Abstract 5012: Regulation of splicing of SEPT9 in health and disease

Abstract

Abstract Septins are members of a highly conserved family of 14 genes whose products are thought to act primarily as scaffolds, recruiting other proteins to particular cellular locations. This gene family shares a complexity of alternative splicing and encodes proteins involved in a number of diverse processes within the cell including cytokinesis, apoptosis and vesicle trafficking, and septins have been implicated in a number of diseases. Work in our laboratory has focussed on the SEPT9 gene and it was mapped it to a region of allelic imbalance on chromosome 17q in sporadic epithelial ovarian tumours. SEPT9 undergoes extensive alternative splicing and can form a possible 18 transcripts due to splicing at the 5’ (v1, v2, v3, v4, v4*, v5) and 3’ (a, b and c) ends, with SEPT9_v4 and SEPT9_v4* differing only in their 5’UTRs thus encoding the same protein. Previously we have shown perturbed expression of the SEPT9_v1 and SEPT9_v4* transcripts in neoplasia. In this study, we sought to determine the expression levels of all SEPT9 transcripts in ovarian tumours using samples from the Scottish Randomised Trial in Ovarian Cancer (SCOTROC) cohort, which compares two chemotherapy regimes. RNA was extracted from 100 FFPE tumour samples and qRT-PCR was performed for each SEPT9 transcript. Results were correlated with clinical data provided from the trial. Statistical significant elevated expression of SEPT9_v1 and SEPT9_v4* (p<0.0001) in tumour tissue compared to normal ovarian tissue was observed, in accordance with our previous data. Furthermore, we detected elevated expression of SEPT9_v5 and SEPT9_vb (p<0.0001) in tumour tissue compared to normal. Further statistical analysis shows correlations of SEPT9_v1, v4*, v5 and vb with tumour stage and SEPT9_v1, v5 and vb with tumour histology. This data suggests that the expression of the various SEPT9 transcripts is independently regulated. Of some note is the difference in 3’UTRs of the 3’ transcripts of SEPT9, with the b and c transcripts sharing the same short 3’UTR (57bases) while the a transcript has a longer 3’UTR (1.938kb). Bioinformatic analysis of the different 3’UTRs of SEPT9 identified a number of potential microRNA binding sites in the 3’UTR of SEPT9_va and only one of note in SEPT9_vb&c, with a number of these microRNAs previously shown to have altered expression in ovarian cancer. Therefore, we wished to determine if the 3’UTRs were important in the regulation of SEPT9. Luciferase assays performed from A2780 ovarian cancer cells show that the various 3’UTRs of SEPT9 drive the expression of reporter genes in a sequence dependent manner and particular microRNAs (hsa-miR-124, has-miR-494 and hsa-miR-199a) may regulate the expression of SEPT9_va. In summary, our data supports the hypothesis that the expression of the splice variants of SEPT9 and their various isoforms is tightly regulated and provides the first evidence that this regulation includes differential action of microRNAs on sites within 3′UTRs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5012. doi:10.1158/1538-7445.AM2011-5012