| Objective:To investigate the effects of low-dose decitabine(DAC)on the phenotypes of HT29 colon cancer cells.Materials and methods:According to whether DAC was used or not,HT29 colon cancer cells were divided into two groups:(1)DAC treatment group:HT29 colon cancer cells were treated with Dulbecco's modified Eagle's medium(DMEM)containing different concentrations of DAC(10-3to 100?M).(2)Control group:HT29 colon cancer cells cultured in DMEM medium.Cell proliferation was detected by cell counting kit-8(CCK-8)assay and colony formation assays.Cell cycle was detected by flow cytometry.Apoptosis was detected by 7-AAD and Annexin V-PE double staining.Continuous variables were compared by t-test,one-way ANOVA,or repeated-measures ANOVA.Graphpad prism 7 software,R3.5.1 software,or SPSS 20.0 software were used for statistical analysis.A P-value<0.05 was considered to indicate statistical significance.Results:CCK-8 assays indicated that DAC,used in the concentration range from10-3to 0.1?M,promoted the proliferation of HT-29 cells in vitro,while it showed no effect on survival in the concentration range from 1 to 10?M;further,it showed an inhibitory effect at concentrations greater than 10?M.Similarly,low-dose DAC(0.1?M)treatment significantly increased the colony formation ability of HT-29cells in vitro and slightly decreased the colony formation ability at a concentration of 1?M.HT-29 cells exhibited a cobblestone shape.Interestingly,following 0.1?M DAC treatment for 48 h,HT-29 cells exhibited a spindle shape with mesenchymal morphology.In addition,treatment with 0.1?M DAC resulted in an increase in the number of cells in the S phase compared to that of the control group(DAC vs control:27.0%vs 14.7%)at the expense of the G1 phase(DAC vs control:57.4%vs 67.6%).The results indicated that low-dose DAC treatment promoted G1/S-specific transcription.The percentage of early apoptotic and total apoptotic cells slightly decreased by?1%when cells were coincubated with low-dose DAC at concentrations ranging from 10-2to 10?M.Conclusion:Low-dose of DAC promotes the proliferation and colony-forming ability of HT29 colon cancer cells,increases the proportion of the S phase,slightly inhibits apoptosis.HT-29 cells exhibit a spindle shape with mesenchymal morphology after low-dose of DAC treatment.Objective: To explore the mechanism by which DAC promoting the proliferation of HT29 colon cancer cells by dynamic transcriptomics analysis.Materials and methods: A total of three microarray datasets,including GSE41364,GSE32323,and GSE22598,were obtained from the Gene Expression Omnibus(GEO)database.Series test of cluster(STC)analysis was performed to study the changes in gene expression and to determine different expression tendencies as a result of increasing concentrations of drug treatment.These genes with the same expression change trends were clustered for further analysis.The data from GSE41364 were used to construct scale-free coexpression networks using the weighted gene coexpression network analysis(WGCNA)algorithm.The hub modules subject to DAC treatment were identified by Pearson's correlation analysis.Fun Rich was used on the most representative trend profile from the STC analysis and hub modules from the WGCNA to identify hub pathways involved in DAC treatment using a community-driven approach.Gene set variation analysis(GSVA)was used to estimate the pathway activity changes over a sample population subject to DAC treatment in an unsupervised manner.Two datasets(GSE32323 and GSE22598)were used to verify the m RNA expression of VIM,CXCL1,VCAM1,E2F3,E2F1,CCNE,PCNA,FOXC1,and BCL2 between the DAC treatment group and the control group.Results: The results of STC analysis showed that with the increase of DAC concentration,the changing trend of transcriptome gene transcription level could be summarized into eight trend patterns,and the overall transcription level showed an upward trend.The trend 6 with the lowest P-value(including 2893 genes,P =3.7e-169)was selected for further pathway enrichment analysis.WGCNA analysis showed that there were 6 modules related to DAC treatment,which were included in further analysis.The functional enrichment analysis suggests that a total of 3signaling pathways were changed subject to DAC treatments:(1)G1/S-specific transcription involved in E2F-mediated regulation of cyclin E-associated events;(2)epithelial-to-mesenchymal transition(EMT);(3)regulation of apoptosis.Among these pathways,GSEA revealed that G1/S-specific transcription was activated by low-dose DAC treatment and was inhibited by treatment with a higher dose.The EMT pathway activity was slightly increased during zero-to high-dose DAC treatment,while apoptosis was consistently inhibited.In both the GSE41364 dataset and the GSE32323 dataset,DAC increased the expression of the markers of these 3 pathways.Conclusion: A total of 3 hub signaling pathways were changed subject to DAC treatments:(1)G1/S-specific transcription involved in E2F-mediated regulation of cyclin E-associated events;(2)EMT;(3)regulation of apoptosis.Objective: Verification of hub gene expression in the G1/S-specific transcription,apoptotic pathway,and EMT pathway.Materials and methods: The dose of 0.1 ?M DAC was selected to stably culture HT29 colon cancer cells.The m RNA expressions of E2F1,E2F3,CCNE1,BCL2,PCNA,FOXC1,VIM,CXCL1,and VCAM1 were detected by real-time quantitative PCR(RT-q PCR).The protein expressions of cyclin E1,PCNA,Bcl-2,and Bax were detected by Western blot.Continuous variables were compared by t-test.Graphpad prism 7 software,R 3.5.1 software,or SPSS 20.0 software was used for analysis.A P-value <0.05 was considered to indicate statistical significance.Results: Verification of hub gene expression in the following pathways was performed:(1)G1/S-specific transcription: in response to 0.1 ?M DAC,the E2F1,E2F3,and CCNE1 m RNA expression was upregulated,while the protein expression of Cyclin E1 and PCNA was upregulated.(2)Endogenous apoptotic pathway: 0.1 ? l DAC increased the expression of BCL2,PCNA,and FOXC1 at the m RNA level and Bcl-2 at the protein level.The expression of Bax protein decreased.(3)EMT pathway: our q PCR assays demonstrated that the m RNA expression of VIM,CXCL1,and VCAM1 increased after 48 hr of 0.1 ?M DAC treatment.Conclusion: low-dose DAC activates the G1/S transformation of HT29 colon cancer cells by activating cyclin E1,and inhibits the endogenous apoptosis pathway of HT29 colon cancer cells by activating Bcl-2 protein.Objective: To select BCL2 as an example to provide a new theoretical explanation for the cancer-promoting effect of DAC.Materials and methods: The expression and methylation data of a DAC-activated gene,BCL-2,in CRC from The Cancer Genome Atlas(TCGA)database were obtained from MEXPRESS.Oncomine was used to perform a meta-analysis to confirm the differential expression levels of BCL2 between colorectal cancer and normal tissues.Bisulfite sequencing PCR(BSP)was performed to detect the methylation level of the BCL2 promoter of HT29 cells subjected to DAC treatment.Results: Based on the transcriptome data of 499 patients with colon cancer in the TCGA database,the expression of BCL2 m RNA in colon cancer was lower than that in normal mucosa(14.4 ± 1.1 vs.16.3 ± 0.3,P < 0.0001).A meta-analysis based on transcriptome data of 19 colorectal cancer datasets in Oncomine further confirmed that the expression of BCL2 m RNA in colorectal cancer tissues was lower than that in normal mucosa(P = 2.27e-6).To discover the underlying upstream regulatory mechanism by which BCL2 levels decreased in colorectal cancer,we analyzed the promoter methylation of BCL2 in colon cancer patients from the TCGA database and found that BCL2 had an increased level of methylation at 12 methylated sites in the promoter region of the colon cancer compared with that of normal colon tissues.The further BSP results showed that the DNA methylation rate of the BCL2 promoter in the 0.1 ?M DAC group was lower than that in the control group(41.1% vs 57.9%).Conclusion: the expression of BCL2 m RNA in colorectal cancer is lower than that in normal mucosa,and its promoter methylation level is higher than that in normal mucosa.The methylation level of BCL2 promoter decreased in HT29 colon cancer cells subjected to DAC treatment. |
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