Hyperstable U1snRNA complementary to the K-ras transcripts induces cell death in pancreatic cancer cells.
In this study, we utilised U1 small nuclear RNA (snRNA) that binds physiologically to the 5' splice site (5'ss) of pre-mRNA, to develop a novel vector system that permits imposed binding of antisense RNA to its target.This revealed that two of the hyperstable U1snRNAs induced cell death after gene transduction, and significantly reduced the number of G418-resistant colonies to less than 10% of the controls.Hyperstable U1snRNA might be a novel approach to express effective antisense RNA in target cells.
Affiliation: Investigative Treatment Division, National Cancer Center Research Institute East, 6-5-1, Kashiwanoha, Kashiwa, Chiba 277-8577, Japan.
- Genes, ras*
- Pancreatic Neoplasms/pathology*
- Point Mutation*
- RNA, Small Nuclear/genetics*/pharmacology
- Blotting, Northern
- Cell Death
- Cell Division
- Colony-Forming Units Assay
- Mice, Inbred BALB C
- Mice, Nude
- Neoplasm Transplantation
- Neoplasms, Experimental/genetics/metabolism/pathology
- Peritoneal Neoplasms/genetics/metabolism
- RNA, Antisense
- Transduction, Genetic
- Tumor Cells, Cultured
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fig1: Construction of the hyperstable U1snRNA vector. (A) In the human U1snRNA gene (Hitomi et al, 1998), six- bases were deleted to generate a NdeI site in the region of the 5′-free end of U1snRNA. The resulting sequence in this region (nU1) is shown with respect to the wild-type sequence. The newly created NdeI site is boxed and the arrowhead indicates the cleavage site. (B) Insertion of the antisense sequence in the U1snRNA gene generates hyperstable U1snRNA, which binds to the target site via the inserted sequence in the 5′-free end of U1snRNA. The secondary structure of U1snRNA is shown as a line drawing. (C) The target sites against the human K-ras transcript are indicated as r1, r2, r3, r4 and r5, aligned to the corresponding K-ras gene structure. The K-ras exons 1, 2, and 3 are indicated by the numbered boxes. The sites of the 20 base-long oligonucleotides used for northern analysis are also indicated as r1-20 for the detection of r1, r2-20 for r2, r4-20 for r3, r4, and r5. The relative lengths of the target sites, exons, and oligonucleotides are fitted to the scale shown above, whereas the intron sequences are shortened. (D) Transient expression of hyperstable U1snRNA in COS-1. The expression plasmid of hyperstable U1snRNA was transfected into COS-1. Three μg of total RNA was separated on a 5% polyacrylamide/7 M urea gel, electroblotted and hybridised with radiolabelled sense (upper panels) or antisense (lower panels) K-ras oligonucleotides corresponding to the target sites, r1 to r5. The position of antisense hyperstable U1snRNA (aU1), sense hyperstable U1snRNA (sU1), and wild-type U1snRNA (wtU1, 164 bases) is indicated. The integrity and load of RNA was examined by hybridizing the same membrane with U2B, an U2snRNA-specific oligonucleotide.
The U1snRNA expression vector has been described elsewhere (Hitomi et al, 1998). The NdeI site was created within the region of the 5′-free end of U1snRNA, using mutated oligomers GAT CTC ATA TGG CAG GGG AGA TAC and TCC CCT GCC ATA TGA GAT CTT GG, generating pEUK/nU1. Antisense and sense oligomers of five target sequences in the K-ras gene (see Figure 1CFigure 1