| Esophageal carcinoma(EC)is the 3rd leading cause of cancer morbidities and 4th cancer mortalities.Esophageal squamous cell carcinoma(ESCC)is the most common histological subtype and accounts for almost 90%of all esophageal cancers in China.This disease has a poor prognosis and does serious harm to human health.The recurrence and metastasis rates of ESCC are very high after surgical treatment and the prognosis is usually poor.The main reason of recurrence and metastasis is likely due to the residual malignant cells in tumor with stem-cell-like potential.These tiny amount cells are called as cancer stem cells(CSCs).CSCs can differentiate to tumor cells through epigenetic events.We postulated that epigenetics play a more important role in regulation of CSCs than genetics.CSCs and non-CSCs have different methylation profiles.The methylation profile of esophageal cancer stem cells is unknown.Side population(SP)cells that represented as substitutes for CSCs were isolated in KYSE150 and EC109 ESCC cell lines.And sphere formation cells were collected from S1 esophageal cancer cells.Methylation analysis was performed using Reduced Representation Bisulfite Sequencing(RRBS).This study using esophageal SP and sphere formation cells as the main material for RRBS sequencing analysis.We filtered the sequencing data,then the high quality clean reads obtained after filtration for further analysis.Alignment results were used for subsequent analysis:using Bismark software to calculate the coverage and mean depth of CGI/promoter region,calculate the methylation level of C cytosine at genome wide scale,draw the methylation maps of the cells,using the differentially methylated region(DMR)in the promoter region of the gene and its expression data to do the KEGG pathway enrichment analysis and GO(gene ontology)analysis.In this study,we found CSCs have different methylation patterns compared with non-CSCs.Importantly,40 DMRs were identified in these three group samples and 13 different methylated genes were detected.DNA methylation may paly an important role in the regulation of CSCs.DNA methyltransferase 1(DNMT1)is a member of the DNA methyltransferase family.It mediates DNA methylation and plays an important role in regulating self-renewal of cancer stem cells(CSCs).However,the function of DNMT1 in CSCs of esophageal squamous cell carcinoma(ESCC)remains unclear.In this study,we isolated the esophageal cancer stem cells from KYSE150 and EC 109 esophageal cancer cell lines,and detected the expression of DNMT1 in esophageal cancer stem cells.To further investigate the effect of DNMT1 in ESCC-CSCs maintenance,we silenced the expression of DNMT1 in KYSE150 and EC 109 ESCC cells using lentivirus-mediated RNA interference(RNAi)or 5-aza-2’-deoxycytidine(5-aza-dC).We evaluated CSCs by SP analysis,sphere formation assay and in vivo xenograft experiment.The malignant phenotypes of KYSE150 and EC109 EC cells were also detected,including proliferation,clone formation,migration and drug resistance abilities.We found a high expression of DNMT1 in both side population(SP)cells and sphere formation cells that represented as substitutes for CSCs in KYSE150 and EC109 ESCC cell lines.In addition,the expression of DNMT1 was decreased during the differentiation from SP to None-SP(NSP)in these ESCC cells.These results suggested that DNMT1 might have a role in regulating self-renewal and/or differentiation of ESCC-CSCs.Ablation of DNMT1 expression in KYSE150 and EC 109 ESCC cells using lentivirus-mediated RNA interference(RNAi)or 5-aza-2’-deoxycytidine(5-aza-dC)resulted in decreased their CSCs by SP analysis,sphere formation assay and xenograft experiment in mice.Meanwhile,ablation of DNMT1 expression inhibited malignant phenotypes in KYSE150 and EC 109 cells,including cell proliferation,colony formation,migration and drug resistance abilities.Also the ablation of DNMT1 expression by Lenti-sh-DNMT1 transfection or 5-aza-dC inhibited the expression of SOX2.The expression of CSC-inhibitory miRNAs such as miR-203,miR-141,miR-200A and miR-200B were up-regulated after the treatment of 5-aza-dC.Thus,our results indicated that DNMT1 was involved in the maintenance of ESCC-CSCs,suggesting that DNMT1 could be a potential target for ESCC,especially ESCC-CSCs,therapy. |
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