Figure S5. Gene expression analysis of LINC00261 co-regulated genes. A. Overlap between H522 differentially expressed genes. Blue circle = Differentially expressed genes in H522 RNAseq. Purple circle = genes co-expressed with LINC00261 in TCGA LUAD (TANRIC). B. DNA damage signaling cascade with genes differentially expressed in A549 shScrambled vs. A549-shLINC00261 (LINC00261 knock down). Pink = upregulated in shLINC00261 samples, green = downregulated. C. Western blotting for additional DDR pathway members in the presence of CMV-LINC00261 or CMV-NEO controls, as indicated. Loading controls were HSP90 or actin, as indicated. Representative blots of three independent experiments shown.
ARTICLE ABSTRACT
Lung cancer is the leading cause of cancer-related death in the United States. Long noncoding RNAs (lncRNA) are a class of regulatory molecules whose role in lung carcinogenesis is poorly understood. In this study, we profiled lncRNA expression in lung adenocarcinoma (LUAD) cell lines, compared their expression with that of purified alveolar epithelial type II cells (the purported cell of origin for LUAD), cross-referenced these with lncRNAs altered in the primary human tumors, and interrogated for lncRNAs whose expression correlated with patient survival. We identified LINC00261, a lncRNA with unknown function in LUAD, adjacent to the pioneering transcription factor FOXA2. Loss of LINC00261 was observed in multiple tumor types, including liver, breast, and gastric cancer. Reintroduction of LINC00261 into human LUAD cell lines inhibited cell migration and slowed proliferation by inducing G2–M cell-cycle arrest, while upregulating DNA damage pathway genes and inducing phosphorylation-mediated activation of components of the DNA damage pathway. FOXA2 was able to induce LINC00261 expression, and the entire locus underwent hypermethylation in LUAD, leading to loss of expression. We have thus identified an epigenetically deregulated lncRNA, whose loss of expression in LUAD promotes the malignant phenotype and blocks activation of the DNA damage machinery, predisposing lung cells to cancer development.
These findings identify LINC00261 as a tumor suppressor that blocks cellular proliferation by activating the DNA damage response and suggest that epigenetic therapy to inhibit DNA methylation may enhance treatment of LUAD.
