Hepatocellular carcinoma (HCC) is the most frequent primary tumor of the liver and the third cause of cancer-related deaths. The identification of candidate molecular targets and biomarkers in HCC clinical practice are needed. The signatures of aberrant long non-coding RNAs (lncRNAs) expression in HCC tissues, their extracellular release and stability had led to their exploration as diagnostic and prognostic tools as well as potential therapeutic targets for HCC. Telomeric-Repeat Containing RNA (TERRA) consists of 100nt-9Kb subtelomeric-derived transcripts able to base-pair with TERC RNA, acting as telomerase allosteric inhibitor. Little is known on the role of lncRNA TERRA in HCC.
By qPCR we measured TERRA expression in tumor and peritumoral (PT) tissues of HCC patients, as well as in plasma and in HCC cells. HCC patients (n.25) did not receive any treatment before surgical resection. By nickel-based isolation method (NBI) we isolated from the conditioned medium (CM) of HA22T/VGH cells the extracellular vesicles (EVs), subsequently analyzed by qNANO instrument.
Global TERRA expression was significantly downregulated in HCC vs PT tissues (p=0.025) and ROC analysis revealed a significant ability to distinguish HCC from PT (p=0.03). Extracellular TERRA transcripts were significantly higher in plasma of HCC patients compared with healthy subjects and logistic regression model strongly evidenced the potential diagnostic ability of circulating TERRA (AUC=0.76; 95% CI=0.624-0.873; p=0.0004). HA22T/VGH cells expressed TERRA, but most of the transcripts are released into the CM, also encapsulated in EVs (mean diameter=185nm, concentration=163x106/ml). Treatment of HCC cells with the multi-kinase inhibitor (KI) sorafenib significantly increased TERRA expression (p=0.001) and decreased (p=0.01) its release in EVs (mean diameter=252nm, concentration=209x106/ml).
Our results provide evidence on TERRA dysregulation in tissues and liquid biopsy of HCC patients, thus focusing on a novel potential non-invasive biomarker of diagnosis and downstream target of the KI. TERRA detected in the EVs of HCC cells open a new field of cancer research to comprehend its role at the extracellular level.
The authors.
Lega Italiana Lotta ai Tumori (LILT), Italian Ministry of University and Research (FFRB), University of Brescia.
All authors have declared no conflicts of interest.