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宫颈癌中候选基因的定量DNA甲基化分析

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

作者信息

Siegel Erin M, Riggs Bridget M, Delmas Amber L, Koch Abby, Hakam Ardeshir, Brown Kevin D

机构信息

Department of Cancer Epidemiology, Division of Population Sciences, Moffitt Cancer Center, 12902 Magnolia Drive, Tampa, FL 33612, United States of America.

Department of Biochemistry and Molecular Biology and UF-Shands Cancer Center, University of Florida College of Medicine, 1200 Newell Drive, Academic Research Building, R3-234, Gainesville, FL 32610, United States of America.

出版信息

PLoS One. 2015 Mar 31;10(3):e0122495. doi: 10.1371/journal.pone.0122495. eCollection 2015.

Abstract

Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2). A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site) per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC) of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003). Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

摘要

宫颈癌中已观察到异常的DNA甲基化;然而,大多数研究使用非定量方法来测量DNA甲基化。本研究的目的是对先前确定为宫颈癌表观遗传沉默靶点的一组特定基因内的甲基化进行定量,并鉴定甲基化水平升高且可区分癌组织与正常宫颈组织的基因。我们纳入了49例宫颈浸润性鳞状细胞癌女性和22例细胞学标本正常的女性。对亚硫酸氢盐修饰的基因组DNA进行扩增,并对10个基因(APC、CCNA、CDH1、CDH13、WIF1、TIMP3、DAPK1、RARB、FHIT和SLIT2)完成定量焦磷酸测序。甲基化指数计算为每个基因(每个序列约4 - 9个CpG位点)分析的所有CpG位点的平均甲基化百分比。定义甲基化>15%为二元切点。检查单个基因或基因组合中甲基化的敏感性、特异性和ROC曲线下面积(AUC)。8个基因的病例组甲基化指数中位数显著高于对照组,而2个基因的甲基化中位数无差异。与HPV和年龄相比,DAPK1、SLIT2、WIF1和RARB的DNA甲基化水平与HPV和年龄的组合显著提高了AUC,从0.79提高到0.99(95%CI:0.97 - 1.00,p值 = 0.003)。焦磷酸测序分析证实,几个基因是宫颈癌中异常甲基化的常见靶点,与单独检测HPV相比,四个基因的DNA甲基化水平似乎提高了识别癌症的特异性。宫颈癌中特定基因如DAPK1、RARB、WIF1和SLIT2的DNA甲基化改变也可能在宫颈癌发生的早期出现,应予以评估。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/a8a4/4380427/fa4a39f023da/pone.0122495.g001.jpg

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