AIM: To observe the gene silencing mediated by the specific shRNA targeted against beta-catenin and its effect on cell proliferation and cycle distribution in the human colon cancer cell line Colo205. METHODS: Two shRNA plasmid vectors against beta-catenin were constructed and transfected into Colo205 cells with Lipofectamine2000. The down-regulations of beta-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two beta-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: These two shRNA vectors targeted against beta-catenin efficiently suppressed the expression of beta-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of beta-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing. CONCLUSION: These specific shRNAs targeted against beta-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against beta-catenin is of potential value in gene therapy of colon cancer.
AIM: To observe the gene silencing mediated by the specific shRNA targeted against beta-catenin and its effect on cell proliferation and cycle distribution in the humancolon cancer cell line Colo205. METHODS: Two shRNA plasmid vectors against beta-catenin were constructed and transfected into Colo205 cells with Lipofectamine2000. The down-regulations of beta-catenin, c-myc and cyclinD1 expressions were detected by RT-PCR and western blot analysis. The cell proliferation inhibitions were determined by MTT assay and soft agar colony formation assay. The effect of these two beta-catenin shRNAs on cell cycle distribution and apoptosis was examined by flow cytometry. RESULTS: These two shRNA vectors targeted against beta-catenin efficiently suppressed the expression of beta-catenin and its down stream genes, c-myc and cyclinD1. The expression inhibition rates were around 40%-50% either at the mRNA or at the protein level. The shRNA-mediated gene silencing of beta-catenin resulted in significant inhibition of cell growth both on the culture plates and in the soft agar. Moreover, the cancer cells showed significant G0/G1 arrest and increased apoptosis at 72 h post transfection due to gene silencing. CONCLUSION: These specific shRNAs targeted against beta-catenin could have a gene silencing effect and block the WNT signaling pathway. They could inhibit cell growth, increase apoptosis, and induce cell cycle arrest in Colo205 cells. ShRNA interference against beta-catenin is of potential value in gene therapy of colon cancer.
Authors: Udit N Verma; Rama M Surabhi; Aurelia Schmaltieg; Carlos Becerra; Richard B Gaynor Journal: Clin Cancer Res Date: 2003-04 Impact factor: 12.531
Authors: S M Powell; N Zilz; Y Beazer-Barclay; T M Bryan; S R Hamilton; S N Thibodeau; B Vogelstein; K W Kinzler Journal: Nature Date: 1992-09-17 Impact factor: 49.962
Authors: John J Tentler; Sujatha Nallapareddy; Aik Choon Tan; Anna Spreafico; Todd M Pitts; M Pia Morelli; Heather M Selby; Maria I Kachaeva; Sara A Flanigan; Gillian N Kulikowski; Stephen Leong; John J Arcaroli; Wells A Messersmith; S Gail Eckhardt Journal: Mol Cancer Ther Date: 2010-10-05 Impact factor: 6.261