Recent Update on the Role of Circular RNAs in Hepatocellular Carcinoma

Abstract

After being overlooked for decades, circular RNAs (circRNAs) have recently generated considerable interest. circRNAs play a role in a variety of normal and pathological biological processes, including hepatocarcinogenesis. Many circRNAs contribute to hepatocarcinogenesis through sponging of microRNAs (miRs) and disruption of cellular signaling pathways that play a part in control of cell proliferation, metastasis and apoptosis. In most cases, overexpressed circRNAs sequester miRs to cause de-repressed translation of mRNAs that encode oncogenic proteins. Conversely, low expression of circRNAs has also been described in hepatocellular carcinoma (HCC) and is associated with inhibited production of tumor suppressor proteins. Other functions of circRNAs that contribute to hepatocarcinogenesis include translation of truncated proteins and acting as adapters to regulate influence of transcription factors on target gene expression. circRNAs also affect hepatocyte transformation indirectly. For example, the molecules regulate immune surveillance of cancerous cells and influence the liver fibrosis that commonly precedes HCC. Marked over- or under-expression of circRNA expression in HCC, with correlating plasma concentrations, has diagnostic utility and assays of these RNAs are being developed as biomarkers of HCC. Although knowledge in the field has recently surged, the myriad of described effects suggests that not all may be vital to hepatocarcinogenesis. Nevertheless, investigation of the role of circRNAs is providing valuable insights that are likely to contribute to improved management of a serious and highly aggressive cancer.

Keywords: HBV; HCC; biomarkers; ceRNA; circRNA; microRNA; signaling pathways.

Figure 1

Schematic illustration of formation of circRNA by back-splicing. (A) Gene transcription generates (B) pre-mRNA sequences, which comprise exons (colored rectangles) and introns (intervening black lines). Alu repeats located within introns may play a role in circularization during back-splicing. (C) Canonical linear splicing of pre-mRNA joins upstream splice donors (GU) with downstream splice acceptors (AG) to link exons, remove introns and form mature mRNA with 5’ cap and 3’ poly(A). (D) Back-splicing, which involves circularization of RNA, entails coupling of downstream donor elements with upstream acceptors. Sequences at the resultant circular splice junctions (red arrowhead) are distinct from the sites of linear splicing (black arrowhead). circRNAs formed by back-splicing typically comprise combinations of exons but may also include introns. Arrow within the circRNAs represents 5’ to 3’ polarity of the pre-mRNA.

Figure 2

Methods for assay of circRNA. (A) Direct sequencing of a reverse transcribed circRNA typically employs a primer that is complementary to an exon. The sequencing reaction does not discriminate between linear RNA and circRNA, but bioinformatics analysis allows identification of circular junctions (red arrowhead). (B) Microarrays make use of probes that straddle unique and defined circularization junctions. Typically random primers are used to generate labeled cRNAs from circRNA templates. Probes span the circular junctions and hybridize to the cRNAs with higher Tms than the partly complementary sequences of linear mature RNA. (C) Reverse transcriptase (RT) quantitative PCR entails specific amplification of sequences derived from circRNAs by using primers that flank the circular junctions. The configuration of the amplifying primers is such that mature linear mRNA is not amplified.

Figure 3

Influence of sponging circRNA on mRNA translation. (A) Overexpression of circRNAs leads to increased sequestration of miRs (red lines). Resultant diminished free miR de-represses mRNA translation. This may lead to overexpression of genes that participate in pathways involving oncogenesis. An example is overexpression of the Wnt/β-catenin pathway that results from circ-DENND4C overexpression and sponging of miR-195-5p. (B) Lowered concentrations of circRNA diminish miR sponging. Increased concentrations of free miRs are available to bind to mRNA cognates, which in turn leads to inhibition of translation that is mediated by the RNA-Induced Silencing Complex (RISC). Hepatocarcinogenesis may be promoted if expression of a tumor suppressor gene is inhibited by this mechanism. An example is the effect of circMTO1 on miR-181b-5p, which leads to diminished expression of Phosphatase and Tensin homolog (PTEN) and promotes fibrosis in premalignant hepatic tissue.