The Research On The Regulation Of SOX17 Gene Methylation In Gastric Cancer Cells

Background and ObjectiveGastric cancer is one of the most common malignant tumors in the world.The incidence and mortality of Gastric cancer ranked the fourth and the second in malignancies respectively.At present,the standard treatment of early gastric cancer is still surgery,but the effect is often not as good as expected.And radical postoperative recurrence of gastric cancer is still as high as 70%.Chemotherapy is the main therapy of advanced gastric cancer.But it also shows a poor effect.Targeted cancer therapy drugs of gastric cancer is very limited,only herceptin is clearly effective.Hence it is urgent for us to further explore the occurrence and development of gastric cancer,and to find new molecular markers to improve the current diagnosis and treatment of gastric cancer.SOX17 gene is a family member of the transcription factor which participates the regulating tissue specificity,organ development,stem cell homeostasis and other important life processes.It also plays an important role in the carcinogenesis process through regulating the cell cycle and cell proliferation.In recent years,people paid more and more attention on the role of SOX17 gene in gastric cancer.In our former sequencing on 10 cases of blood samples of advanced gastric cancer patients,we found high frequency mutation of SOX17 gene.Then after three cycle chemotherapy treatments the patients with SOX17 mutations got partial response and SOX17 gene mutation is not detected again.with the clinical prognosis improved.The results show that the gene mutation and clinical prognosis is closely related.Based on the previous research results and the recent research progress,we focus on SOX17 gene in order to explore the possible epigenetic mechanism of gastric cancer development and the impact of cell signal pathway and cell proliferation and apoptosis.MethodsReal-time PCR and Western Blot was performed to detect the expression of SOX17 m RNA and protein in three gastric cancer cell lines(AGS,MGC-803,MKN45)and normal gastric epithelial cell line(GES-1)were detected by.PCR(MSP)was used to detect the methylation status of the promoter region of SOX17 gene of the above four cell lines.After treating AGS cell lines with classic DNA demethylation drug 5-Aza –Cd R,CCK-8 method was used to observed the proliferation of cells.The apoptosis rate was detected by Annexin V FITC / PI flow cytometry.Real-time PCR was used to detect the expression of SOX17,DNMT1,DNMT3 A and DNMT3 B.Western-blotting was used to identify the expression of SOX17 and ?-catenin.Results1.The relative expression of SOX17 gene m RNA in three gastric cell lines as AGS,MGC-803,MKN-45 and normal gastric epithelial cell lines GES-1 were 0.7498 ±0.0279,0.0402 ± 0.0026,0.0183 ± 0.0004 and 1 ± 0.0 respectively.Compared with GES-1,the expression of SOX17 gene was significantly decreased in three gastric cancer cells(P <0.05),and the expression of AGS cells was the lowest.The expression of SOX17 protein was detected by Western Blot at the protein level.The results showed that the expression of SOX17 protein was consistent with the change of m RNA level.2.The difference of methylation of SOX17 gene promoter region in four cell lines:complete methylation in AGS and MKN45,partial methylation of MGC-803,and complete unmethylation of GES-1.3.After treating AGS cell lines with 5-Aza – Cd R,the methylation status of SOX17 gene promoter region changed gradually: the methylation band gradually weakened to disappear,and the non-methylated band gradually increased.That is said 5-Aza-Cd R can successfully reverse the methylation of SOX17 gene promoter region,making it non-methylated state.And the effect gradually increased with the increase of drug concentration,in a concentration-dependent manner.At the same time,the expression of SOX17 m RNA was also up-regulated(P <0.05),and the greater the effect of drug concentration was about.The up-regulation of SOX17 gene protein was consistent with the change of m RNA level after drug intervention.After 10?mol / L 5-Aza-Cd R treatment,The expression of DNMT3 A and DNMT3 B m RNA was down-regulated,but the difference was not statistically significant(P> 0.05).DNMT1 m RNA expression was significantly down-regulated(P = 0.03 <0.05)4.After treatment at different concentrations and different time of 5-Aza-Cd R,the proliferation of AGS cells was inhibited.In 1?mol / L group and 5?mol / L group the inhibition rate of AGS cells was increased with the prolongation of time The growth inhibition rate of AGS cells increased with the increase of drug concentration(P <0.05),and the concentration of AGS cells was in a concentration-dependent manner.FCM showed that G1 phase cells gradually increased and the cells were arrested in G0 / G1 phase with the increase of drug concentration after 48 hours of AGS cell line.The difference was statistically significant(P <0.05).Annexin V assay showed that the apoptotic rate increased with the increase of drug concentration compared with the control group,but the difference was not statistically significant(P >0.05).The expression of ?-catenin protein decreased with the increase of drug concentration after48 h of AGS cells treated with different concentrations of 5-Aza-Cd R.Conclusion1.The expression of SOX17 gene in gastric cancer cell lines was significantly lower than that in normal gastric mucosa epithelial cells,and the promoter region Cp G island was highly methylated.(Combined with clinical follow-up,associated with poor prognosis-no clinical specimens or large sample high-throughput sequencing data can not compare clinical outcomes).2.5-Aza-Cd R may reverse the SOX17 gene hypermethylation by down-regulating the expression of DNMT1,thereby restoring the expression of SOX17 gene and inhibiting the activity of WNT pathway by down-regulating the expression of ?-catenin.3.5-Aza-Cd R can inhibit the proliferation of AGS cells in a concentration and time-dependent manner.It is mainly related to inhibition of cell cycle G1-S conversion and block cell cycle in G0 / G1 phase,which does not cause obvious apoptosis.5-Aza-Cd R inhibition of gastric cancer cell proliferation may be due to methylation of silencing genes re-expression of interfering with cell cycle.