Method Of MS-HRM And Its Preliminary Application In Genes Promotor Methylation Of Colorectal Cancer

Background:Colorectal cancer is one of the most popular digestive cancers over the world, and is characterized of poor prognosis with high rate of mortality. Current studies show that both genetics and epigenetics attribute to the occurrence and development of cancers. Multi-factors and multi-steps involve in the occurrence of colorectal cancer. With the development of epigenetics, DNA methylation is revealed to be strongly associated with the initiation and progression of colorectal cancer. As a consequence, the detection of multiple genes methylation profile plays an important role in the early diagnosis、treatment and evaluation of prognosis in CRC.Objective:The aim of the present study is to establish a stable method of MS-HRM to detect quantitatively MGMT、CDH13、h MLH1、DAPK and SEPT9 methylation and analyze the relationship between multiple genes methylation profile and colorectal cancer.Methods:1. Establishing the protocol of MS-HRM based on HRM: With the innovative temperature control and date acquisition technology of Light Cycler 480, the standard curves to detect genes methylation was constructed and the reaction mixture、the PCRand melting programs were optimized.2. Detecting colorectal tumour related genes methylation profile: MGMT、CDH13、h MLH1、DAPK and SEPT9 methylation were detected quantitatively in colorectal tumour tissue、normal tissue and colorectal peripheral blood by MS-HRM in high throughput.3. Analyse of the relationship between multiple genes promotor hyper- methylation and colorectal cancer: We analysed MGMT、CDH13、h MLH1、DAPK and SEPT9 methylation between colorectal tumour and normal tissue and its significance in relation to the clinical-pathological features of colorectal cancer. The impact of concordant detection of multiple genes methylation profile in the diagnosis of CRC was also evaluated.Results:1. The optimized reaction mixture、PCR and melting program were applied to detect quantitatively multiple genes methylation by MS-HRM in high throughput.2. MGMT gene methylation was significantly higher in tumour tissue than in normal tissue(P<0.05).No significant correlation was observed between the MGMT methylation levels and patients’ age、gender and tumor location. However, MGMT methylation was significantly higher in DNA from colorectal cancer patients with high Dukes stage and differentiation(P<0.05). CDH13、h MLH1 and DAPK methylation levels were also higher in tumour tissue than in normal tissue(P<0.05), however, there is no difference between SEPT9 methylation in tumour and normal tissue. No statistical correlation was found between SEPT9、CDH13、h MLH1 and DAPK methylation and some of the clinical factors, including age, gender, tumour location, Dukes stage and differentiation.3. The sensitivity and specificity of CDH13 、 hMLH1 、 DAPK and MGMTmethylation concordant detection in the diagnosis of CRC is 78.8% and 90.9%, with the AUC of 0.848, which has statistical significance.Conclusions:1. The present study successfully established the method of MS-HRM to detect multiple genes methylation profile and also confirmed its advantage and feasibility both in technology and practice.2. MGMT methylation level has a significant correlation with tumour Dukes stage and differentiation, which may serve as a promising biomarker for the evaluation of the progression of colorectal cancer.3. The MS-HRM analysis of a panel of genes methylation including CDH13、h MLH1、DAPK and MGMT has a good performance, which may offer a good alternative in the detection of colorectal cancer.