ODANI Shoji Niigata University, Institute of Science and Technology, Professor, 自然科学系, 教授 (60018702)
OHKOSHI Shogo Niigata University, Medical and Dental Hospital, Lecturer, 医歯学総合病院, 講師 (70231199)
IGARASHI Masato Niigata University, Medical and Dental Hospital, Fellow, 医歯学総合病院, 医員 (00401745)
須田 剛士 新潟大学, 大学院・医歯学総合研究科, 助手 (10361916)
|Budget Amount *help
¥3,500,000 (Direct Cost : ¥3,500,000)
Fiscal Year 2006 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 2005 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 2004 : ¥2,500,000 (Direct Cost : ¥2,500,000)
In Japan, hepatocellular-carcinoma (HCC) is one of the most prevalent human cancers. The diagnosis and treatment for HCC are still difficult although several therapeutic modalities and serological tumor markers, such as -alpha-fetoprotein (AFP), have been developed. We have already shown that the relative amount of the Lens culinaris agglutinin (LCA)-reactive species of AFP is significantly greater in HCC than in non-neoplastic liver diseases and that the higher proportion of (LCA)-reactive species of AFP is significantly associated with the tumor invasiveness. The molecular basis of the LCA-reactive species of AFP is the fucosylation at the innermost N-acetylglucosamine residue of the biantennary sugar chain of AFP. Thus, it is supposed that these phenotypic changes must be provided by GDP-L-Fuc: N-acethy1-13-D-glucosaminide: al-6 fucosyltransferase (αFT), which catalyzes the addition of fucose from GDP-fucose through an α1-6 linkage to the reducing end of N-acetylglucosamine residue
of N-linked oligosaccharides of glycoproteins. Accordingly, we attempt to perform the treatment of HCC by the regulation of αFT in the present project.
For this issue, we attempted to establish αFT knockdown cells aiming to evaluate the effects of αFT activity on cellular glycosylation profile and cell phenotype. According to analysis of secondary structure and BLAST search for αF : GenBank D89289, the sequence consisting of 19 nucleotides from the position 1002 was selected as a target to induce RNA interference against aFT. Synthetic DNAs of 64 base pairs designed to form a small hairpin RNA.including the target was cloned using pSUPER.retro plasmids (OligoEngine, Seattle, USA), which-involve a puromycin resistance gene. The plasmids were transfected to HepG2 cells by lipofection, and positive cells were selected by exposing to puromycin. Although the clones carrying empty plasmids or the target sequence with G to A transition at the middle have been established, no clones carrying the target sequence has been established.
In order to clarify the reasons for the failure to establishαFT knockdown cells, we took the same strategy targeting HBV in huH-1 cells and established HBV knockdown cells. There was no significant difference of cell viability among the cells with and without pSUPER.retro plasmids. Immunohisotochemical studies revealed marked reduction of HBx gene products in the knockdown cells and in the liver tissues of HBx transgenic mice, in which the same constructs were delivered by hydrodynamic gene delivery. The concentration of HBsAg and the level of HBV expression were suppressed in the knockdown cells to less than 5 and 50 % of those in cells carrying the target with A to G transition at the middle, respectively. Furthermore, lipofection of 2'-methoxyethyl phosphorothioate RNAs comprised of 13 nucleotides from the position 861 in αFT open reading frame transiently diminished the fucosylation of AFP in HepG2 cells. Those results suggest that the RNAi system we employed works properly and targeting αFT is not critical in terms of cell viability. Because a seed locator program, http://www.dharmacon.com/seedlocatoridefault.aspx, detected multiple matches in more than thousands cellular genes with the target sequence we selected for RNA interference against αFT, it is highly possible that off-target effects led to the difficulty in the establishment of αFT knockdown cells. Circumvention of the off-target effects should be demanded for a successful knockdown of αFT in RNA interference system. Less