Feasibility of testing ppENK gene methylation in stool with methylation-specific polymerase chain reaction assay in pancreatic cancer diagnosis

Abstract

Objective To evaluate the feasibility of testing the high methylation of ppENK gene in stool with methylation-specific polymerase chain reaction (MSP) assay in pancreatic cancer diagnosis.Methods Twenty-four fresh stool samples of pancreatic cancer patients and six healthy control samples were collected.The methylation status of ppENK gene in all the stool samples was detected by MSP assay.The positive rate of wild-type ppENK gene in all the stool samples was determined by polymerase chain reaction (PCR).And 10 non wild-type ppENK gene negative pancreatic cancer samples were collected,and K-ras gene mutation was detected by PCR-restriction fragment length polymorphism (RFLP).The single cell suspension of pancreatic cancer PC3 cell line was added into stool sample from the same healthy individual,the positive rate of ppENK gene methylation was detected by MSP assay.The minimum number of pancreatic cancer cell was calculated when methylation was positive.Results The rate of methylation detection in 30 samples was 0 (0/30); and the rate of non-methylation detection was 10％ (3/30).The rate of wild-type ppENK detection was 6.7％ (2/30).By PCR-RFLP assay,eight were successfully amplified and seven had mutation in 12th code of K-ras gene in 10 selected wild-type ppENK gene negative pancreatic cancer samples.The minimum number of pancreatic cancer cells needed for ppENK methylation band positive detected by MSP was 50 cell/ml.Conclusion Detecting ppENK gene methylation status in stool samples of pancreatic cancer patients by MSP assay has not yet become the method of pancreatic cancer screening and diagnosis. Key words: Polymerase chain reaction; Polymorphism, restriction fragment length; DNA methylation; Feces; Pancreatic neoplasms; Enkephalins; Genes, K-ras; Mutation