Abstract 4081: Antisense against U6 induces cytotoxicity in cancer cells

Abstract

Abstract Background: The U6 snRNA is one of the most conformationally dynamic RNAs in the spliceosome. In preparation for spliceosome assembly, the U6 3′ intramolecular stem-loop unwinds and the exposed sequences pair with U4 RNA to form the U4/U6 small nuclear ribonucleoprotein complex. After assembly of the spliceosome on an intron, spliceosome activation occurs, during which U4 RNA is unwound from U6 RNA. U6 then base-pairs with U2 RNA to form the catalytically active form of the spliceosome. Upon activation of the spliceosome the “U6-ISL” structure forms to position important nucleotides of the U6 snRNA at the 5′splice site so they can base-pair with the pre-mRNA at the 5′ prime splice site. Here we report the cytotoxic activity against cancer cells treated with antisense DNAs against U6, especially those targeting U6-ISL. Methods: The functional activity of U6 antisense oligos was tested by cytotoxic and apoptosis assays. In the cytotoxic assay, U6 antisense oligos were reverse transfected into various mammalian cell lines, and the cytotoxic activity was measured by cell-titer blue 72 hr post-transfection. In the apoptosis assay, U6 antisense oligos are reverse transfected into various mammalian cell lines, and the apoptosis was measured by the release of caspase 3/7 72 hr post-transfection. To evaluate the knockdown ability of antisense oligos, the expression level of U6 snRNA was measured by Taqman Gene Expression Assay. The localization of U6 antisense oligos in tissue assay was studied by ISH. The effects of U6 antisense oligos on cellular protein and mRNA profile were also examined using microarray technology. Results: About 100 U6 antisense oligos were synthesized. Several U6 antisense oligos were identified to significantly induce cytotoxic activity of HT29 and other cancer cell lines but not in normal cells, such as hepatocytes. No significant apoptosis activity was induced by any U6 antisense oligos. The identified U6 antisense oligos localized at the nucleus and knocked down the U6 snRNA in vitro, suggesting that U6 antisense oligos target to U6 snRNA and lead to the downregulation of U6 snRNA. mRNA microarray and antibody array showed that U6 oligos regulate various mRNA and protein expression in cancer cells. The most effective U6 antisense oligos were those targeting the U6-ISL. Conclusions: Antisense against U6, especially those targeting U6-ISL region, were effective in U6 knockdown and cytoxicity against cancer cells in vitro. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4081.