A lincRNA connected to cell mortality and epigenetically-silenced in most common human cancers

Immortality is an essential characteristic of human carcinoma cells. We recently developed an efficient, reproducible method that immortalizes human mammary epithelial cells (HMEC) in the absence of gross genomic changes by targeting 2 critical senescence barriers. Consistent transcriptomic changes associated with immortality were identified using microarray analysis of isogenic normal finite pre-stasis, abnormal finite post-stasis, and immortal HMECs from 4 individuals. A total of 277 genes consistently changed in cells that transitioned from post-stasis to immortal. Gene ontology analysis of affected genes revealed biological processes significantly altered in the immortalization process. These immortalization-associated changes showed striking similarity to the gene expression changes seen in The Cancer Genome Atlas (TCGA) clinical breast cancer data. The most dramatic change in gene expression seen during the immortalization step was the downregulation of an unnamed, incompletely annotated transcript that we called MORT, for mortality, since its expression was closely associated with the mortal, finite lifespan phenotype. We show here that MORT (ZNF667-AS1) is expressed in all normal finite lifespan human cells examined to date and is lost in immortalized HMEC. MORT gene silencing at the mortal/immortal boundary was due to DNA hypermethylation of its CpG island promoter. This epigenetic silencing is also seen in human breast cancer cell lines and in a majority of human breast tumor tissues. The functional importance of DNA hypermethylation in MORT gene silencing is supported by the ability of 5-aza-2′-deoxycytidine to reactivate MORT expression. Analysis of TCGA data revealed deregulation of MORT expression due to DNA hypermethylation in 15 out of the 17 most common human cancers. The epigenetic silencing of MORT in a large majority of the common human cancers suggests a potential fundamental role in cellular immortalization during human carcinogenesis.细胞永生性是人类癌细胞的本质特征。我们近期开发了一种高效且可重复的方法，可在不引发显著基因组改变的前提下，通过靶向两大关键衰老屏障实现人类乳腺上皮细胞（human mammary epithelial cells, HMEC）的永生化。通过对4名供体来源的同基因正常有限增殖停滞前、异常有限增殖停滞后以及永生化HMEC进行微阵列分析，我们鉴定出了与细胞永生性相关的一致性转录组变化。共计277个基因在从增殖停滞后阶段向永生化阶段转变的细胞中呈现一致性表达改变。对受影响基因进行基因本体（Gene Ontology）分析后发现，细胞永生化过程中多个生物学过程发生显著改变。这些与永生化相关的表达变化模式，与癌症基因组图谱（The Cancer Genome Atlas, TCGA）临床乳腺癌数据集的基因表达变化高度相似。在永生化过程中，最显著的基因表达变化为一个未命名、注释尚不完全的转录本的下调，我们将其命名为MORT（取自mortality，即“衰老/存活受限”之意），因其表达与细胞有限寿命的衰老表型紧密相关。本研究证实，MORT（ZNF667-AS1）在迄今检测过的所有正常有限寿命人类细胞中均有表达，而在永生化HMEC中则表达缺失。MORT在细胞衰老与永生化分界点的基因沉默，源于其CpG岛启动子区域的DNA高甲基化修饰。这种表观遗传沉默现象同样存在于人类乳腺癌细胞系以及多数人类乳腺肿瘤组织中。5-氮杂-2'-脱氧胞苷（5-aza-2′-deoxycytidine）可重新激活MORT的表达，这一结果进一步验证了DNA高甲基化在MORT基因沉默中的功能性重要性。对TCGA数据集的分析显示，在17种最常见的人类癌症中，有15种存在因DNA高甲基化导致的MORT表达失调。MORT在绝大多数常见人类癌症中的表观遗传沉默现象，提示其在人类癌变过程中的细胞永生化环节可能发挥基础性调控作用。