通过定量甲基化特异性PCR和焦磷酸测序检测肺癌细胞系KEAP1基因的甲基化模式密度
背景: KEAP1 / NRF2途径是抗氧化剂和细胞应激反应的关键调节剂,与肿瘤的发展和肿瘤对治疗的抗性有关。在实体瘤中,作为启动子甲基化的 KEAP1 沉默是KEAP1 / NRF2轴复杂调控的一部分,但其预后作用在肺癌中仍有待解决。 方法:通过分析来自不同组织学的25种细胞系,我们对位于 KEAP1 启动子区域的13个CpG进行了详细的甲基化密度图。使用定量甲基化特异性PCR(QMSP)和焦磷酸测序评估甲基化状态,并比较两种测定的性能。 结果:在三分之一的细胞系中检测到 KEAP1 启动子区域的甲基化,并且其对调节 KEAP1 mRNA水平的影响通过体外5-氮杂胞苷治疗对肺癌,小肺癌和腺癌细胞系的证实。即使焦磷酸测序显示两个不同的启动子CpGs亚岛(P1a和P1b)具有不同的甲基化密度模式,QMSP和焦磷酸测序也显示出较高的一致性结果。 结论:我们的结果证实了甲基化对肺癌多种组织学中 KEAP1 转录控制的影响,并建议焦磷酸测序是研究启动子中CpGs甲基化模式的最佳方法 KEAP1 区域。此方法必须在肺癌患者队列中进行验证,以阐明 KEAP1 的表观遗传去调控在肺癌中的预后价值。**
Methylation Density Pattern of KEAP1 Gene in Lung Cancer Cell Lines Detected by Quantitative Methylation Specific PCR and Pyrosequencing
BACKGROUND: The KEAP1/NRF2 pathway is the key regulator of antioxidants and cellular stress responses, and is implicated in neoplastic progression and resistance of tumors to treatment. KEAP1 silencing by promoter methylation is widely reported in solid tumors as part of the complex regulation of the KEAP1/NRF2 axis, but its prognostic role remains to be addressed in lung cancer. METHODS: We performed a detailed methylation density map of 13 CpGs located into the KEAP1 promoter region by analyzing a set of 25 cell lines from different histologies of lung cancer. The methylation status was assessed using quantitative methylation specific PCR (QMSP) and pyrosequencing, and the performance of the two assays was compared. RESULTS: Hypermethylation at the promoter region of the KEAP1 was detected in one third of cell lines and its effect on the modulation KEAP1 mRNA levels was also confirmed by in vitro 5-Azacytidine treatment on lung carcinoid, small lung cancer and adenocarcinoma cell lines. QMSP and pyrosequencing showed a high rate of concordant results, even if pyrosequencing revealed two different promoter CpGs sub-islands (P1a and P1b) with a different methylation density pattern. CONCLUSIONS: Our results confirm the effect of methylation on KEAP1 transcription control across multiple histologies of lung cancer and suggest pyrosequencing as the best approach to investigate the pattern of CpGs methylation in the promoter region of KEAP1. The validation of this approach on lung cancer patient cohorts is mandatory to clarify the prognostic value of the epigenetic deregulation of KEAP1 in lung tumors.
pmid: 31159323 Int J Mol Sci 影响因子: 4.183 发表日期: 20190531 官网 免费下载
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