The Study Of PCDH8Gene, And Promoter Methylation In Human Colorectal Cancer

Chapter1:PCDH8mRNA, PCDH8promoter methylation and PCDH8protein in colorectal cancer cell linesPurpose:Colorectal cancer is one of the world-wide malignancy leading to highest morbidity and mortality. In China, colorectal cancer is the3rd frequentest malignancy, and the4th cause of death among all of the malignancy. The study of DNA methylation in colorectal cancer has become a hotspot research in recent years, and aberrant methylation has been considered as an alternative mechanism, which is responsible for inactive tumor suppressor gene. Therefore, DNA methylation is likely to play an very important role in the development and progression of colorectal cancer. Low expression and methylation of the promoter in protocadherin8(PCDH8) gene have been detected in gastric cancer, breast cancer and bladder cancer, in which PCDH8is confirmed a novo tumor suppressor gene. However, none reserch has been done in colorectal cancer about PCDH8. In the part, we studied the expression of PCDH8in the cell lines of colorectal cancer.Methods:1.The expression of PCDH8mRNA was detected by RT-PCR in the human normal colonic epithelial cell line (NCM460) and four human colorectal cancer cell lines;2. PCDH8protien was detected by Werstern Blotting in human normal colonic epithelial cell line and four human colorectal cancer cell lines;3.PCDH8methylation was evaluated by methylation-specific PCR in human normal colonic epithelial cell line and four human colorectal cancer cell lines.Results:1. PCDH8mRNA was stablely expressed in the human normal colonic epithelial cell line (NCM460), PCDH8mRNA was nearly not detected in the human colorectal cancer cell lines HT29and HCT116, PCDH8mRNA was lowly expressed in he human colorectal cancer cell lines SW480and SW620.2. PCDH8protein was lowly expressed in all of the four human colorectal cancer cell lines,while it was high expressed in the human normal colonic epithelial cell line.3. PCDH8promoter methylation was showed in all of the four human colorectal cancer cell lines, while none PCDH8promoter methylation was detected in the human normal colonic epithelial cell line.Conclusions:PCDH8gene is lowly expressed in all of the four human colorectal cancer cell lines, which is likely to be associated with the PCDH8promoter methylation. Maybe, it is the one of the reason for the development and progression of colorectal cancer. Chapter2:PCDH8protein and PCDH8promoter methylation in colorectal cancer tissuesPurpose:To explore the PCDH8protein and PCDH8promoter methylation in the human colorectal cancer tissues, human colonic adenoma tissues and adjacent non-tumor tissues;.Methods:1.PCDH8protien was detected by Immunohistochemical staining in human colorectal cancer tissues,human colonic adenoma tissues and adjacent non-tumor tissues;2. PCDH8promoter methylation was evaluated by methylation-specific PCR (MSP) in human colorectal cancer tissues,human colonic adenoma tissues and adjacent non-tumor tissues;3. Applicate SPSS13.0software and chi-square test statistics method to explore the possible correlation between PCDH8protein and clinicopathologic charters of colorectal cancer;4. Applicate SPSS13.0software and chi-square test statistics method to explore the possible correlation between PCDH8promoter methylation and clinicopathologic charters of colorectal cancer.Results:1. Immunohistochemical staining showed that PCDH8protein was frequently detected in adjacent non-tumor tissues (94.12%,32/34), human colonic adenoma tissues (80%,16/120), and human colorectal cancer tissues (30.77%,16/68). Statistically, there was no difference in expression of the PCDH8protein between adjacent non-tumor tissues and human colonic adenoma tissues(P=0.321). However, there were statistically significant differences between colorectal cancer tissues and adjacent non-tumor tissues (P<0.01), and between human colorectal cancer tissues and human colonic adenoma tissues (P<0.01).2. MSP analysis showed that PCDH8gene promoter methylation was frequently detected in human colorectal cancer tissues (82.35%,56/68) and human colonic adenoma tissues (70%,14/20), but in adjacent non-tumor tissues only2of32(5.88%) were showed PCDH8gene promoter methylation. Statistically, there was no difference in methylation of the PCDH8gene promoter between human colorectal cancer tissues and human colonic adenoma tissues(P=0.402). However, there were statistically significant differences between colorectal cancer tissues and adjacent non-tumor tissues (P<0.01), and between human colonic adenoma tissues and adjacent non-tumor tissues (P<0.01).3. PCDH8protein expression was not significantly correlated with sex, age of diagnose, size of tumor, TNM stage, tumor differentiation, lymph node metastasis in colorectal cancer(P>0.05).4. PCDH8promoter methylation was statistical significantly correlated with size of tumor, in colorectal cancer(P<0.01),while not significantly correlated with sex, age of diagnose, TNM stage, tumor differentiation, lymph node metastasis in colorectal cancer(P>0.05). ConcIusions:PCDH8gene promoter methylation was frequently detected in human colorectal cancer tissues and human colonic adenoma tissues, which is likely to be an early stage in colorectal cancer. Maybe, it is the one of the reason for the development and progression of colorectal cancer.