Bhat Samatha, Kabekkodu Shama Prasada, Varghese Vinay Koshy, Chakrabarty Sanjiban, Mallya Sandeep P, Rotti Harish, Pandey Deeksha, Kushtagi Pralhad, Satyamoorthy Kapaettu
1 Department of Cell and Molecular Biology, School of Life Sciences, Manipal University, Manipal, India.
2 Department of Obstetrics & Gynaecology, Kasturba Medical College, Manipal University, Manipal, India.
Tumour Biol. 2017 Mar;39(3):1010428317694573. doi: 10.1177/1010428317694573.
Multicomponent molecular modifications such as DNA methylation may offer sensitive and specific cervical intraepithelial neoplasia and cervical cancer biomarkers. In this study, we tested cervical tissues at various stages of tumor progression for 5-methylcytosine and 5-hydroxymethylcytosine levels and also DNA promoter methylation profile of a panel of genes for its diagnostic potential. In total, 5-methylcytosine, 5-hydroxymethylcytosine, and promoter methylation of 33 genes were evaluated by reversed-phase high-performance liquid chromatography, enzyme-linked immunosorbent assay based technique, and bisulfate-based next generation sequencing. The 5-methylcytosine and 5-hydroxymethylcytosine contents were significantly reduced in squamous cell carcinoma and receiver operating characteristic curve analysis showed a significant difference in (1) 5-methylcytosine between normal and squamous cell carcinoma tissues (area under the curve = 0.946) and (2) 5-hydroxymethylcytosine levels among normal, squamous intraepithelial lesions and squamous cell carcinoma. Analyses of our next generation sequencing results and data from five independent published studies consisting of 191 normal, 10 low-grade squamous intraepithelial lesions, 21 high-grade squamous intraepithelial lesions, and 335 malignant tissues identified a panel of nine genes ( ARHGAP6, DAPK1, HAND2, NKX2-2, NNAT, PCDH10, PROX1, PITX2, and RAB6C) which could effectively discriminate among the various groups with sensitivity and specificity of 80%-100% (p < 0.05). Furthermore, 12 gene promoters (ARHGAP6, HAND2, LHX9, HEY2, NKX2-2, PCDH10, PITX2, PROX1, TBX3, IKBKG, RAB6C, and DAPK1) were also methylated in one or more of the cervical cancer cell lines tested. The global and gene-specific methylation of the panel of genes identified in our study may serve as useful biomarkers for the early detection and clinical management of cervical cancer.
多组分分子修饰如DNA甲基化可能提供敏感且特异的宫颈上皮内瘤变和宫颈癌生物标志物。在本研究中，我们检测了肿瘤进展不同阶段的宫颈组织中5-甲基胞嘧啶和5-羟甲基胞嘧啶水平，以及一组基因的DNA启动子甲基化谱，以评估其诊断潜力。总共通过反相高效液相色谱、基于酶联免疫吸附测定的技术和基于亚硫酸氢盐的下一代测序评估了33个基因的5-甲基胞嘧啶、5-羟甲基胞嘧啶和启动子甲基化。鳞状细胞癌中5-甲基胞嘧啶和5-羟甲基胞嘧啶含量显著降低，受试者工作特征曲线分析显示，（1）正常组织与鳞状细胞癌组织之间的5-甲基胞嘧啶存在显著差异（曲线下面积=0.946），（2）正常组织、鳞状上皮内病变和鳞状细胞癌之间的5-羟甲基胞嘧啶水平存在显著差异。对我们的下一代测序结果以及来自五项独立发表研究的数据（包括191例正常组织、10例低级别鳞状上皮内病变、21例高级别鳞状上皮内病变和335例恶性组织）进行分析，确定了一组九个基因（ARHGAP6、DAPK1、HAND2、NKX2-2、NNAT、PCDH10、PROX1、PITX2和RAB6C），它们能够以80%-100%的敏感性和特异性有效区分不同组（p<0.05）。此外，在测试的一种或多种宫颈癌细胞系中，12个基因启动子（ARHGAP6、HAND2、LHX9、HEY2、NKX2-2、PCDH10、PITX2、PROX1、TBX3、IKBKG、RAB6C和DAPK1）也发生了甲基化。我们研究中确定的这组基因的整体和基因特异性甲基化可能作为宫颈癌早期检测和临床管理的有用生物标志物。
