Reactivation of oncogenes involved in G1/S transcription and apoptosis pathways by low dose decitabine promotes HT29 human colon cancer cell growth in vitro.

backgroundTo examine the effects of low-dose decitabine (DAC) on the proliferation of HT-29 cell lines, and to explore the central mechanism by which low-dose DAC affects HT-29 cell proliferation using a systematic biological approach.

methodsFirst, we examined the global effects of DAC on cell proliferation, the cell cycle, and apoptosis in HT29 colon cancer cells. Then, a series test of cluster (STC) analysis and weighted gene coexpression network analysis (WGCNA) were employed to identify critical pathways involved in the response to DAC treatment using 3 datasets from the GEO database. Finally, the expression changes and promoter methylation levels of hub genes were further confirmed by in vitro experiments.

resultsLow-dose DAC (less than 1 µM) promoted the proliferation and colony formation ability of HT-29 cell lines. The results of the system-level analysis, including STC analysis, WGCNA, and Gene set variation analysis (GSVA), showed that DAC modulated 3 critical pathways: G1/S-specific transcription involved in E2F-mediated regulation of Cyclin E-associated events, apoptosis pathways, and EMT pathways. Subsequent in vitro experiments showed that low-dose DAC (0.1 µM) promoted G1/S-specific transcription and decreased apoptosis rates. Then, several regulatory hub oncogenes in these 3 pathways, CCNE1, E2F1, BCL2, PCNA, FOXC1, VIM, CXCL1, and VCAM1, were further confirmed to be activated by DAC at either the mRNA or protein level. We chose the oncogene BCL2 as an example and detected its methylation status and the effect of low-dose DAC on BCL2 expression. Data from TCGA and Oncomine databases demonstrated that BCL2 was decreased in colon cancer compared with normal mucosa. Further analysis showed that BCL2 had an increased degree of promoter methylation in 12 methylated sites in colon cancer compared with normal colon tissues. Bisulfite sequencing PCR showed that low-dose DAC decreased the methylation rate at the BCL2 promoter region.

conclusionsWe concluded that low-dose DAC treatment resulted in a cancer-promoting effect in HT29 cell lines. Mechanistically, high methylation levels at the promoter region of oncogenes with dominant effects in CRC, such as BCL2 in HT29, might play a role in suppressing CRC by inhibiting oncogene expression. Low-dose DAC treatment triggered BCL2 expression by decreasing its promoter methylation level, thereby resulting in cancer promotion.