Functional characterization of hypoxia-regulated lncRNAs in Non-Small Cell Lung Carcinomas

Abstract

Hypoxia triggers the activation of several signaling pathways inducing a complex transcriptomic response with multiple consequences on cell survival, migration, invasion and metabolic reprogramming. This regulatory network integrates specific feedbacks and checkpoint signaling loops including epigenetic factors and non-coding RNAs. However, while a large set of miRNAs have been reported as crucial molecular regulators of the hypoxic response, the precise functional characterization of hypoxia-regulated lncRNAs is still in its infancy. Our study aimed at comprehensively characterize the whole set of hypoxia-regulated lncRNAs in lung adenocarcinoma. Using a combination of experimental profiling approaches in both tumor cell lines and patient samples as well as exploring in silico TCGA datasets, we identified a validated signature of lncRNAs correlated to i) the hypoxic status of tumors and ii) the overall survival of patients. Molecular characterization of a subset of these “hypoxa-lcnRNAs” using RNA-Seq, RT-PCR, northern blot and single molecule RNA FISH pointed to 2 interesting candidates with different subcellular localization pattern: i) NLUCAT1, a large nuclear transcript composed of 6 exons and ii) LINC01116, a short cytosolic transcript composed of 3 exons. We next used CRISPR/Cas9-mediated invalidation and transcriptomic analyses as well as RNA-pulldown approaches to functionally characterize their function and mode of action. While NLUCAT1 was mainly identified as a regulator of the NRF2-mediated anti-oxidant response with an impact on cisplatin resistance, LINC01116 was mostly associated to the regulation of cellular morphology, cell-to-cell contact and cell invasion through an alpha catenin gene network. Overall, our study strongly supports the central role of non-coding RNAs in the cellular response to hypoxia and provides the first molecular characterization of 2 hypoxa-lncRNAs contributing to an aggressive phenotype in hypoxic tumors