Emerging roles of circular RNAs in liver cancer

Abstract

Hepatocellular carcinoma and cholangiocarcinoma are the most common primary liver tumours, whose incidence and associated mortality have increased over recent decades. Liver cancer is often diagnosed late when curative treatments are no longer an option. Characterising new molecular determinants of liver carcinogenesis is crucial for the development of innovative treatments and clinically relevant biomarkers. Recently, circular RNAs (circRNAs) emerged as promising regulatory molecules involved in cancer onset and progression. Mechanistically, circRNAs are mainly known for their ability to sponge and regulate the activity of microRNAs and RNA-binding proteins, although other functions are emerging (e.g. transcriptional and post-transcriptional regulation, protein scaffolding). In liver cancer, circRNAs have been shown to regulate tumour cell proliferation, migration, invasion and cell death resistance. Their roles in regulating angiogenesis, genome instability, immune surveillance and metabolic switching are emerging. Importantly, circRNAs are detected in body fluids. Due to their circular structure, circRNAs are often more stable than mRNAs or miRNAs and could therefore serve as promising biomarkers - quantifiable with high specificity and sensitivity through minimally invasive methods. This review focuses on the role and the clinical relevance of circRNAs in liver cancer, including the development of innovative biomarkers and therapeutic strategies.

Keywords: ASO, antisense oligonucleotide; CCA, cholangiocarcinoma; CLIP, cross-linking immunoprecipitation; EMT, epithelial-to-mesenchymal transition; EVs, extracellular vesicles; HCC, hepatocellular carcinoma; HN1, haematopoietic- and neurologic-expressed sequence 1; IRES, internal ribosome entry sites; NGS, next-generation sequencing; QKI, Quaking; RBP, RNA-binding protein; RISC, RNA-induced silencing complex; TAM, tumour-associated macrophage; TSB, target site blockers; biomarker; cancer hallmarks; cholangiocarcinoma; circRNA; circRNA, circular RNA; hepatocellular carcinoma; miRNA, microRNA; shRNA, small-hairpin RNA; snRNP, small nuclear ribonuclear proteins.

Fig. 1

The biogenesis of circRNA. CircRNAs are generated by a non-canonical back-splicing, where a 5’ donor splice site (5’ss) of a downstream exon (here, exon 3, E3) will react with a 3’ acceptor splice site (3’ss) of an upstream exon (here, exon 2, E2), resulting in the formation of a unique back-splice junction that does not exist in linear RNA (here, the 5’ extremity of E2 with the 3’ extremity of E3). So far, 3 main mechanisms spearhead the RNA circularisation. (A) Lariat intermediate back-splicing. The back-splicing process occurs after alternative splicing generates an intermediate structure called a lariat, which includes the excluded exons (here, E2 and E3) and which can be secondly spliced. (B) Intronic base pairing. A direct physical proximity between distant splice sites allows the back-splicing process. This proximity is mediated by a complementary base pairing between the introns flanking the circRNA. (C) RBP interaction or dimerisation. Here, the physical proximity between 5’ss and 3’ss is mediated by RBP interaction/dimerisation located on the exons flanking the circularisation junction. These 3 main mechanisms result in the formation of either EcircRNA, EIcircRNA or IcircRNA. CircRNA, circular RNA; EcircRNA, exonic circRNA; EIcircRNA, exonic-intronic circRNA; IcircRNA, intronic circRNA; RBP, RNA-binding protein.

Fig. 2

CircRNA functions. (A) miRNA sponging: Numerous circRNA harbour miRNA response elements. Therefore, by sponging miRNA, circRNA act as competitive endogenous RNA, preventing miRNA post-transcriptionally binding to and repressing their natural targets. (B) RBP sponging: CircRNA display specific protein binding motifs offering them the capability to sequester RBP, regulate their activity and influence their localisation. (C) IRES/m6A-mediated translation: CircRNA containing IRES or with m6A epitranscriptomic modifications can be translated in a cap-independent manner. (D) Protein scaffolding: Proteins can be recruited by circRNA, facilitating enzymatic reactions. (E) Translational regulation: The translation initiation complex factors PABP and eIF4G are able to bind to circRNA. In this case, the interaction negatively regulates the translation initiation process. (F) Immunity: The endogenous m6A modification is necessary to distinguish self and non-self circRNA like those coming from viruses. (G) Transcription regulation: EIcircRNA regulate transcription by interacting with U1 snRNP and promoting the transcription of their parental genes. (H) Splicing competition. Spliceosome machinery can foster circRNA biogenesis under specific conditions leading to the reduction of linear mRNA production. CircRNA, circular RNA; EIcircRNA, exonic-intronic circRNA; IRES, internal ribosome entry sites; miRNA, microRNA; RBP, RNA-binding protein.

Fig. 3

CircRNA functions in liver cancer onset and progression. The upper part gathers the most described functions driven by circRNAs in liver cancer, while the lower part pinpoints potential emerging regulatory functions of circRNAs. Pro-oncogenic circRNAs are indicated in red and tumour suppressor circRNAs are indicated in green. CircRNAs deregulated in iCCA are marked by an asterisk. CircRNA, circular RNA; iCCA, intrahepatic cholangiocarcinoma.

Fig. 4

CircRNAs as innovative biomarkers and therapeutic targets in liver cancer. The left panel highlights tumour-suppressive and pro-tumourigenic circRNAs acting on well-known cancer hallmarks. The right panel highlights circRNAs as innovative biomarkers and therapeutic targets using ASOs. ASO, antisense oligonucleotide; circRNA, circular RNA; HCC, hepatocellular carcinoma; iCCA, intrahepatic cholangiocarcinoma.