Identification of lncrnas deregulated in hepatocellular carcinomaNew key players in tumour growth

Abstract

Hepatocellular carcinoma is the most common primary liver cancer, the sixth most common cancer and the fourth cause of cancer-related death in the world. The incidence of HCC is very similar to its mortality, as most patients are diagnosed at advanced stages when only systemic therapies, such as chemotherapy and molecular targeted therapies, can be administered. However, patients develop resistance to these treatments and their median survival hardly reaches a year. In this thesis, we have found that long non-coding RNAs (lncRNAs) can be a valuable therapeutic alternative. We have selected a collection of lncRNAs upregulated in HCC tumours of three independent cohorts whose patients were exposed to different risk factors. The increased expression of these lncRNAs associates with high alpha-fetoprotein levels in serum, the presence of vascular invasion and advanced histological grades, all of them indicators of poor prognosis. Moreover, they are upregulated in aggressive tumours, in which TP53 gene is mutated and TERT is overactivated, producing high chromosomal instability. All these hints lead us to think that the upregulation of these lncRNAs may be used as a predictor of an unfavourable outcome. These lncRNAs are predicted to contribute to cancer hallmarks, favouring cell division, DNA damage repair and gene expression related processes. Besides, their expression is modulated by key proto-oncogenic factors, such as Myc, c-Jun, c-Fos or Stat3, among others. We evaluated the role of these lncRNAs in liver cancer cell proliferation by inhibition assays. We have developed a high-throughput screening using a CRISPR-Cas9 genome editing technique known as HITI (Homology-Independent Targeted Insertion) to impair the full transcription of the lncRNAs. The lncRNAs that could not be assessed using this technique because of potential off-target effects at neighbouring coding genes, were evaluated with antisense oligonucleotides (ASOs). Altogether, we have identified six different candidates whose expression contributes to hepatoma cell growth. Interestingly, we observed that the expression of these six lncRNAs was cell-cycle deregulated, specially at S and G2 phases. In fact, one of these lncRNAs is a SCAT or S-phase Cancer Associated Transcript, which are lncRNAs characterized for being upregulated in S-phase in a tumour-specific manner that act as scaffolds to mediate the interaction of transcription factors and the promoter of oncogenes. Our lncRNA candidate may be necessary for the transcriptional activity and turnover of FoxM1, which directs the transcription of protein coding genes that are necessary during the mitotic phase and for a successful cell-cycle progression.