Therefore, we focused on ribozyme-based functional screening. Ribozymes are small catalytic RNA molecules comprising target recognition sequences and a catalytic center with RNase activity. Ribozyme libraries have been used to identify genes that are associated with several pathways [12]. Additionally, OZ1, a vector containing a ribozyme targeting selleckchem the reading frames of HIV-1, was used in a clinical trial [13]. In the present study, we hypothesized that if the genes sensitizing HCC cells to 5-FU are identified, they could be applied to IFN-��/5-FU therapy and used as prognostic markers. To confirm this hypothesis, we used ribozymes to perform reverse genetics-based functional screening to identify genes that augment the efficacy of IFN-��/5-FU therapy.

Materials and Methods Ethics Statement All animal experiments were approved by the Institutional Animal Care and Use Committee of Tottori University (the permit number: 10-Y-54). The mice received humane care in accordance with the study guidelines for the care and use established by the Tottori University. All mice were kept under pathogen-free conditions and were maintained in a temperature-controlled room with a 12 h light/dark illumination cycle. Materials and Cell Culture IFN-�� and 5-FU were provided by MSD (Tokyo, Japan) and Kyowa Hakko Kirin Co., Ltd. (Tokyo, Japan), respectively. HepG2, HuH7, and HLF human HCC cells were obtained from the Japanese Collection of Research Bioresources and maintained in Dulbecco��s modified Eagle��s medium (Nissui Pharmaceutical Co., Ltd.

, Tokyo, Japan) supplemented with 10% fetal bovine serum (MBL, Nagoya, Japan), L-glutamine, and glucose in a humidified atmosphere of 5% CO2 at 37��C. The Water Soluble Tetrazolium Salt (WST) Assay The IFN-�� and 5-FU antitumor effects were assessed using the WST assay (Seikagaku Corporation, Tokyo, Japan). The cells were cultured in various concentrations of IFN-�� and 5-FU for 72 h. The viability of cells treated with dimethyl sulfoxide was defined as 100%. Screening for Genes Involved in Enhancing the Effect of 5-FU We constructed a plasmid DNA (pDNA) library expressing ribozyme genes (Figure S1 and Table S1). Screening was performed on the basis of the ability of 5-FU to eliminate cells expressing ribozymes that target unrelated chemosensitive genes, as described in File S1.

Subcutaneous Xenograft Model in Mice Four-week-old male non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). We subcutaneously transplanted 6.8��106 cells HepG2 cells in 0.1 mL PBS into the right flank of each mouse. The mice were randomly assigned to the 4 following groups; (i) Brefeldin_A LacZ-adenovirus and PBS (instead of IFN-�� and 5-FU); (ii) LacZ-adenovirus and IFN-��/5-FU; (iii) TGFBR2-adenovirus and IFN-��/5-FU; and (iv) EXT1-adenovirus and IFN-��/5-FU.