Decoy and antisense RNA oligonucleotides targeting the EGFR exon 3 splice site suppress proliferation of A172 glioblastoma cells

Short RNA oligonucleotides directed at the 3′ splice site region of EGFR exon 3 reduced the proliferation of A172 glioblastoma cells, while the same oligonucleotides had no detectable effect on A549 lung adenocarcinoma cells. Glioblastoma carries EGFR amplification in roughly a third of IDH–wildtype cases, and the A172 line harbours a tandem duplication in the EGFR tyrosine kinase domain. Because exon definition in human genes depends on RNA–binding proteins that engage exonic sequence near splice sites, we used the SpliceAid database to identify candidate RNA–binding proteins at EGFR exons 2 and 3. Y–box binding protein 1 (YB–1, encoded by YBX1), reported to drive glioblastoma growth through its RNA–binding function, scored highly immediately adjacent to the exon 3 3′ splice site. We designed four 2′-modified RNA oligonucleotides carrying a nuclear localisation signal: two antisense oligonucleotides against the exon 3 region (asRNA1, spanning the splice site; asRNA2, downstream of it), a decoy complementary to the asRNA2 target (dRNA2), and a decoy carrying the YB–1 consensus binding sequence (dYB–1). In A172 cells, all four reduced EGFR protein in a single Western blot experiment, and asRNA1, asRNA2 and dRNA2 reduced proliferation relative to scrambled RNA at days 1 to 3; dYB–1 produced a comparable reduction at day 3. None of these effects was seen in A549 cells. YB–1 protein level and subcellular distribution were unchanged by the oligonucleotides, arguing against an indirect route through altered YB–1 abundance. These observations are preliminary. Every experiment reported here was performed once: the proliferation data come from single plates with three wells per group, analysed by Dunnett′s test against the scrambled–RNA control, and the Western blot, RT–PCR and immunocytochemistry panels are single runs presented as representative observations. Direct binding of YB–1 to the targeted EGFR sequence has not been demonstrated. We post this work as a preprint so that the design strategy and the initial observations are available while these questions remain open.