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外周血Septin9基因甲基化检测对肺癌诊断的意义及对肺癌细胞A549生物学特性的影响
作者:华海琴  郑小妹  吴大平 
单位:儋州市人民医院 肿瘤科, 海南 儋州 571700
关键词:肺癌 甲基化 增殖 凋亡 迁移 侵袭 
分类号:R734.2
出版年·卷·期(页码):2022·50·第五期(573-580)
摘要:

目的:探讨肺癌患者外周血Septin9基因甲基化状态与临床病理特征的关系,及其对人肺癌A549细胞生物学行为的影响。方法:选取104例肺癌患者为研究对象,96例健康人员为对照,采用甲基化特异聚合酶链式反应(PCR)法检测两组外周血中Septin9基因甲基化情况,并计算Septin9基因甲基化阳性率,分析Septin9基因甲基化阳性率与肺癌临床病理特征的关系。采用5、10、20 μmol·L-1 5-aza-dC处理人肺癌细胞系A549,以正常培养的A549细胞作为对照组,采用实时荧光定量PCR和蛋白质印迹(Western blot)检测细胞中Septin9 mRNA和蛋白表达,通过MTT法检测细胞的增殖活性,流式细胞仪检测细胞的凋亡情况,Transwell实验检测细胞的迁移与侵袭能力。结果:与对照组相比,肺癌患者外周血中Septin9基因甲基化阳性率显著升高。Septin9基因甲基化阳性与肺癌患者的TNM分期、分化程度、淋巴结转移有关,Septin9基因甲基化阳性与肺癌患者的性别和年龄无关。经5-aza-dC处理后,A549细胞中Septin9 mRNA和蛋白表达水平均显著上调,且呈浓度依赖性;5-aza-dC能显著抑制A549细胞增殖,呈浓度与时间依赖性;在5-aza-dC处理后,A549细胞的凋亡率呈浓度依赖性地显著增加;同时,5-aza-dC显著抑制了A549细胞的迁移与侵袭,且均呈浓度依赖性。结论:肺癌外周血Septin9基因甲基化阳性率增加,该指标与肺癌肿瘤分期、分化程度和淋巴结转移有关,5-aza-dC处理能通过恢复A549细胞中Septin9表达来抑制肺癌细胞增殖、迁移与侵袭,并诱导细胞凋亡。

Objective:To investigate the relationship between the methylation status of Septin9 gene in peripheral blood of patients with lung cancer and the clinicopathological characteristics, and its effect on the biological behavior of human lung cancer A549 cells. Methods:One hundred and four patients with lung cancer and 96 healthy people were respectively divided into research group and control group. The methylation-specific PCR method was used to detect the methylation of Septin9 gene in the peripheral blood of the two groups, and the positive rate of Septin9 gene methylation was calculated. The relationship between the positive rate of Septin9 gene methylation and the clinicopathological characteristics of lung cancer was analyzed. The human lung cancer cell line A549 was treated with 5, 10, and 20 μmol·L-1 5-aza-dC, and the normally cultured A549 cells were used as controls. Real-time fluorescent quantitative PCR and Western blot were used to detect the expression of Septin9 mRNA and protein in cells. Cell proliferation activity, cell apoptosis and cell migration and invasion ability were detected by MTT method, flow cytometry and Transwell experiment respectively. Results:Compared with the control group, the positive rate of Septin9 gene methylation in peripheral blood of lung cancer patients was significantly increased, which was related to the TNM stage, degree of differentiation and lymph node metastasis, while it was not related to the gender and age of lung cancer patients. After 5-aza-dC treatment, the expression levels of Septin9 mRNA and protein in A549 cells were significantly up-regulated in a concentration-dependent manner. 5-aza-dC could significantly inhibit the proliferation of A549 cells in a concentration- and time-dependent manner. After 5-aza-dC treatment, the apoptotic rate of A549 cells was significantly increased in a concentration-dependent manner and at the same time, 5-aza-dC significantly inhibited the migration and invasion, all being concentration-dependent. Conclusion:The positive rate of methylation of Septin9 gene in peripheral blood of lung cancer is increased, which is related to tumor stage, degree of differentiation and lymph node metastasis. 5-aza-dC treatment can inhibit lung cancer cell proliferation, invasion and migration by restoring Septin9 expression in A549 cells, and induce cell apoptosis.

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