Effect of β-catenin gene silencing by shRNA on biologic characteristics of human esophageal carcinoma cells

Abstract

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. 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. 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). 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.