Human Stool TFPI2 Gene Methylation Analysis In The Diagnosis Of Colorectal Cancer

Colorectal cancer, including colon and rectal cancer, is a common digestive system cancer. The incidence of the cancer accounted for the third place in the world, and the second place in Western developed countries.In China, with the improvement of living standards and dietary changes in the structure in recent years, the incidence of colorectal cancer is rising gradually, which has leapt to the 3rd-5th place. And, according to forecasts, China's colorectal cancer incidence and mortality rate will increase in the future. Treatments of colorectal cancer were not satisfactory, and the prevention is hard to implement effectively at present. In addition, screening,early diagnosis and early treatment are possible, because colorectal cancer has more obvious precancerous lesions and early lesions, which last more than ten years.In recent years, studies have clarified that the occurrence of colorectal cancer is a multi-step,multi-factor and a variety of genetically modified synergistic process. The programs of gene expression are often changed. Studies have shown that epigenetic is one of important ways to change modification of gene expression. Epigenetic is a modification of the genome, which dose not changes the genomic DNA sequence but can be transmitted to progeny cells in the process of cell division. DNA methylation and histone acetylating modification are the main forms of epigenetic modification. DNA methylation is often the early phase in the development of tumor, and can easily be detected in the stool of patients. Therefore, the detection of colorectal cancer-related genes methylation status in stool may become a new way of colorectal cancer screening and early non-invasive diagnosis.The product of TFPI2 gene encodes is a broad-spectrum serine protease inhibitor, which is widely distributed in human's normal tissues such as liver, pancreas, kidney, heart, skeletal muscle and prostate, can strongly inhibit plasmin, trypsin, matrix metalloproteinase's 1(matrix metalloproteinase's, MMP-1) and 3 (MMP-3) activation in exterior, is involved in extracellular matrix (extracellular matrix, ECM) remodeling process to maintain the structural integrity of ECM and then to regulate of tumor cell invasion and metastasis, and plays an important role in a series of physiological and pathological processes, such as the invasion and metastasis of tumor cells. When the TFPI2 gene appears methylation, it can fall gene expression from the transcriptional level, thereby reducing its regulation ability of tumor cell invasion and metastasis and promoting the occurrence and development of malignant tumors.ObjectiveIn this paper, methylating-specific PCR(MSP) technique was used to analyses 30 cases of colorectal cancer patients and 30 normal controls stool TFPI2 gene methylation status, and stool occult blood test was done for the same stool samples with immunochemical method, which was used as a comparison to evaluate the feasibility of TFPI2 gene methylation in colorectal cancer as a non-invasive diagnosis method.Materials and methods60 cases including 30 colorectal cancers and 30 normal controls were randomly selected from the patients, who admitted to the first affiliated hospital of Zhengzhou University to take colonoscopy from May to September,2009.30 colorectal cancer patients,17 male and 13 female, male to female ratio of 1.3:1, age between 21 and 82 with an average age of 57. As for 30 cases of normal controls including 15 males and 15 females, male to female ratio of 1:1, age range 32 to 79 years, with an average age of 54 years, all colonoscopy were negative. Stool specimens were collected before colonoscopy or surgery from the 60 patients, then sent to the laboratory immediately and preserved in -70℃refrigerator. All patients admitted to hospital or clinic examination were detected by immunochemical stool occult blood. After pretreatment of specimens, DNA was extracted with QIAamp DNA Stool mini kit(QIAGEN,Germany) and saved in -20℃refrigerator. Then DNA was modified by Bisulfite and purified according to The MethylampTM One-Step DNA Modification Kit (EPIGENTEK, USA) operation instructions and saved in -70℃refrigerator. The methylation status of genes TFPI2 was detected with the DNA with bisulfate modification as the template of MSP. The results of the experiment were processed by statistical software SPSS 17.0, with gene frequencies obtained through direct counting method and each group rate compared by the X2 test. The test results signify statistically with P<0.05.Results1 Among 30 stool specimens of colorectal cancer patients,19 specimens (63.3%) were detected to show TFPI2 gene methylation. Meanwhile, no one detected TFPI2 gene methylaion among 30 normal controls.2 Among 30 stool specimens of colorectal cancer patients,11 patients with stool occult blood positive (36.7%), the results of TFPI2 gene methylaion of these patients were positive too. With TFPI2 methylation positive rate (63.3%) and stool occult blood testing positive rate (36.7%) compared, X2=4.267, P<0.05.3 TFPI2 gene methylation and patients sex, age and tumor location have no signification correlation (P>0.05).4 The positive rates of TFPI2 gene methylation in Dukes stages A,B and C,D,47.1% and 84.6% respectively, were significantly different(p<0.05). Secondly, poorly differentiated tumor tissue TFPI2 gene methylation positive rate was 100%. In well-differentiated tumor tissue TFPI2 gene methylation positive rate was 47.6%. The difference is significant (p<0.05).Conclusion 1 TFPI2 gene methylation is a common change in stool cells of colorectal cancer patients, and TFPI2, as a tumor suppressor gene, may be one of molecular markers which are used non-invasive diagnosis for colorectal cancer.2 TFPI2 gene methylation status arid colorectal cancer patient's age, sex and tumor location have no signification correlation.3 The correlation between TFPI2 gene methylation status and colorectal cancer patient's Dukes staging and tumor differentiation degree is obvious.