Epigenetic regulation of CDH1 exon 8 alternative splicing in gastric cancer

Xiao-Wei Li, Bing-Yu Shi, Qing-Lan Yang, Jie Wu, Hui-Min Wu, Yu-Feng Wang, Zhi-Jiao Wu, Yi-Mei Fan, Ya-Ping Wang

The tumor suppressor gene CDH1 is critical for intercellular adhesion. In our previous work, we reported a nonfunctional CDH1 transcript that lacks the final 83 base pairs of exon 8 (1054del83). In this work, we probed the role of histone epigenetic modifications as well as DNA methylation in selection of this isoform. RT-qPCR was used to detect CDH1 RNA expression. Methylation of CDH1 was analyzed by bisulphite sequencing PCR. ChIP assay was performed to show histones level. Cell lines were treated with DNA methyltransferase inhibitor AZA, HDAC inhibitor TSA, or siRNA oligonucleotides to test regulation of CDH1 splicing. Greater CDH1 1054del83 transcripts were observed in gastric cancer (GC) cell lines than human gastric mucosal epithelial cell line GES-1. All the cell lines showed significant methylation pattern at the CpG sites of CDH1 exon 8. AZA treatment did not influence selection of 1054del83 transcripts. A significant decrease in acetylation for histones H3 and H4K16Ac in an internal region of the CDH1 gene surrounding the alternative exon 8 were detected in GC cell lines. Treatment with TSA preferentially expressed the correctly spliced transcript and not the exon 8 skipped aberrant transcripts, showing that histone acetylation was involved in the splicing regulation. SiRNA-mediated knockdown of SETD2 (The specific methyltransferase of H3K36) decreased exclusion of exon 8, suggesting that the presence of this mark correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. However, CDH1 splicing was not affected by SRSF2 knockdown. H3K36me3 correlates with increased skipping of the final 83 base pairs of CDH1 exon 8. Histone acetylation was involved in the splicing regulation as well.