopeptidase 9 via PI3K/AKT signaling in human gastric carcinoma cell MKN-45
Ling-Yun Guo, Yu-Min Li, Liang Qiao, Tao Liu, Yuan-Yuan Du, Jun-Qiang Zhang, Wen-Ting He, Yong-Xun Zhao, Dong-Qiang He
Ling-Yun Guo, Yuan-Yuan Du, Jun-Qiang Zhang, Yong-Xun Zhao, Dong-Qiang He, the Second Clinical Medical School of Lanzhou University, the Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China
Yu-Min Li, Key Laboratory of Digestive System Tumors, Department of General Surgery, the Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China
Liang Qiao, Storr Liver Unit at the Westmead Millennium Institute, the University of Sydney at Westmead Hospital, Westmead, NSW 2145, Australia
Tao Liu, Wen-Ting He, Key Laboratory of Digestive System Tumors, the Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China
Yong-Xun Zhao, Department of Oncological Surgery, the First Hospital of Lanzhou University, Lanzhou 730000, Gansu Province, China
Author contributions: Guo LY, Zhang JQ and Zhao YX performed the majority of experiments; Liu T and He WT provided vital reagents and analytical tools and were also involved in revising the manuscript; Guo LY, Du YY and He DQ analyzed and interpreted data; Guo LY, Li YM and Qiao L designed the study, wrote and revised the manuscript.
Supported by The National Natural Science Foundation of China, No. 30870364; Fund for Key Laboratory of Digestive System Tumors, Gansu Province, No. lzujbky-2011-t03
Correspondence to: Yu-Min Li, Professor, Key Laboratory of Digestive System Tumors, Department of General Surgery, the Second Hospital of Lanzhou University, Lanzhou 730030, Gansu Province, China. liym@lzu.edu.cn
Telephone: +86-931-8943092 Fax: +86-931-8943092
Received: September 14, 2012
Revised: November 7, 2012
Accepted: November 24, 2012
Published online: December 28, 2012
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: Notch2, Stomach, Cancer, Invasion, Epithelial mesenchymal transition, Matrix metallopeptidase 9, RNA interference