Examination of genome-wide methylation changes in lung cancer cell lines A549 (A) and HTB56 (H) [RRBS-Seq experiments]. Homo sapiens

Cancer cell phenotypes are partially determined by epigenetic specifications such as DNA methylation. Metastasis development is a late event in cancerogenesis and might be associated with epigenetic alterations. Here, we analyzed genome wide DNA methylation changes that were associated with pro-metastatic phenotypes in non-small cell lung cancer with Reduced Representation Bisulfite Sequencing. DNMT-inhibition by 5-Azacytidine at low concentrations reverted the pro-metastatic phenotype. 5-Azacytidine led to preferential loss of DNA methylation at sites that were DNA hypermethylated during the in vivo selection. Changes in DNA methylation persisted over time. Keywords: Methylation profiling by high throughput sequencing Overall design: We studied genome-wide methylation changes in lung cancer cell lines A549 (A) and HTB56 (H). We generated NSCLC lines with highly increased propensity to form tumor nodules in murine lungs after intravenous injections. In addition to the normal cell lines (0R) we analyzed the methylome of the the cell lines after three rounds of in vivo selection towards a highly metastatic phenotype (3R). Next we studied changes in the methylome of highly metastatic cell lines after DNA Methyltransferase inhibition by 5-Azacytidine treatment at low concentrations (250 nM & 1000 nM) for 6 days. During treatment cells were supplemented with fresh medium every 48 hours. After 6 days of 5-Azacytidine exposure, cells were washed three times with PBS to wash out the drug. The cells were released for additional 7 days in regular medium. We followed up the DNA methylation changes at day 13 of the experiment.

癌细胞表型部分由表观遗传特征（如DNA甲基化）决定。肿瘤转移是肿瘤发生进程中的晚期事件，且可能与表观遗传改变密切相关。本研究采用简化亚硫酸氢盐测序（Reduced Representation Bisulfite Sequencing），分析了非小细胞肺癌中与促转移表型相关的全基因组DNA甲基化变化。低浓度5-氮杂胞苷（5-Azacytidine）介导的DNA甲基转移酶（DNMT, DNA Methyltransferase）抑制可逆转该促转移表型。5-氮杂胞苷可优先使体内筛选过程中发生DNA高甲基化的位点出现甲基化丢失。DNA甲基化的改变可长期维持。
关键词：高通量测序甲基化谱分析
实验设计：本研究以肺癌细胞系A549（标记为A）与HTB56（标记为H）为研究对象，分析其全基因组甲基化变化。我们通过静脉注射构建了在小鼠肺内形成肿瘤结节的倾向性显著升高的非小细胞肺癌细胞系。除原始亲本细胞系（0R）外，我们还分析了经过三轮体内筛选以获得高转移表型的细胞系（3R）的甲基化组。随后，我们对高转移细胞系施加低浓度（250 nM与1000 nM）5-氮杂胞苷处理以抑制DNA甲基转移酶活性，持续6天。处理期间，每48小时为细胞更换新鲜培养基。经5-氮杂胞苷处理6天后，用磷酸盐缓冲液（PBS）洗涤细胞三次以清除药物。随后将细胞置于常规培养基中继续培养7天，并在实验第13天追踪细胞的DNA甲基化变化。