Human Stool RASSF1A、RUNX3、hMLH1Gene Methylation Analysis In The Screening And Diagnosis Of Colorectal Cancer

Colorectal cancer (CRC) is a common gastrointestinal cancer.5-yearsurvival rate after active treatment of early diagnosis of CRC can be more than90%, while the vast majority of patients have progressed to advanced stage,the survival rate is less than10%. Therefore, early detection, early diagnosishas a very important significance for CRC. Currently diagnosis of CRCmethods such as FOBT or colorectal microscopy lack of specificity, sensitivityor patient compliance, limiting its wide application in CRC screening andearly diagnosis. Because of steady existence of stool shedding cells in thestool, in2009the American Gastroenterological Association of CRC screeningguidelines has made it clear that detecting stool DNA methylation is arecommended screening methods.RASSF1A is a tumor suppressor gene. Currently aberrant methylation ofRASSF1A causing to transcriptional silencing can be detected in a variety oftumors, especially in colorectal cancer. Methylation rates is up to81%.RUNX3is a tumor suppressor gene. RUNX3protein is a very importanttranscription factor of TGF-β signaling pathway downstream.It plays anegative regulatory role in cell differentiation, cell cycle regulation, apoptosis.Methylation or allelic deletion of RUNX3gene can cause abnormalexpression. Now many studies have shown that RUNX3expression is relatedto gastrointestinal cancer. hMLH1gene is one of the most important MMR. Incancer cells, the inactivation of gene function is due to be highly methylatedCpG islands. Kuismanenu studied36cases of sporadic MSI CRC and found30cases (92%) occurred hMLH1hypermethylation. It shows that hMLH1methylation plays a major role in the occurrence of sporadic CRC. Objective:To study the use of methylation analysis of RASSF1A, RUNX3andhMLH1gene in stool and tissue from patients with CRC, colorectal adenomasand colorectal polyps as well as from normal controls by MSP as anon-invasive screening and diagnosis tool for CRC, and it’s relationship withthe clinicopathological characteristics of CRC patients．Methods:1Stool and tissue from the four groups above who were admitted to thesecond and fourth Hospital of Hebei Medical University during January2013to october2013were collected. Record Clinico-Pathologic Features of thesepatients.2After stool DNA isolated by the stool DNA isolation kit and tissue DNAby the phenol chloroform method. MSP was applied to analyze the promoterhypermethylation of RASSF1A,RUNX3and hMLH1gene in the stool andtissue DNA．Results:1DNA metylation analysis in stoolStool DNA Were isolated from stool samles of147Patients and at last145samples verify existence of the human genome and completed the MSPincluding56patients with CRC and27patients with colorectal adenomas and32patients with colorectal polyps and26patients in control group．TheRASSF1A,RUNX3and hMLH1gene promoter hypermethylation in CRCwere53.57％,58.93％,50.00％,respectively,and the specificity were86.52％，84.27％,89.89%respectively. At least one methylated gene was detected in91.07%patients with CRC with specificity of75.28%.The RASSF1A,RUNX3and hMLH1gene promoter hypermethylation in colorectal adenomas were33.33％,37.04％,25.93％respectively,and at least one methylated gene wasdetected in66.67%patients with colorectal adenomas.The RASSF1A,RUNX3and hMLH1gene promoter hypermethylation in advanced adenomas were38.89％,44.44％,33.33％respectively,and at least one methylated gene wasdetected in77.78%patients with advanced adenomas.The RASSF1A,RUNX3 and hMLH1gene promoter hypermethylation in colorectal polyps were9.38％,9.38％,6.25％respectively,and at least one methylated gene was detected in9.38％patients with colorectal polyps.The RASSF1A,RUNX3and hMLH1gene promoter hypermethylation in control group were0％,3.33%,0％respectively, and only onemethylated RUNX3gene was detected in controlgroup.145cases of stool specimens for detection of FOBT, there were a totalof18cases of positive test results, including17patients with CRC (positiverate of30.36%), and1patient with advanced adenoma (positive rate of3.70%).There was statistical difference between two methods(P <0.001).The promoter hypermethylation of RASSF1A,RUNX3and hMLH1genein CRC stool correlate with tumor size, depth of invasion，but didn’t correlatewith other Clinico-Pathologic Features（except for RUNX3with degree ofdifferentiation）．The promoter hypermethylation of RASSF1A,RUNX3and hMLH1genein adenoma stool correlate with adenoma size,intraepithelial neoplasia type,but no correlation with tissue types.There were statistical differences between the CRC and the colorectalpolyps groups，the CRC and the normal control groups, the colorectaladenoma and the colorectal polyps groups(except for hMLH1),but there wereno statistical differences between the CRC and the colorectal adenomagroups(except for hMLH1),the colorectal polyps and the normal controlgroups.Methylated RASSF1A,RUNX3and hMLH1were detected in42.86％,57.14％,42.86%of early CRC and advanced adenoma，respectively．And atleast one methylated gene was detected in80.36％patients with early CRCand advanced adenoma.2DNA metylation Analysis in tissue145samples completed the MSP including56patients with CRC and27patients with colorectal adenomas and32patients with colorectal polyps and26patients in normal control group．The RASSF1A,RUNX3and hMLH1gene promoter hypermethylation in CRC were55.36％,60.71％,51.79％, respectively, and the specificity were83.15%％,77.53％,87.64%respectively.At least one methylated gene was detected in92.86％patients with CRC withspecificity of69.66％.The RASSF1A, RUNX3and hMLH1gene promoterhypermethylation in colorectal adenomas were40.74％,44.44％,33.33％,respectively,and at least one methylated gene was detected in70.37％patientswith colorectal adenomas.The RASSF1A,RUNX3and hMLH1gene promoterhypermethylation in advanced adenomas were50.00％,55.56％,38.89％respectively,and at least one methylated gene was detected in83.33%patientswith advanced adenomas.The RASSF1A, RUNX3and hMLH1gene promoterhypermethylation in colorectal polyps were12.50％,25.00％,6.25％,respectively,and at least one methylated gene was detected in25.00％patientswith colorectal polyps.No methylated gene was detected in control group.The promoter hypermethylation of RASSF1A,RUNX3and hMLH1genein CRC tissue correlate with tumor size, depth of invasion, but didn’t correlatewith other Clinico-Pathologic Features（except for RUNX3with degree ofdifferentiation）．There were statistical differences between the CRC and the colorectalpolyps groups，the CRC and the normal control groups,the colorectal adenomaand the colorectal polyps groups(except for hMLH1),but there were nostatistical differences between the CRC and the colorectal adenomagroups(except for hMLH1),the colorectal polyps and the normal controlgroups.3Comparison of DNA methylation between stool and tissueRASSF1A, RUNX3, hMLH1gene methylation between colorectalcancer tissues and faeces, there was no statistical difference between stool andtissue with CRC about RASSF1A, RUNX3, hMLH1gene methylation. Andthere was no statistical difference between stool and tissue with colorectaladenoma about RASSF1A, RUNX3, hMLH1gene methylation.Conclusion:1RASSF1A, RUNX3, hMLH1gene methylation rates were increasedgradually in normal controls, colorectal polyps, colorectal adenomas and CRC.2The promoter hypermethylation of RASSF1A,RUNX3and hMLH1gene in CRC correlate with tumor size, depth of invasion(except RUNX3intissue)，but didn’t correlate with other Clinico-Pathologic Features（except forRUNX3with degree of differentiation）．3The rate of combined detection of RASSF1A, RUNX3, hMLH1genemethylation in early colorectal cancer and advanced adenomas stool is80.36%,and it can be used as one of the method of colorectal cancer screening andearly diagnosis.