Original Article
Copyright ©2014 Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jul 28, 2014; 20(28): 9497-9505
Published online Jul 28, 2014. doi: 10.3748/wjg.v20.i28.9497
Down-regulation of FoxM1 inhibits viability and invasion of gallbladder carcinoma cells, partially dependent on inducement of cellular senescence
Jie Tao, Xin-Sen Xu, Yan-Zhou Song, Kai Qu, Qi-Fei Wu, Rui-Tao Wang, Fan-Di Meng, Ji-Chao Wei, Shun-Bin Dong, Yue-Lang Zhang, Min-Hui Tai, Ya-Feng Dong, Lin Wang, Chang Liu
Jie Tao, Xin-Sen Xu, Kai Qu, Rui-Tao Wang, Fan-Di Meng, Ji-Chao Wei, Shun-Bin Dong, Lin Wang, Chang Liu, Department of Hepatobiliary Surgery, the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Yan-Zhou Song, Department of General Surgery, Lianyungang First People’s Hospital, Lianyungang 222002, Jiangsu Province, China
Qi-Fei Wu, Department of Thoracic Surgery, the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Yue-Lang Zhang, Department of Radiology, the First Affiliated Hospital of Medical College, Xi'an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Min-Hui Tai, Ya-Feng Dong, Department of Obstetrics and Gynecology, University of Kansas School of Medicine, KA 66160, United States
Author contributions: Tao J and Xu XS contributed equally to this work; Tao J, Xu XS and Song YZ performed the majority of experiments such as transfection, MTT assay, and invasion assay; Qu K and Wu QF performed the senescence experiment; Wang RT participated in the data analysis; Meng FD and Wei JC constructed the recombinant adenovirus; Dong SB performed cell culture and shRNA selection; Zhang YL and Tai MH performed data analysis; Dong YF and Wang L were involved in editing the manuscript; Liu C designed the study.
Supported by Science Foundation of the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, No. 2010YK1
Correspondence to: Chang Liu, MD, PhD, Department of Hepatobiliary Surgery, the First Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China. liuchangdoctor@163.com
Telephone: +86-29-85323900 Fax: +86-29-82654746
Received: December 18, 2013
Revised: February 14, 2014
Accepted: April 8, 2014
Published online: July 28, 2014
Processing time: 220 Days and 2.7 Hours
Abstract

AIM: To investigate the effect of knockdown of Forkhead box M1 (FoxM1) on the proliferation and invasion capacities of human gallbladder carcinoma (GBC)-SD cells.

METHODS: Four FoxM1 shRNAs were transfected into GBC-SD cells with Lipofectamine 2000 to select the appropriate shRNA for down-regulation of FoxM1. A recombinant lentivirus for shFoxM1 (Lv-shFoxM1), which expresses FoxM1-specific shRNA, and a negative control carrying green fluorescent protein, which expresses a scrambled RNA, were constructed. After transfection with the recombinant adenovirus and screened with puromycin, RT-PCR and Western blot were utilized to evaluate the inhibition efficiency. Cell viability was evaluated by MTT assay, and cell migration and invasion were assessed using the Transwell system. Cells were suspended in serum-free medium and seeded into Transwell inserts either uncoated (for migration assay) or coated (for invasion assay) with growth factor-reduced Matrigel. To verify the involvement of FoxM1 in the senescence of tumor cells, staining of senescence β-galactosidase (SA β-gal), the widely used biomarker of cellular senescence, was also performed.

RESULTS: After successful transfection of four FoxM1 small interfering RNAs (shRNAs) with Lipofectamine 2000, the shF1822 was selected as the most appropriate shRNA according to its obvious inhibitory effect. The recombinant adenovirus was then constructed with the shF1822 and successfully transfected into the GBC-SD cells, resulting in the significant inhibition of FoxM1 expression at both the mRNA and protein levels, compared with the negative control (P < 0.05). After transfection, down-regulation of FoxM1 significantly inhibited cell viability according to the MTT assay (P < 0.05). In addition, Transwell migration and invasion assays also suggested the suppression of invasion ability of the transfected cells. SA β-gal staining showed that down-regulation of FoxM1 could induce more senescent GBC cells (P < 0.05), suggesting the possible involvement of the senescence process of the FoxM1-deficient cells in GBC.

CONCLUSION: FoxM1 is functionally involved in viability of GBC cells, partially dependent on the inducement of cellular senescence, and is a potential target for GBC therapy.

Keywords: Forkhead box M1; Gallbladder carcinoma; Senescence; Viability; Invasion

Core tip: Gallbladder cancer is characterized by early metastases, thus it is in urgent need to identify novel therapies to enhance the therapeutic effect. We have previously reported that Forkhead box M1 (FoxM1) expression was closely correlated with gallbladder carcinoma differentiation, Nevin stage and metastasis, indicating the potential roles of FoxM1 in gallbladder carcinoma. In this study, by regulating the expression of FoxM1 with small interfering RNAs, we demonstrated the impact of FoxM1 on cellular viability in a human gallbladder carcinoma cell line, which was probably via the regulation of cellular senescence, revealing FoxM1 as a potential target for gallbladder carcinoma therapy.