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.
目的： 探讨DNA甲基转移酶3b(DNMT3b)表达与SEPT9基因甲基化的关系，以及二者在结直肠癌诊治中的应用前景。 方法： 收集75例结直肠癌及癌旁组织、68例结直肠高级别内瘤变组织(简称癌前病变组织)和高级别内瘤变旁组织(简称癌前病变旁组织)，采用焦磷酸测序检测SETP9基因甲基化水平，采用实时荧光定量聚合酶链反应检测SEPT9 mRNA的表达，采用免疫组化方法检测SETP9和DNMT3b的蛋白表达。采用脂质体介导法，将DNMT3b siRNA和阴性对照siRNA转染HT-29细胞，设DNMT3b siRNA 15 nmol/L组、DNMT3b siRNA 30 nmol/L组、阴性对照siRNA 15 nmol/L组、阴性对照siRNA 30 nmol/L组和空白对照组。采用焦磷酸测序检测各组SEPT9基因甲基化和DNMT3b mRNA表达水平。 结果： 结直肠癌组织、癌旁组织、癌前病变组织和癌前病变旁组织中，SEPT9基因甲基化率分别为(76.8±6.5)%、(14.4±2.6)%、(34.6±5.0)%和(7.4±1.2)%，结直肠癌组织最高(P<0.001)；SEPT9mRNA的相对表达量分别为0.18±0.03、0.89±0.41、0.69±0.41和1.01±0.21，结直肠癌组织最低(P<0.001)，而癌旁组织、癌前病变组织及癌前病变旁组织差异无统计学意义(P>0.05)；SEPT9蛋白表达阳性率分别为12.0%(9/75)、53.3%(40/75)、55.1%(38/69)和62.3%(43/69)，结直肠癌组最低(P<0.001)，而癌旁组、癌前病变组及癌前病变旁组差异无统计学意义(P>0.016 7)；DNMT3b蛋白表达阳性率分别为56.3%(45/75)、26.7%(20/75)、46.4%(32/69)和33.3%(23/69)，结直肠癌组最高(P<0.001)，但与癌前病变组差异无统计学意义(P>0.016 7)。体外实验显示，空白对照组、阴性对照siRNA 15 nmol/L组、阴性对照siRNA 30 nmol/L组、DNMT3b siRNA 15 nmol/L组和DNMT3b siRNA 30 nmol/L组中，DNMT3b siRNA 30 nmol/L组DNMT3b mRNA表达量最低，与其他各组差异均有统计学意义(均P<0.05)，SEPT9基因甲基化率最低，但与DNMT3b siRNA 15 nmol/L组差异无统计学意义(P>0.05)。 结论： 在结直肠癌病变进程的不同阶段，DNMT3b的表达与SEPT9基因甲基化水平有明显的相关性，DNMT3b高表达可能是SEPT9基因甲基化前重要的分子事件，在结直肠癌早期病变诊治中具有重要的潜在应用价值。.
Keywords: Colorectal neoplasms; DNA methyltransferase 3b; High-grade intraepithelial neoplasia; Methylation; Precancerous lesion; SEPT9.