To Explore The Diagnostic Value Of Gene Promoter Methylation In Fecal Exfoliated Cells For Early Colorectal Cancer

Background:The morbidity and mortality of colorectal cancer were ranked third and fifth respectively in China.With the improvement of people's living standards,the morbidity and mortality of colorectal cancer in China is increasing in recent years.Lacking specific symptoms for early colorectal cancer,most patients had been on the advanced stage when they were in hospital,and their 5-year survival rate is less than10%.Therefore,early diagnostic methods of high accurateness are significant for reducing the mortality of colorectal cancer.Fecal occult blood test,colonoscopy and sigmoidoscopy are the most common screening modalities,however the test performance of FOBT are not satisfied,and colonoscopy or sigmoidoscopy has suboptimal screening compliance because of its invasive property.Many studies aimed to identify molecular markers for the development of highly accurate,non-invasive screening tests for early CRC.DNA methylation is an enzyme-induced inheritable chemical modification.Abnormal DNA methylation of tumor suppressor genes down-regulates the expression of tumor suppressor gene.This mechanism affects gene expression more frequently during tumor's outgrowth and development.Therefore,DNA methylation is more likely to be potential tumor biomarkers to achieve high sensitivity and specificity in tumor screening.Li H etc.used methylation chips to do genome-wide scans on the matched pairs of colorectal cancer and precancerous tissue,covering4010 genes,and found 297 CpG dinucleotides in 211 genes in cancer tissue showed methylation phenomenon.The found candidate genes were verified in paired colorectal cancer tissues,and three new genes(PHOX2B,FGF12 and GAD2)methylation was found to have at least four probes positive.Highly methylatedPHOX2 B,FGF12 and GAD2 have better ability to distinguish cancer and non-cancerous tissue than vimentin and SEPT9's.This revealed the possibility of using these three gene methylation assays for early diagnosis of colorectal cancer.In this study,GAD2 gene methylation was chosen to be examined in the population to assess the possibility of screening for colorectal cancer as biomarkers.Purpose:To investigate the enrichment efficiency of fecal exfoliated cells of the improved fecal exfoliated cells enrichment method,and to evaluate the diagnostic value of GAD2 gene promoter methylation in fecal exfoliated cells for early colorectal cancer.Method:Use the improved enrichment methods to enrich the exfoliated cells in the colonic diarrhea effusion of the high-risk patients in the General Hospital of Gastrointestinal Endoscopy Center during July 2015 to June 2016.The tumor tissue and precancerous tissue were also collected by Disposable biopsy forceps.DNA were extracted in the fecal exfoliated cells samples,the tumor tissue and the precancerous tissue.Examine housekeeping gene ?-action expression by real-time PCR to evaluate the effectivity of the improved enrichment method for fecal exfoliated cells and verify the methylation rate of GAD2 gene promoter in fecal samples by the methylation Specific PCR,and calculate the sensitivity and specificity of GAD2 methylation for CRC diagnosis.Result:Among 93 patients,95.7% patients had positive ?-actin gene expression in fecal samples,95.7%,85.7%,100%,100%,100% and 95.7% patients show positive ?-actin gene expression in the group of high-risk adenomas,stage ?cancer,stage I?cancer,stage ? cancer,stage ? cancer and normal respectively.The positive rate of the methylation of GAD2 gene promoter in tumor tissue was 76.6%,and 17.0%in non-tumor tissue.The methylation rate of GAD2 gene promoter in fecal samples of stage I,stage ?,stage ? and stage ? was 58.3%,75.0%,76.5% and 100.0%,respectively.The total sensitivity of GAD2 gene promoter methylation for all colorectal cancer was 73.3%,and the specificity was 95.5%.Conclusion:The enrichment efficiency of fecal exfoliated cells with improved enrichment method is satisfied,providing a technical base for further fecal molecular biology detection.The methylation of GAD2 gene in fecal exfoliated cells has acceptable diagnostic significance for colorectal cancer.