Subhash Santhilal, Kanduri Meena
Department of Medical Genetics, Institute of Biomedicine, Gothenburg University.
Department of Clinical Chemistry and Transfusion Medicine, Institute of Biomedicine, Gothenburg University;
J Vis Exp. 2017 Jun 16(124):55773. doi: 10.3791/55773.
The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.
由于人们越来越有兴趣了解长链非编码RNA（lncRNA）在癌症发生和发展过程中的机制功能，其在癌症中的作用正日益受到关注。尽管如此，lncRNA和癌症中重复序列的整体表观遗传调控尚未得到充分研究，尤其是在慢性淋巴细胞白血病（CLL）中。本研究聚焦于一种独特的方法：使用甲基结合域（MBD）蛋白基于免疫沉淀捕获双链甲基化DNA片段，随后进行二代测序（MBD-seq）。本研究使用了属于两个预后亚组的CLL患者样本（5个IGVH突变样本 + 5个IGVH未突变样本）。分析显示，与正常健康对照相比，有5800个高甲基化和12570个低甲基化的CLL特异性差异甲基化基因（cllDMG）。重要的是，这些结果鉴定出了几个具有潜在预后价值的CLL特异性差异甲基化lncRNA、重复元件和蛋白质编码基因。这项工作概述了一个用于MBD-seq的详细方案以及为使用CLL患者样本全面分析高CpG富集区域的整体甲基化谱而开发的生物信息学流程。最后，使用焦磷酸测序验证了一个蛋白质编码基因和一个lncRNA，焦磷酸测序是一种用于分析CpG甲基化水平的高度定量方法，以进一步证实MBD-seq方案的结果。
