Relationship of DNMT3b expression and SEPT9 methylation in theprogression of colorectal carcinogenesis

Abstract

Objective: To investigate the relationship between the expression of DNA methyltransferase 3b (DNMT3b) and the methylation of SEPT9 gene, and their application prospects in the diagnosis and treatment of colorectal cancer. Methods: Seventy-five cases of colorectal cancer and adjacent tissues, 68 cases of colorectal high-grade internal neoplasia tissues (referred to as precancerous tissues) and high-grade internal adjacent neoplasia tissues (referred to as adjacent precancerous tissues) were collected. Pyrosequencing was used to detect the methylationlevel of SETP9. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to examine the mRNA expressionof SEPT9 and immunohistochemistry(IHC) was applied to detect the protein expressions of SETP9 and DNMT3b. Liposome-mediated method was used to transfect DNMT3b siRNA and negative control siRNA into HT-29 cells. Five groups including DNMT3b siRNA 15 nmol/L group, DNMT3b siRNA 30 nmol/L group, negative control siRNA 15 nmol/L group, negative control siRNA 30 nmol/L group and blank control group were set up. Pyrosequencing was applied to determine the methylation level of SEPT9 and mRNA expression of DNMT3b in each group. Results: The methylation rates of SEPT9 gene in colorectal cancer tissues, adjacent tissues, precancerous tissues and adjacent precancerous tissues were (76.8±6.5)%, (14.4±2.6)%, (34.6±5.0)% and (7.4±1.2)%, respectively, which was highest in colorectal cancer tissue (P<0.001). The relative expressions of SEPT9 mRNA were 0.18±0.03, 0.89±0.41, 0.69±0.41 and 1.01±0.21, respectively, which was lowest in colorectal cancer tissue (P<0.001), while there were no statistically significant differences in adjacent tissues, precancerous tissues and adjacent precancerous tissues (P>0.05). The positive rates of SEPT9 protein expression were 12.0% (9/75), 53.3% (40/75), 55.1% (38/69) and 62.3% (43/69), which was lowest in the colorectal cancer tissue (P<0.001), while there were no statistically significant differences in the adjacent group, precancerous group and adjacent precancerous group (P>0.016 7). The positive rates of DNMT3b protein expression were 56.3% (45/75), 26.7% (20/75), 46.4% (32/69) and 33.3% (23/69), respectively, which was highest in colorectal cancer tissue (P<0.001), while without statistically significant difference from the precancerous tissue (P>0.016 7). Experiments in vitro showed that DNMT3b mRNA expression was lowest in DNMT3b siRNA 30 nmol/L group among five groups and was statistically different from other groups (all P<0.05). Meanwhile, the methylationrate of SEPT9 gene was lowest in this group, but without statistically significant difference from the DNMT3b siRNA 15 nmol/L group (P>0.05). Conclusions: The expression of DNMT3b is significantly correlated with the methylation level of SEPT9 gene in different stages of colorectal cancer. The high expression of DNMT3b may be an important molecular event before SEPT9 gene methylation and it may have an important potential application value in the diagnosis and treatment of early colorectal cancer.