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.永生是人类癌细胞的核心特征之一。本团队近期开发了一种高效且可重复的方法，通过靶向两类关键衰老屏障，在无明显基因组改变的条件下实现人乳腺上皮细胞（human mammary epithelial cells, HMEC）的永生化。我们对4名个体来源的同基因正常有限增殖停滞前期、异常有限增殖停滞后期以及永生化HMEC开展微阵列分析，鉴定出与永生化状态相关的一致性转录组学变化。最终鉴定出277个在细胞从增殖停滞后期向永生化状态转变过程中发生一致性表达改变的基因。对差异基因的基因本体论（Gene Ontology, GO）分析显示，永生化过程中多个核心生物学过程发生了显著改变。这些与永生化相关的表达谱变化，与癌症基因组图谱（The Cancer Genome Atlas, TCGA）收录的临床乳腺癌数据集的基因表达特征高度相似。永生化步骤中最显著的基因表达变化，是对一个尚未命名、注释尚不完整的转录本的下调；我们将该转录本命名为MORT（取自mortality，即“存活寿命”），因该基因的表达与细胞具有有限增殖寿命的衰老死亡表型紧密相关。本研究证实，MORT（ZNF667-AS1）在目前已检测的所有正常有限寿命人类细胞中均有表达，而在永生化HMEC中表达完全缺失。MORT基因在增殖/永生化边界处的沉默，源于其CpG岛启动子的DNA高甲基化修饰。这种表观遗传沉默同样存在于人类乳腺癌细胞系，以及大部分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，这一实验结果佐证了DNA高甲基化在MORT基因沉默中的功能性重要地位。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的细胞永生化阶段可能发挥关键性的基础作用。