title: [Effect of β-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells].
abstract: OBJECTIVE
  To study the effects of short hairpin RNA (shRNA) mediated gene silencing of β-catenin on the biological characteristics of esophageal carcinoma cells, and to provide theoretical and experimental evidence for the gene therapy of esophageal carcinoma through target inhibition of β-catenin gene.
  
  
  METHODS
  Single strand DNA was synthesized according to the hairpin RNA sequence, and then subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter of β-catenin (pGen-3-CTNNB1). One additional construct of random siRNA (pGen-3-con) without homologous to any human genes was constructed in a similar fashion as control.Positive clones were identified and verified by restriction cleavage and DNA sequencing analyses. pGen-3-CTNNB1 and pGen-3-con were then transfected into esophageal carcinoma cell line Eca-109 with liposome, respectively. Positive colonies were selected with G418. Expression of β-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells were examined by Western blotting, immunofluorescence and RT-PCR, respectively. Xenograft tumor model was used to compare the tumorigenesis of three different cells.Expressions of β-catenin in all tumor tissues were examined by immunohistochemistry staining. The invasive abilities of three different cells were examined with transwell invasion filter and Matrigel.
  
  
  RESULTS
  β-catenin expression levels were found markedly decreased in Eca-109 cells transfected with pGen-3-CTNNB1. In vivo, transfection with β-catenin shRNA greatly impeded the tumor growth, pGen-3-con (1.18 ± 0.13) g, Eca-109 (1.38 ± 0.21) g, pGen-3-CTNNB1 (0.42 ± 0.09) g, P < 0.05. Immunohistochemistry staining showed a significantly decreased expression of β-catenin in β-catenin shRNA transfected cells than in random shRNA transfected and nontransfected cells (P < 0.05). The infiltration abilities of esophageal carcinoma cells were significantly suppressed, pGen-3-con (81 ± 5)/HPF, Eca-109 (77 ± 6)/HPF, pGen-3-CTNNB1 (41 ± 4)/HPF, P < 0.01; along with significantly decreased migration abilities, pGen-3-con (73 ± 5)/HPF, Eca-109 (69 ± 5)/HPF, pGen-3-CTNNB1 (38 ± 4)/HPF (P < 0.05).
  
  
  CONCLUSIONS
  There are abnormal expression of β-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109. RNA interference targeting β-catenin gene suppresses the growth of xenograft tumorigenesis in nude mouse and the invasiveness and metastatic capability of esophageal carcinoma cells.
authors.affiliation: Central Laboratory, Heping Hospital Affiliated to Changzhi Medical University, Changzhi 046000, Shanxi Province, China.
authors.forename: Jin-Sheng
authors.initials: JS
authors.lastname: Wang
authors.forename: Ai-Fang
authors.initials: AF
authors.lastname: Ji
authors.forename: Ji-Fang
authors.initials: JF
authors.lastname: Wen
authors.forename: Hong-Zheng
authors.initials: HZ
authors.lastname: Ren
mesh_terms: D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D045744:Cell Line, Tumor; D002465:Cell Movement; D004938:Esophageal Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D005260:Female; D020868:Gene Silencing; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D010957:Plasmids; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics*; D011897:Random Allocation; D015398:Signal Transduction; D014162:Transfection; D047368:Tumor Burden; D051153:Wnt Proteins / Q000378:metabolism; D051176:beta Catenin / Q000235:genetics* / Q000378:metabolism / Q000502:physiology
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051153:Wnt Proteins; D051176:beta Catenin