Analysis Of SEPTIN5,8,9Methylation And Its Clinical Significance In Colorectal Cancer And Gastric Cancer

[Objective]tablish a reliable and simple method of DNA methylation analysis based onMS-HRM technology. SEPTIN5, SEPTIN8and SEPTIN9methylation statues and theirclinical significance in colorectal cancer or gastric cancer were analysised in this paper.[Methods]1. A new Bis-DNA（bisulfite treated DNA）purification method was established. Themethod was compared and verified with Promega Wizard DNA Clean-up System.2. Picogreen was used as the MS-HRM dye in the analysis. Three plasmids withinsertes were used in the analysis to assess the sensitivity of picogreen.（The lengthof insert DNA was91bp. There was only one base difference between plasmid2andplasmid3. The sequencing results of3plasmids were showed in appendix B.）3. The new Bis-DNA purification method was combined with MS-HRM technology toestablish a method that can assay DNA methylation quickly and reliably.4. To verify the method，methylation of SEPTIN9promotor of colorectal tumor tissue samples from34patients were analyzed.5. The methylation levels of gastric cancer cell lines SGC7901and AGS,9samples ofgastric cancer tissues and corresponding adjacent normal tissues,73samples ofgastric cancer of somatic cell embeddied paraffine,39samples of pre-cancerous lesiontissues were analysed.6. BSP technology was applied to find out the concrete methylated cytosine in thepromoter of SEPTIN5and SEPTIN8[Results]1. A simple and reliable method to purify Bis-DNA was successfully established. Therecovery of DNA purification following bisulfite treatment by the new method wasmore than63%. While the recovery of DNA purification of Promega Wizard DNAClean-up System was lower than40%. 2. The sensitivity of HRM using Picogreen dye was as high as0.5%. One basemethylation out of91can be distinguished by using Picogreen dye.3. Methylation of SEPTIN9promotor was observed in97.0%of colorectal tissuesamples (33/34), which is the same as reported.4. The aberrant SEPTIN9methylation was detected in2cell lines,59（72.0%）samplesof gastric cancer tissues,29（74.3%）samples of pre-cancerous lesion tissues. Whileonly2of9（22.2%）samples of corresponding adjacent normal tissues existedSEPTIN9methylation. No significant correlation was observed between the SEPT9methyaltion and patients’ age, TNM stage, clinical stage.(χ~2=3.83,χ~2=1.041,χ~2=0.242;P=0.051, P=0.308, P=0.623, respectively). However, significantcorrelation was observed between the SEPTIN9methyaltion and differentiation,gender, Lauren classification (χ~2=13.098,χ~2=6.184, χ~2=10.152;P=0.0001，P=0.013, P=0.001, respectively).5. No methylated cytosine was found out in the promoter of SEPTIN5. But4methylated CpG sites were detected in-200to-5region of SEPTIN8promoter.Demethylation of these sites was observed after MTA treatment.[Conclusion]1. The new method can be applied to analyze DNA methylation, and it is a reliable andsimple method.2. Aberrant SEPTIN9methylation may promote tumorigenesis of gastric cancer. And itis a promising bio-marker for detection of gastric cancer.3. Hypemethylation of SEPTIN8in RKO cell line indicates that SEPTIN8may play animportant role in colorectal cancer. MTA can reverse the methylated modification ofSEPTIN8.