Epigenetic Regulation Of SOX17in Esophageal Cancer

Background: Esophageal cancer is the eighth most malignant tumor in theworld. Esophageal squamous cell carcinoma is the major type in China. Even throughimproved therapeutic procedure, the overal5year survival is still below10%. Themechanism of esophageal carcinogenesis remains unclear. Abnomal epigeneticchange was regarded as an important reason of esophageal cancer. SRY-boxcontaining gene17(SOX17) is a development related gene, which was found to be apotential tumor suppressor in colorectal cancer and hepatocellular carcinoma.However the epigenetic changes and the function of SOX17remain to be elucided inesophageal cancer.Objective: To explore the possibility of epigenetic changes of SOX17asesophageal cancer early detection marker, promoter region methylation was analyzedin esophageal cancer cell lines, normal esophageal mucosa, different grades ofesophageal dysplasia and advance esophageal cancer. The regulation of SOX17expression by DNA methylation and miRNA was also explored in this study. Thefunction of SOX17and the role in WNT signaling pathway were analyzed in ourstudy to find possible esophageal cancer therapeutic targets.Material and Methods: Eight esophageal cancer cell lines (SKGT4, YSE2,KYSE30, KYSE70, KYSE140, KYSE150, KYSE180and KYSE450),9cases ofnormal esophageal mucosa,60cases of esophageal dysplasia and169cases ofprimary esophageal cancer were included in this study. Expression of SOX17wasdetected by semi-quantitive RT-PCR before and after5-aza-2’-deoxycytidine(5-aza-dc) treatment. The methylation status was detected by methylation specificPCR (MSP). The association of SOX17methylation and clinical factors ofesophageal cancer patients was analyzed by SPSS13.0software. P<0.05was regardedas significant difference. Expression of SOX17and β-catenin was analyzed byimmunohistochemistry (IHC) in39cases available paired of primary esophagealcancer and adjacent tissue paraffin samples. Colony formation assay was performedto explore the effect of SOX17on esophageal cancer proliferation. Luciferase reporter assay and western blot were employed to examine the potential role ofSOX17in WNT signaling pathway. TargetScan and microRNA (miRNA) relatedexperiments were applied to find the candidiate miRNAs which regulated SOX17expression.Results: Promoter region complete methylation of SOX17was detected inSKGT4, KYSE30, KYSE140and KYSE150cell lines. And partial methylation wasfound in KYSE70, KYSE180and KYSE450cell lines. Unmethylation was detectedin YSE2cell line. Normally expressed SOX17was found in YSE2cell line.Signisicantly reduced expression of SOX17was found in KYSE70, KYSE180andKYSE450cell lines. No expression of SOX17was found in SKGT4, KYSE30,KYSE140and KYSE150cell lines. Increased or restored SOX17expression wasfound in partial or complete methylated cell lines after5-aza-dc treatment. Nochanges of SOX17expression was found in YSE2cells before and after5-aza-dctreatment. These results suggest that SOX17expression is silenced or reduced bypromoter region hypermethylation. Methylation of SOX17was exhibited in0%ofnormal esophageal mucosa,42%of esophageal dysplasia and65%of primaryesophageal cancer. It indicates that SOX17is frequently methylated in esophagealcancer and with a progression tendency during esophageal carcinogenesis. The aboveresults suggest that methylation changes of SOX17may serve as a potentialesophageal cancer early detection marker. SOX17methylation was associated withalcohol history of esophageal cancer patients, the result demonstrated that alcoholmay cause esophageal carcinogenesis. In SOX17methylated primary esophagealcancer samples, β-catenin was expressed in nuclear and cytoplasma. While in SOX17unmethylated adjacent esophageal tissue samples, β-catenin expressed mainly incellular membrane. The results suggest that SOX17expression may induce β-cateninre-distribution in esophageal cancer. Restoration of SOX17expression inhibitedcolony formation, TCF luciferase reporter activity and WNT signal downstreamtargets expression. It further suggests that SOX17may suppress esophageal cancerproliferation by inhibiting WNT signaling pathway. Expression of SOX17wasdownregulated by miRNA-141, which suggested that microRNA may regulateSOX17expression in esophageal cancer. Conclusions: SOX17is frequently methylated in esophageal cancer and with aprogression tendency during esophageal carcinogenesis, which suggests thatmethylation of SOX17may serve as a potential esophageal cancer early detectionmarker. Alcohol may cause SOX17methylation and further lead to esophagealcarcinogenesis. Expression of SOX17was regulated by promoter regionhypermethylation and miRNA-141in esophageal cancer. SOX17may suppressesophageal cancer proliferation by inhibiting WNT signaling pathway.