Original Article
Copyright ©2013 Baishideng Publishing Group Co., Limited. All rights reserved.
World J Gastroenterol. Jun 28, 2013; 19(24): 3792-3801
Published online Jun 28, 2013. doi: 10.3748/wjg.v19.i24.3792
Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells
Hai-Jian Zhang, Deng-Fu Yao, Min Yao, Hua Huang, Li Wang, Mei-Juan Yan, Xiao-Di Yan, Xing Gu, Wei Wu, Shao-Lin Lu
Hai-Jian Zhang, Deng-Fu Yao, Min Yao, Li Wang, Mei-Juan Yan, Wei Wu, Shao-Lin Lu, Research Centre of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Hua Huang, Department of Pathology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Xiao-Di Yan, Xing Gu, Department of Clinical Oncology, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China
Author contributions: Zhang HJ and Yao M contributed equally to this work, designed the research study, conducted the experiments, analysed the data, and wrote the paper; Wang L, Yan MJ and Gu X carried out the in vitro experiments; Wu W and Yan XD performed the in vivo experiments; Huang H conducted the immunohistochemical and hematoxylin and eosin staining analyses; Lu SL critically reviewed the manuscript; Yao DF is the guarantor.
Supported by The Society Development of Nantong, No. HS2012034; the Jiangsu Health Projects, No. BL2012053 and No. K201102; the Priority Academic Program Development of Jiangsu, and the International S and T Cooperation Program of China, No. 2013DFA32150
Correspondence to: Deng-Fu Yao, MD, PhD, Professor, Research Centre of Clinical Medicine, Affiliated Hospital of Nantong University, 20 West Temple Road, Nantong 226001, Jiangsu Province, China. yaodf@ahnmc.com
Telephone: +86-513-85052297 Fax: +86-513-85052254
Received: March 13, 2013
Revised: April 30, 2013
Accepted: May 18, 2013
Published online: June 28, 2013
Processing time: 107 Days and 1.7 Hours
Abstract

AIM: To investigate the effects of Annexin A2 (ANXA2) silencing on invasion, migration, and tumorigenic potential of hepatoma cells.

METHODS: Human hepatoma cell lines [HepG2, SMMC-7721, SMMC-7402, and MHCC97-H, a novel human hepatocellular carcinoma (HCC) cell line with high metastasis potential] and a normal hepatocyte cell line (LO2) were used in this study. The protein and mRNA expression levels of ANXA2 were analysed by western blotting and real-time polymerase chain reaction, respectively. The intracellular distribution profile of ANXA2 expression was determined by immunofluorescence and immunohistochemistry. Short hairpin RNA targeting ANXA2 was designed and stably transfected into MHCC97-H cells. Cells were cultured for in vitro analyses or subcutaneously injected as xenografts in mice for in vivo analyses. Effects of ANXA2 silencing on cell growth were assessed by cell counting kit-8 (CCK-8) assay (in vitro) and tumour-growth assay (in vivo), on cell cycling was assessed by flow cytometry and propidium iodide staining (in vitro), and on invasion and migration potential were assessed by transwell assay and wound-healing assay, respectively (both in vitro).

RESULTS: The MHCC97-H cells, which are known to have high metastasis potential, showed the highest level of ANXA2 expression among the four HCC cell types examined; compared to the LO2 cells, the MHCC97-H expression level was 8-times higher. The ANXA2 expression was effectively inhibited (about 80%) by ANXA2-specific small hairpin RNA (shRNA). ANXA2 expression in the MHCC97-H cells was mainly localized to the cellular membrane and cytoplasm, and some localization was detected in the nucleus. Moreover, the proliferation of MHCC97-H cells was obviously suppressed by shRNA-mediated ANXA2 silencing in vitro, and the tumour growth inhibition rate was 38.24% in vivo. The percentage of MHCC97-H cells in S phase dramatically decreased (to 27.76%) under ANXA2-silenced conditions. Furthermore, ANXA2-silenced MHCC97-H cells showed lower invasiveness (percentage of invading cells decreased to 52.16%) and suppressed migratory capacity (migration distance decreased to 63.49%). It is also worth noting that shRNA-mediated silencing of ANXA2 in the MHCC97-H cells led to abnormal apoptosis.

CONCLUSION: shRNA-mediated silencing of ANXA2 suppresses the invasion, migration, and tumorigenic potential of hepatoma cells, and may represent a useful target of future molecular therapies.

Keywords: Annexin A2; Small hairpin RNA; Hepatocellular carcinoma; Invasion; Migration; Tumorigenic potential

Core tip: The overexpression of annexin A2 (ANXA2) is closely related to the high metastasis potential and invasion ability of HCC cells, and ANXA2 deficiency inhibits the invasion, migration, and tumorigenic potential of hepatocellular carcinoma (HCC) cells, which not only provides further insight into the pathogenesis of HCC but also provides a potential predictive biomarker for HCC prognosis and a potential therapeutic target.