| Colorectal Cancer (CRC) is a common gastrointestinal malignancy whichthreatens the health of human. It is the second leading death cause of cancer-related diseases in western developed countries after lung cancer and the fourth to sixth death cause in China. The mortality rate of CRC has been rising over the past two decades in urban and developed rural area of China. The increasing rate of colon cancer is higher than that of rectal cancer. With the changes of life style and diet structure, the risk of CRC will be increasing continuously. Therefore, it is very important to extensively study the mechanisms of colorectal cancer and the strategies of prevention and control.Carcinogenesis is known to be a long-term and multi-step process involving the actions of various cancer associated genes, including activation of oncogenes and silence of tumor suppressor genes. Epigenetics, which refers to heritable changes in gene expression that occur without a change in DNA sequence, is a new mechanism of tumorigenesis besides genetic alteration. DNA methylation of CpG islands within promoter region is a powerful molecular mechanism of epigenetic silencing. Many important tumor suppressor genes such as p16, ER, BRCA1,hMLH1, etc., are inactivated through this mechanism. There are different patterns of DNA methylation in CpG islands of a specific gene between normal and cancer tissue. Therefore, new cancer-related genes can be screened by comparing the differences of the DNA methylation status of cancer with that of normal tissue. This is a new way to study the new potential tumor suppressor genes. Objective To study the aberrant methylation status in the early stage of colorectal cancer and screen some potential cancer-related genes involved in carcinogenesis of colorectal cancer.Material and methodsEnrich the methylated CpG islands of genome with methylated CpG island amplification (MCA) Genomic DNA was digested with endonuclease Sma I and its isoshizomers Xma I respectively. Then adaptor RXMA24/12 was ligated with the digested DNA by T4 DNA ligase and the methylated DNA was amplified by PCR with RXMA24 as a primer.Screen the differentially methylated sequence with representational differential analysis (RDA) The adaptor of the MCA amplicon from mucosa adjacent to colon cancer (MACC) was excised by Xma I digestion and ligated with a new adaptor RMCA24/12. This new ligation DNA was used as the tester amplicon. The driver amplicon was prepared by removing the adaptor from MCA amplicon of normal colon tissue by Sma I digestion. Then the tester amplicon was competitively hybridized with driver amplicon in the proportion of 1:50 to 100. After hybridization, the homoduplex of tester was amplified with RMCA24 as primer. For the second round RDA, the former adaptor was excised from the substraction product, ligated to a new adaptor (JXMA24/12) and rehybridized. The differentially methylated DNA was obtained after four rounds of RDA. Clone, sequencing and blast search analysis The differentially methylatedDNA were cloned into pGEM T-easy vector, and the positive inserts were sequenced with automated DNA sequencer. Then sequence similarities were identified with BLAST program.Dot blot analysis To confirm the substraction of MCA-RDA, tester and driver amplicon, 1st to 4th RDA products were dot into the positive charge nylon membrane and hybridized with Digoxigenin labeled fragment A12 as a probe. Reversed transcription PCR and immunohistochemistry (IHC) To study the expression of Staufen gene at mRNA level in colorectal cancer, adenoma, corresponding MACC and normal colonic tissues, semi-quantitative RT-PCR was performed. Relative expression level of Staufen gene was adjusted with the expression of G3PDH as an internal control. The relative expression level of Staufen gene was defined as the odds of net intensity of Staufen to that of G3PDH. The expression of Staufen at protein level was studied with IHC. Matched rank test was used to compare the statistical differences among groups.RESULTS33 d...
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