By Victoria Zhang
Within the non-coding RNA family, circular RNAs(circRNAs) is a novel endogenous class that forms a covalently closed ring structure without 3’ and 5’ ends. Due to their lack of free ends, circRNAs have high stability and resistance to RNase degradation.1 This high stability enables them to be evolutionarily conserved and present widely in eukaryotic cells.2 Importantly, the aberrant expression of circRNAs has been found to associate with diseases like cancer with high specificity to cell type and/or developmental stage.3 This is why circRNA is gaining growing attention as therapeutic targets or biomarkers in cancer. For these purposes, the critical roles played by circRNA in cancer progression is studied in recent years.
Currently, circRNA have been found to affect gene expression in cancer at multiple levels through the following four mechanisms:
circRNAs as miRNA sponges
circRNAs can act as competitive endogenous RNAs(ceRNAs) as they have multiple miRNA response elements (MREs). MREs are complementary to target miRNAs and can competitively bind to them to prevent them from attaching to their mRNA targets, thus regulating the downstream gene expression.4 Numerous circRNAs have been implicated in binding cancer-associated miRNAs.
For example, in breast cancer, a circ_0006528-miR-7–5p axis has been discovered. There is a negative correlation between circRNA circ_0006528 and miRNA miR-7–5p, and the target of miR-7–5p is protein Raf1 which is part of PI3K/Akt signalling pathway which mediates drug resistance of cancer cells. The expression of circRNA could aid the development of drug resistance in breast cancer cells.5
The circRNA-miRNA-mRNA axis has also been found to be involved in the invasion and migration of tumours. In hepatocellular carcinoma (HCC), circ_000839 negatively regulates miR-200b, which has an inverse correlation with its target RhoA. The network contributes to the suppression of the invasion and migration in HCC.6 Also, the circHIAT1/miR-195-5p/29a-3p/ 29c-3p/CDC42 network has been found in clear cell renal cell carcinoma (ccRCC) development. circHIAT1 expression can deregulate miR-195-5p/ 29a-3p/29c-3p expression and lead to increased CDC42 expression that enhances ccRCC cell migration and invasion.7
Circular RNAs also affect cell cycles by modulating key cell cycle modulators like p53, CDKs, and cyclins. For instance, circTP63 is upregulated in lung squamous cell carcinoma (LUSC) tissues and its upregulation promote cell proliferation by competitively binding to miR-873-3p and prevents the decrease of FOXM1 level, promoting cell proliferation.8
Another circRNA, circAGFG1, may sponge miR195-5p to relieve the repression of cyclin E1 expression, leading to tumorigenesis and the development of triple-negative breast cancer progression.9
circRNAs also play a role in the loss of apoptosis in cancer cells. circCCDC66 is overexpressed in lung adenocarcinoma and inhibit both miRNA-33b and miR-93. Thus, the inhibition of the MYC oncogene is relived, and apoptosis is inhibited in this way 10. Bcl2, the anti-apoptotic protein, is a target for miR-143, which circUBAP2 and hsa_circ_0001892 both compete to inhibit. Respectively, their expression reduces apoptosis in osteosarcoma and breast cancer cells.10
Protein interactome
Although miRNA sponging is a more common way for circRNAs to regulate cellular pathways, they have also been proposed to serve as a platform for protein interaction. For example, circFoxo3 can form a ternary complex with cell cycle-related proteins like CDK2 and p21 to strengthen their interaction and block cell cycle progression.11 In another case, circNfix can enhance the interaction between two proteins Ybx1 and Nedd4l and inhibit cyclin A2 and cyclin B1 expression by inducing ubiquitination-mediated Ybx1 degradation.12 A subfamily of circRNA can bind to IMP3 protein, which has a role in post-transcriptional regulation and is an oncofetal and tumour marker.11
Transcriptional regulation
circNOL10 is able to suppress the progression of the cell cycle by suppressing ubiquitination of a transcription factor called sex comb on midleg‐like 1 (SCML1). This ultimately inhibits cell proliferation and facilitates the apoptosis of lung cancer cells, which inhibits lung cancer development.13
There are also Exon-intron circular RNAs(EIciRNAs) like circEIF3J and circPAIP2 that can enhance gene expression by associating with transcriptional related proteins. They might hold factors such as small nuclear RNA U1 snRNP through RNA-RNA interaction and form a complex that further interacts with the Pol II transcription complex at the promoters.14
Translation regulation
A number of circRNAs can behave as mRNA brakes and protein scaffolds to modulate gene expression. Circ-MALAT1 can serve both as a microRNA sponge and mRNA translation brake by forming a ternary complex with both ribosomes and mRNA. The mRNA braking mechanism allows the cell to maintain a specific cell state, and for Circ-MALAT1, it facilitates hepatocellular cancer stem cell self-renewal.15
Novel discoveries have found that circRNA can even code for a protein within their exons containing start codon. circPPP1R12A carried an open reading frame (ORF) that codes for a functional protein circPPP1R12A-73aa. That can activate the Hippo-YAP signalling pathway and promote the growth and metastasis of colon cancer.16
Being far more stable than linear RNA, circRNAs with a critical role in cancer are ideal biomarkers and therapeutic targets. circRNAs are highly preserved in blood plasma and have a half-life of over 48 hours. Using liquid biopsy sampling and reverse transcriptase-digital droplet polymerase chain reaction (RT-ddPCR), the whole tumour can be screened, and lower concentrations of circRNAs can be more effectively and accurately detected than traditional PCR.4 circRNAs have been discovered as prognostic and diagnostic biomarkers for gastric cancer. Overexpression of circERBB2 correlates with higher cancer recurrence and lower survival rates in patients.2 Another example is hsa_circRNA_002059 as a potential diagnostic biomarker because its expression is correlated with distal metastasis, TNM stage, gender and age in gastric cancer patients.11 Other types of cancer, including lung cancer, liver cancer, breast cancer, and colorectal cancer, have seen circRNAs as possible novel biomarkers.4
Although clinical application of circRNAs as therapeutic targets is still under development, current techniques like CRISPR-Cas13 have been used to successfully knock down circRNAs in mouse embryos without impacting their related mRNAs. RNA interference and antisense oligonucleotides have been used to target ncRNAs in tumours for knockdown and has the potential to be used on circRNAs to regulate cancer progression. Further studies on circRNA mechanisms using RNA sequencing and knockdown techniques can facilitate the future application of circRNAs in combination with other chemotherapies to more effectively treat cancer.12
Overall, circRNAs are a novel class of RNAs regulating cells’ biological functions, especially in diseases like cancer, where they have promising applications as biomarkers and targets for therapeutic interventions with the progression of research techniques.
References:
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(7) Wang K, Sun Y, Tao W, Fei X, Chang C. Androgen receptor (AR) promotes clear cell renal cell carcinoma (ccRCC) migration and invasion via altering the circHIAT1/miR-195-5p/29a-3p/29c-3p/CDC42 signals. Cancer letters. 2017; 394 1-12. 10.1016/j.canlet.2016.12.036.
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(10) Zhong Y, Du Y, Yang X, Mo Y, Fan C, Xiong F, et al. Circular RNAs function as ceRNAs to regulate and control human cancer progression. Molecular cancer. 2018; 17 (1): 79. 10.1186/s12943-018-0827-8.
(11) Ng WL, Mohd Mohidin TB, Shukla K. Functional role of circular RNAs in cancer development and progression. RNA biology. 2018; 15 (8): 995-1005. 10.1080/15476286.2018.1486659.
(12) Wei X, Juan L, June H, Lingzhi W, Jia-Rong H, Gautam S, et al. Circular RNAs in cell cycle regulation: Mechanisms to clinical significance. Cell proliferation. 2021; e13143. 10.1111/cpr.13143.
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(14) Li Z, Huang C, Bao C, Chen L, Lin M, Wang X, et al. Exon-intron circular RNAs regulate transcription in the nucleus. Nature structural & molecular biology. 2015; 22 (3): 256-264. 10.1038/nsmb.2959.
(15) Chen L, Kong R, Wu C, Wang S, Liu Z, Liu S, et al. Circ‐MALAT1 Functions as Both an mRNA Translation Brake and a microRNA Sponge to Promote Self‐Renewal of Hepatocellular Cancer Stem Cells. Advanced science. 2020; 7 (4): 1900949-n/a. 10.1002/advs.201900949.
(16) Zheng X, Chen L, Zhou Y, Wang Q, Zheng Z, Xu B, et al. A novel protein encoded by a circular RNA circPPP1R12A promotes tumor pathogenesis and metastasis of colon cancer via Hippo-YAP signaling. Molecular cancer. 2019; 18 (1): 47. 10.1186/s12943-019-1010-6.
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