| BACKGROUND:A number of studies suggest that the expression of miR-9 in colorectal cancer is significantly lower than that in normal tissue.The increase of miR-9 level can inhibit tumor growth in vitro,and its abnormal expression can cause tumor invasion and metastasis.Gene methylation is a modulatory mode of miR-9 expression.Methylation-specific PCR(MSP)has been reported to detect methylation of miR-9-1 gene promoter region of colorectal cancer.The detection of gene methylation,pyrosequencing is the gold standard,but the comprehensive operation and cost,etc.,is not suitable for clinical application,MSP is widely recognized methylation detection method.However,with the high resolution dissolution curve(HRM),fluorescence probe methylation specific PCR(Methylight)and other methods,MSP is still the most appropriate detection method has not been related to the study.Purpose:1.To analyze the merits and demerits of miR-9-1 gene promoter methylation in MSP,HRM and Methylight of colorectal cancer,the optimal method was selected.2.To further study the changes of miR-9-1 methylation in different stages of colorectal cancer.Methods:1.The methylation detection method of miR-9-1 gene promoter region of MSP,HRM and Methylight colorectal cancer was compared with the standard of pyrosequencing.The optimal method and optimum condition were selected.2.The methylation status of miR-9-1 promoter region in 79 colorectal cancer specimens was detected by the optimal method.The relationship between methylation status and colorectal cancer was analyzed according to the staging of the patients.Results:1.The total detection rate of MSP method relative to pyrosequencing method was 70%,the positive detection rate was 100%and the negative detection rate was 57.14%.HRM technology in the course of the experiment found that its repeated experimental results vary greatly,considering its not suitable for complex samples in the detection of gene methylation.The total detection rate,positive detection rate and negative detection rate of Methylight method relative to pyrophosphate method were 100%.Methylight method,which uses the HotStart Taq enzyme methylation-specific PCR to amplify the combination of fluorescent probes,may be a more clinically applicable method.2.The rate of miR-9-1 methylation was increased in 79 cases of colorectal cancer(P<0.05),but there was no significant differenceConclusion:1.Methylight method of methylation-specific PCR combined with fluorescent probe is the best method for the detection of specific DNA methylation in paraffin tissue,which facilitates the subsequent DNA methylation detection2.MiR-9-1 gene promoter region methylation may be associated with advanced colon cancer,for the subsequent expansion of the sample study miR-9-1 gene promoter methylation and colorectal cancer tumor invasion and metastasis of the relationship reference. |
