[Frontiers in Bioscience 17, 1589-1598, January 1, 2012]

SNCG gene silencing in gallbladder cancer cells inhibits key tumorigenic activities

Shenghua Han1,2, Feifei She2,3, Dong Wang1,Xiangqing Yao1, Lei Jiang1,Yanling Chen1,2

1Department of Hepatobiliary Surgery, Union Hospital, Fujian Medical University, Fuzhou 350001, China, 2 Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou 350001, China, 3Research Center for Molecular Medicine, The Key Laboratory of Infection and Oncology of Universities in Fujian Province, Fujian Medical University, Fuzhou 350004, China

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell lines
3.2. Establishment of NOZ cells stably expressing SNCG-shRNA
3.3. Semi-quantitative RT-PCR
3.4. Western blot analysis
3.5. Cell proliferation analysis
3.6. Colony formation assay
3.7. Transwell cell invasion assay
3.8. Flow cytometry in analysis of the cell cycle and apoptosis
3.9. Antitumor effects of SNCG gene silencing in vivo
3.10. Statistical analysis
4. Results
4.1. Generation of NOZ cells stably expressing SNCG-shRNA
4.2. SNCG gene silencing leads to a decrease in cell proliferation
4.3. SNCG gene silencing inhibits cell colony formation in NOZ cells
4.4. SNCG gene silencing decreases invasion of NOZ cells
4.5. SNCG-silencing in NOZ cells treated with paclitaxel induces G2/M arrest
4.6. SNCG-silencing in NOZ cells increases paclitaxel-induced apoptosis
4.7. In vivo antitumor effects of SNCG-silencing on NOZ cells
5. Discussion
6. Acknowledgements
7. References

1. ABSTRACT

We recently determined that synuclein-gamma (SNCG) is highly expressed in human gallbladder cancer (GBC), and its abnormal expression is associated with tumor aggressiveness. To investigate the effects of SNCG gene silencing on the tumorigenic profiles of the GBC cell line, NOZ, short-hairpin RNA (shRNA) interference was employed. Specifically, the SNCG transcript was targeted by SNCG-shRNA lentiviral particles designed to silence SNCG gene expression. Following selection of NOZ cells stably expressing SNCG-shRNA, SNCG expression was examined by western blot and semi-quantitative RT-PCR analyses. Phenotypic hallmarks of gallbladder carcinogenesis were assayed by CCK-8, soft agar (colony formation), modified Boyden-Chamber (invasion), and flow cytometry (cell-cycle and apoptosis) assays. Our results showed that SNCG gene silencing in NOZ cells inhibited cell growth, colony formation, and invasion. In addition, it directly increased the effectiveness of paclitaxel in inducing G2/M cell-cycle arrest and cell apoptosis. Data from our in vivo study showed a decrease in tumor growth and weight in mice injected with SNCG-silenced NOZ cells. Together, these findings suggest that SNCG plays an important role in the progression of GBC.