title: Annexin A2 silencing inhibits invasion, migration, and tumorigenic potential of hepatoma cells.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Research Centre of Clinical Medicine, Affiliated Hospital of Nantong University, Nantong 226001, Jiangsu Province, China.
authors.forename: Hai-Jian
authors.initials: HJ
authors.lastname: Zhang
authors.forename: Deng-Fu
authors.initials: DF
authors.lastname: Yao
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.forename: Hua
authors.initials: H
authors.lastname: Huang
authors.forename: Li
authors.initials: L
authors.lastname: Wang
authors.forename: Mei-Juan
authors.initials: MJ
authors.lastname: Yan
authors.forename: Xiao-Di
authors.initials: XD
authors.lastname: Yan
authors.forename: Xing
authors.initials: X
authors.lastname: Gu
authors.forename: Wei
authors.initials: W
authors.lastname: Wu
authors.forename: Shao-Lin
authors.initials: SL
authors.lastname: Lu
mesh_terms: D017306:Annexin A2 / Q000235:genetics* / Q000502:physiology; D063646:Carcinogenesis / Q000473:pathology*; D006528:Carcinoma, Hepatocellular / Q000473:pathology* / Q000503:physiopathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000502:physiology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D020868:Gene Silencing / Q000502:physiology*; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D008113:Liver Neoplasms / Q000473:pathology* / Q000503:physiopathology; D009361:Neoplasm Invasiveness / Q000473:pathology / Q000503:physiopathology; D034741:RNA, Small Interfering / Q000494:pharmacology
chemical_list: D017306:Annexin A2; D034741:RNA, Small Interfering
keywords: Annexin A2; Hepatocellular carcinoma; Invasion; Migration; Small hairpin RNA; Tumorigenic potential