Methylation status of Septin 9 in peripheral blood and its relationship with colorectal cancer
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摘要:目的
检测结直肠癌(CRC)患者外周血胞裂蛋白9(Septin 9)的甲基化状态, 分析Septin 9甲基化与CRC的关系。
方法选取120例CRC患者为研究对象，分析其外周血Septin 9甲基化状态与临床资料的关系; 分离CRC患者和健康人群的血清外泌体，采用该外泌体处理CRC细胞，测定CRC细胞的增殖和凋亡情况。
结果共计1586例体检人群接受了Septin 9筛查，其中Seprtin 9甲基化阳性56例，阳性率为3.5%。Sepetin 9阳性者中，胃肠状态异常者(肠胃炎症或者息肉)20例(35.7%), 而Septin 9阴性者胃肠状态异常率仅为5.4%(83/1530), 提示Septin 9在外周血中的甲基化状态与胃肠状态密切相关。120例CRC患者中, Septin 9甲基化阳性率为78.4%(73/93);在不同分化程度的比较中，高分化、中分化、低分化CRC患者的Septin 9甲基化阳性率依次为52.6%(10/19)、79.7%(59/74)、88.9%(24/27), 低分化CRC患者外周血Septin 9甲基化阳性率高于高分化组，差异有统计学意义(P=0.006)。外周血Septin 9甲基化的灵敏度为72.3%, 特异度为92.5%。Western blot结果显示CRC患者外泌体中Septin 9表达较健康人群降低，外泌体中Septin 9甲基化水平较正常人群上升，差异均有统计学意义(P=0.009、0.003)。本研究结果显示, CRC患者血清外泌体可促进细胞增殖，对细胞凋亡有抑制作用，差异有统计学意义(P < 0.05或P < 0.01)。
结论Septin 9可作为CRC早期筛查的标志物, CRC患者血清外泌体诱导的CRC增殖、凋亡抵抗可能是Septin 9的作用机制。
Abstract:ObjectiveTo detect the methylation status of Septin 9 in the peripheral blood of patients with colorectal cancer (CRC) and analyze the relationship between the methylation of Septin 9 and CRC.
MethodsA total of 120 patients with CRC were selected as research objects, and the relationship between the methylation status of Septin 9 in the peripheral blood and the medical materials was analyzed; the serum exosomes isolated from patients with CRC and healthy people were used to treat the CRC cells, and the proliferation and apoptosis of CRC cells were detected.
ResultsA total of 1 586 people with health examinations were screened for Septin 9, and 56 of whom were positive for methylation of Septin 9, with a positive rate of 3.5%. Among patients with Septin 9 positive, there were 20 cases (35.7%) with abnormal gastrointestinal status (gastrointestinal inflammation or polyps), while only 5.4% (83/1 530) of patients with Septin 9 negative had abnormal gastrointestinal status, which indicated that the methylation status of Septin 9 in the peripheral blood was closely related to the gastrointestinal status. Among 120 CRC patients, the positive rate of methylation of Septin 9 was 78.4% (73/93); in the comparison of different differentiation degrees, the positive rates of Septin 9 methylation in patients with highly differentiated, moderately differentiated and poorly differentiated CRC were 52.6% (10/19), 79.7% (59/74) and 88.9% (24/27) respectively, and the positive rate of Septin 9 methylation in peripheral blood of patients with poorly differentiated CRC was significantly higher than that of patients with highly differentiated CRC (P=0.006). The sensitivity and specificity of Septin 9 methylation in the peripheral blood were 72.3% and 92.5% respectively. Western blot results showed that the expression of Septin 9 in the exosomes of CRC patients was significantly lower than that in the normal people, while the methylation level of Septin 9 in the exosomes was significantly higher than that in the healthy people (P=0.009, 0.003). The results of this study showed that serum exosomes of CRC patients could significantly promote cell proliferation and inhibit cell apoptosis (P < 0.05 or P < 0.01).
ConclusionSeptin 9 can be used as a marker for early screening of CRC, and the mechanism of Septin 9 may be the proliferation and apoptosis resistance of CRC induced by serum exosomes in patients with CRC.
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Keywords:
- colorectal cancer /
- Septin 9 /
- exosomes /
- carcinoembryonic antigen /
- carbohydrate antigen 199 /
- diagnosis
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图 2 CRC患者血清外泌体中Septin 9表达及甲基化状态
A: Western Blot检测外泌体标志物CD63; B: Western Blot检测外泌体中Septin 9表达; C: 特异性甲基化PCR检测外泌体中Septin 9甲基化状态。与正常人群相比, **P<0.01; 与CRC相比, ##P < 0.01。
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图 3 CRC患者血清外泌体对CRC细胞系Saos2增殖和凋亡的影响
A: CCK-8检测CRC血清外泌体对细胞增殖的影响; B: 流式法检测CRC患者血清外泌体对细胞凋亡的影响。两者比较, **P < 0.01。
表 1 1 586例正常人群Septin 9甲基化与胃肠状态的相关性
指标 分类 Septin 9甲基化状态 阳性(n=56) 阴性(n=1 530) 性别 男 43 982 女 13 548 胃肠状态 正常 36 1 447 异常 20 83 
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表 2 CRC患者Septin 9检测情况[n(%)]
指标 分类 Septin 9阳性(n=93) Septin 9阴性(n=27) 性别 男 67(79.8) 17(20.2) 女 26(72.2) 10(27.8) 年龄 ≤60岁 51(71.8) 20(28.2) >60岁 42(85.7) 7(14.3) 分化程度 高分化 10(52.6)** 9(47.4) 中分化 59(79.7) 15(20.3) 低分化 24(88.9) 3(11.1) 低分化比较, ** P < 0.01。
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表 3 CRC患者Septin 9、CEA、CA199的灵敏度和特异度比较
指标 灵敏度(95%CI) 特异度(95%CI) 曲线下面积 Septin 9 72.3(60.5~80.5) 92.5(75.5~95.5) 0.87 CEA 42.2(32.2~55.6) 75.0(65.5~87.5) 0.59 CA199 20.5(12.1~27.1) 45.0(37.7~55.5) 0.24
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