Decoding the functions of long noncoding RNAs in hepatocellular carcinoma

Abstract

Liver cancer is an aggressive malignancy representing the third leading cause of cancer deaths worldwide. Despite a variety of small molecule inhibitors along with the availability of FDA-approved chemotherapeutic drugs, the clinical outcome of HCC patients remains poor. The exquisite complexity of the human genome portends a daunting task for HCC treatment. Recent advancement in biotechnologies has led to the discovery of a massive landscape of regulatory elements in the human genome, amongst the most extensive of which are the genomic loci encoding long noncoding RNAs (lncRNAs). LncRNAs, the formerly invisible part of the genome, have come to the spotlight in recent years. Evidence accumulated over the past few years shows that lncRNAs interface with various proteins and nucleotide species to collectively shape the outcome of a wide array of biological processes, ranging from gene transcription to protein translation and signal transduction. LncRNAs have emerged as critical regulators in key cancer pathways. Thousands of lncRNAs have been shown to be differentially expressed in HCC tumors by high-throughput sequencing, but only a minority of them have been functionally characterized. Herein, we sought to address the functional roles of lncRNAs in HCC progression by genome-wide CRISPR activation library screening in xenograft mouse models. The library contains 96,958 sgRNAs targeting 10,504 lncRNA transcriptional start sites (TSS), with 10 sgRNAs tiling the proximal promoter of each lncRNA TSS. We identified 1603 positively selected lncRNAs, among which, 485 lncRNAs displayed significant differential expression in human HCC. In addition to the previously well-characterized lncRNAs, we also identified a wealth of novel lncRNA candidates. These lncRNA candidates were frequently overexpressed in human HCC and correlated with poor survival and elevated risk of tumor recurrence. Systematic transcriptomic data analysis and clinical investigation revealed that patients with high expression of these lncRNAs candidates were highly correlated with biological processes associated with aggressive tumor behaviour. Detailed characterization of a nuclear-specific lncRNA, Cancer Susceptibility 11 (CASC11) and a cytoplasmic lncRNA, Uncharacterized LOC730101 (LOC730101), demonstrated their important roles in cell proliferation and tumor growth. Mechanistic investigation on CASC11 revealed that CASC11 was preferentially bound to CASC11/MYC shared promoter region and fine-tuned the transcriptional activity of MYC in a cis-regulatory manner, which in turn, affected cancer cell hallmarks through dysregulating MYC and its downstream pathways associated with cell cycle progression. In addition, we found that the CASC11/MYC axis regulated G1/S phase progression through modulating CDK4 and CDK6. We also revealed that LOC730101 dysregulated a subset of genes associated with inflammatory response and epithelial-and-mesenchymal transition, thereby promoting HCC tumor growth and metastasis. Taken together, our study demonstrated the power of CRISPR/dCas9 activation screening for identifying functional lncRNAs that promoted HCC progression in a high-throughput manner, providing a rationale for targeting these lncRNAs clinically. Finally, in view of the rapid development in the field of lncRNA studies, we review the current state of lncRNA studies and highlight the works to be done in the coming years in the hope of inspiring the future goal towards the development of novel therapeutic strategies for HCC patients.