Circular RNAs in hepatitis B virus-induced hepatocellular carcinoma: A comprehensive review and recent advances

Abstract

Circular RNAs (circRNAs) are a class of stable and versatile non-coding RNAs that are pivotal in the occurrence and development of some diseases, particularly tumors. Hepatitis B virus (HBV)-induced hepatocellular carcinoma (HCC) is a liver disease with substantial global impact. Despite efforts towards adequate management, the survival of patients with HBV-induced HCC has been consistently low. circRNAs regulate various physiological activities of HBV-induced HCC. This review aims to elucidate the biogenesis of circRNAs and the pathophysiology of HBV-induced HCC and comprehensively analyze the applications of circRNAs in oncology and therapeutics. In addition, this review summarizes past research achievements on circRNAs in HBV-induced HCC. Finally, the limitations of existing methodologies and circRNA research in HBV-induced HCC have been discussed to provide a blueprint for future investigations.

Keywords: Biomarker; Hepatitis B virus; Hepatocellular carcinoma; Therapy; circRNA.

Figure 1

Part 1: The process of HBV infection and replication in cells: (A) HBV enters the liver cell via fusion and endocytosis. (B, C) The HBV genome is released into the cytoplasm and delivered to the nucleus. (D–F) Host proteins repair gaps in rcDNA to form cccDNA, which is transcribed into various RNAs, translating diverse proteins such as HBsAg, HBeAg, HBcAg, and HBx. (G–J) The multiple components assemble into an intact HBV released outside the cell. Part 2: The influence of proteins from HBV in HCC tumorigenesis: (A) Wnt combines with LRP and the frizzled receptor to inhibit the function of the Axin/APC/CK1α/GSK-3β complex by interacting with Dishevelled. The Axin/APC/CK1α/GSK-3β complex cannot phosphorylate β-catenin to promote its decomposition. (B) HBx mediates epigenetic modifications of RNAs, such as circRNAs. (C) HBx activates TCF in the Wnt/β-catenin signaling pathway. (D) HBx boosts RNA replication. (E, F) RNAs participate in the Wnt/β-catenin signaling pathway and activate the pathway. (G–I) HBx represses the function of the Axin/APC/CK1α/GSK-3β complex; therefore, β-catenin accumulates in the cytoplasm and enters the nucleus. (J) HBsAg increases the expression of LEF. (K, S) Multiple mechanisms facilitate the combination of β-catenin and TCF/LEF, regulating gene expression to promote the proliferation, drug resistance, and stemness of HBV-HCC cells. (L–R) The products, such as HBsAg, HBeAg, HBcAg, and HBx, from the HBV-infected HCC cells increase the expression of PD-1, CD244, CTLA4, IL-10, and PD-L1, and decrease the expression of IFN-γ, TNF-α, and activating receptors on many immune cells. Part 3: Four stages of HBV-HCC development: The process by which a healthy liver becomes HBV-HCC includes the following four stages: uninfected liver, chronic hepatitis B infection, cirrhosis, and HBV-HCC; however, a healthy liver does not progress to cirrhosis in all patients. HCC, hepatocellular carcinoma; HBV, hepatitis B virus; rcDNA, relaxed circular DNA; cccDNA, covalently closed circular DNA; HBeAg, hepatitis B virus e antigen; HBsAg, hepatitis B surface antigen; HBcAg, hepatitis B core antigen; HBx, HBV x protein; PD-1, programmed death-1; CTLA4, cytotoxic T-lymphocyte associated protein 4; IL-10, interleukin 10; PD-L1, programmed cell death ligand 1; IFN-γ, interferon-gamma; TNF-α, tumor necrosis factor alpha; APC, adenomatous polyposis coli; CK1α, casein kinase 1α; Axin, Axis inhibition protein; GSK-3β, glycogen synthase kinase-3β; TCF, T-cell factor; LEF, lymphoid enhancer factor.

Figure 2

Biogenesis and functions of circRNAs. (A, B) Formation mechanisms of circRNAs: intron-pairing model, RNA-binding protein (RBP)-inducing model, debranching escape model, and exon-skipping model. Intron-pairing model: The flanking introns of an exon contain complementary sequences, facilitating back-splicing and circRNA formation by bringing splice sites closer. RBP-inducing model: RBPs bind to flanking intronic sequences and get them in proximity, enabling back-splicing and circularization. Debranching escape model: When a pre-mRNA undergoes splicing to remove introns and join exons, 2′–5′ phosphodiesters at the intron's branch points are conducive to circRNA cyclization. Exon-skipping model: During exon skipping, exons form lariats, producing circRNAs by back-splicing.(C) Types of circRNAs: exonic circRNAs, intronic circRNAs, exon-intron circRNAs, intergenic circRNAs, and antisense circRNAs., , (D–K) The behavior mechanisms of circRNAs: regulating transcription, binding to miRNA, protein scaffolding, translation, producing pseudogenes, stabilizing RNA molecules, facilitating nucleus transport, and modulating alternative splicing. (L) Generation of mRNA from pre-mRNA. (M, N) Release and reception of circRNAs via microvesicles. (O) Biological functions of circRNAs in cancers.

Figure 3

Flow chart of the study exploring the functions of circRNAs in HBV-HCC: Sample collection from HBV-HCC tissues and adjacent normal tissues with HBV or from HBV-infected cells, HBV-HCC cells, and non-HBV HCC cells. (1) Extraction of total RNAs from samples and purification of circRNAs by RNase R digestion and high-performance liquid chromatography (HPLC). (2) Analysis and verification of differentially expressed circRNAs by microarray and quantitative reverse transcription PCR. (3) circRNA localization by fluorescence in situ hybridization (FISH) and sequencing analysis. (4) Verification of circRNAs in large samples. (5, 6) Prediction of ceRNA networks by computers and functions of circRNAs with GO and KEGG pathway enrichment analysis. (7, 8) Cell and animal assays were performed to confirm the ceRNA network and function of circRNAs. HCC, hepatocellular carcinoma; HBV, hepatitis B virus.