Background: Dysregulation of erythropoietin-producing hepatoma (Eph) proteins in human cancers is extensively documented but not clear in colorectal cancer (CRC). In this study, we aimed to investigate the role of Notch signaling pathway and epigenetic modification of EPHB2 and EPHB4 expression in serrated neoplasia development. 
Methods: The expression of EPHB2 and EPHB4 in CRC clinical specimens and cell lines were determined by immunohistochemistry, Western blot, and real-time PCR. Cell proliferation and invasion were evaluated by MTT and chamber kits, luciferase assay and co-immunoprecipitation were used to detect the transcriptional regulation and protein–protein interactions, respectively. The immunofluorescence assay was employed to confirm the subcellular location of Notch intracellular domain (NICD), and chromatin immunoprecipitation assay was implied to detect the modification types of H3K4me3 and H3K27me3. Mice xenograft model was used to detect the in vivo effects of EPHB2 and EPHB4 genes on cell growth. 
Results: In CRC clinical specimens and cell lines, we found that EPHB2 was significantly decreased, while EPHB4 was elevated in the CRC tissues, and these aberrant expression manners correlated with worse overall survival rates in the clinic. When the EPHB2 and EPHB4 expressions were manipulated by overexpression or knockdown in the SW620 cells, the cell proliferation and invasion were obviously suppressed, whereas EPHB2 knockdown or EPHB4 overexpression showed the opposite phenotypes. We also found that Notch signaling pathway was abnormally activated and treatment of Notch signaling ligand human Jagged1 peptide downregulated EPHB2 and upregulated EPHB4 in the SW620 cells, as well as promoted the chromatin modification protein Jumonji domain-containing protein-3 (JMJD3) cytonuclear trans-localization with the NICD, which indicated that NICD brought JMJD3 to the EPHB4 enhancer region to decrease the H3K27me3 level. 
Conclusion: Taken together, we provide a new mechanistic option in understanding the role of Notch signaling and the roles of EPHB2 and EPHB4 in CRC.
Keywords: Notch signaling, Eph gene, histone methylation, colorectal cancer