Epigenetic regulation of CDH1 exon 8 alternative splicing in gastric cancer

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: 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.

Fig. 1

a Splice sites prediction by bioinformatic analysis. Capital letters show CDH1 exon 8 and lowercases, the flanking intron sequences. The italic characters indicate SRSF2 motifs flanking donor site 1. b Schematic diagram of alternative splicing of CDH1 exon 8

Fig. 2

Expression of CDH1 normal transcript (a), CDH1 1054del83 transcript (b), and ratio of expression value of CDH1 1054del83 transcript vs CDH1 normal transcript (c) in human gastric mucosal epithelial cell line GES-1 and the GC cell lines SGC-7901, BGC-823 and MGC80-3. The expression level in GES-1 was set to 1. The star * means P < 0.05, indicating statistically significant

Fig. 3

Methylation status of CDH1 exon 8 in human gastric mucosal epithelial cell line GES-1 (a) and the GC cell lines SGC-7901 (b), BGC-823 (c) and MGC80-3 (d). DNA isolated from cells shows a high C content at all CpGs attributable to reduced bisulfite modification because of complete methylation of the DNA

Fig. 4

Histone modification patterns of CDH1 in SGC-7901 (a) and BGC-823 (b) compared to GES-1. The graphs demonstrate fold differences of distinct histone modifications at different region of CDH1 between SGC-7901 or BGC-823 and GES-1 cells

Fig. 5

The relative folds of ratios of CDH1 1054del83 transcript vs CDH1 normal transcript after AZA or TSA treatment. The values were calculated as (2^{-ΔCt (1054del83 transcript-actin)}/2^{-ΔCt (normal transcript-actin)}). Relative folds of ratios of untreated cell lines serve as 1. The star * means P < 0.05, indicating statistically significant

Fig. 6

The level change of CDH1 normal transcript and CDH1 1054del83 transcript after TSA treatment in SGC-7901 (a) and BGC-823 (b). The values were calculated as 2^{-ΔCt (1054del83 transcript-actin)} or 2^{-ΔCt (normal transcript-actin)}. The values of untreated cell lines were set to 1. The star * means P < 0.05, indicating statistically significant

Fig. 7

CDH1 expression after siSETD2 treatment. a Interference efficiency of the three kinds of siSETD2 in GES-1 cells. The values were calculated as 2^{-ΔCt (SETD2-actin)}. The star * means P < 0.05, indicating statistically significant. b The relative folds of ratios of CDH1 1054del83 transcript vs CDH1 normal transcript after siSETD2-003 treatment. The values were calculated as (2^{-ΔCt (1054del83 transcript-actin)}/2^{-ΔCt (normal transcript-actin)}). Relative folds of ratios of untreated cell lines serve as 1

Fig. 8

CDH1 expression after siSRSF2 treatment. a Interference efficiency of the three kinds of siSRSF2 in GES-1 cells. The values were calculated as 2^{-ΔCt (SRSF2-actin)}. The star * means P < 0.05, indicating statistically significant. b The relative folds of ratios of CDH1 1054del83 transcript vs CDH1 normal transcript after siSRSF2-001 treatment. The values were calculated as (2^{-ΔCt (1054del83 transcript-actin)}/2^{-ΔCt (normal transcript-actin)}). Relative folds of ratios of untreated cell lines serve as 1