Evidence of the important biological functions of circRNA – to help you understand circRNA better
Case 1: circRNA acts as a miRNA sponge or combines with RNA binding protein to function
The sponging of miRNAs by circRNAs is the most studied function of circular RNAs. The tissue specificity of circRNAs (including cancer tissues) raised a question about identifying potential expression profiles of circRNAs and their relation to cancer progression. In their study, Wang et al. acquired sequencing data from gallbladder cancer tissues. After a closer examination, the authors identified upregulated circRNAs, and correlation analyses showed that circFOXP1 is associated with the most aggressive cancer progression scenario. The results of overexpression studies supported the hypothesis that high circFOXP1 expression results in more intensive tumor growth via the Warburg effect promotion. The proposed mechanism for this phenomenon is the interaction of circFOXP1 with RNA-binding protein PTBP1 (improvement of translocation) and subsequent sponging of miR-370 (targets PKLR). PTBP1 promotes the mRNA expression of the PKRL gene, which induces tumor growth and proliferation. The findings were supported by RNA pull-down assay, LC-MS, western blotting, and immunofluorescence. Thus, circRNAs are new key players in cancer metabolism, and they can serve as potential therapeutic targets.
Case 2: circRNA can be translated
One of the lesser-known mechanisms of circRNA is through translation. The research by Mahmoudi et al. shows that some of the circRNAs can interact with mRNAs and induce expression (demonstrated in human neuroblasts). To verify the hypothesis, the loss-of-function of one of the circRNAs disrupted the expression of mRNAs, responsible for the gene function (EXOC6B). This circEXOC6B loss was sufficient enough to produce drastic effects such as whole pathway disruption. Ribo-Seq (also known as ribosome profiling) data also suggested that many of the circRNAs have signatures of potential for polypeptide production such as ORFs or conserved domains. This finding supports the hypothesis that circRNAs cannot be considered “junk” genome anymore and they may result in new protein-like products.
Case 3: circRNA as a molecular marker and oncology target
The unique sponging properties of circRNAs sparked an interest in oncology studies. For example, circRNA MTO1 suppressed hepatocellular carcinoma progression by acting as a sponge of miR-9 and circRNA HIPK3 sponges several miRNAs resulting in slowing the tumor progression. The additional study of hepatocellular carcinoma (HCC) revealed another perspective on circRNA as facilitating the development of novel anticancer treatments. Shi et al. previously reported the function of circPABPC1, circSMARCA5, circASAP1, and cirtMAT2B in HCC, however, they suggest that circRNA may act rather as a facilitator of protein interactions. For example, specific circRNAs were found to mediate integrin signaling (one of the key processes in cancer growth) such as linking β1 integrin to the proteasome for degradation. This is another example of how ubiquitous circRNA functions may be – signaling is one of them.
In a similar clinical study, RNA sequencing analysis of tissues from over 600 patients with advanced-stage colon cancer allowed collecting enough data to establish a classification mechanism. According to the circRNA-based score, the patients are divided into high-risk and low-risk groups. The high-risk group was determined by the prevalence of circRNAs that are playing a role in cancer progression. The roles of individual circRNAs were supported by loss-of-function assays. The addition of circRNAs as new molecular targets deepens our understanding of cancer origin and patient response to anti-cancer therapies.
Scientists try to harvest the unique qualities of circRNA such as sponging and stability to devise new treatment options (artificial circRNA-like molecules). This approach proved itself effective by silencing several oncogenes (KLF17, CDH1, LASS2) and inhibited tumor migration, invasion, proliferation, and colony formation. It was possible to silence all three genes simultaneously and achieve impressive results just by harnessing circRNA properties for novel drug development.
Case 4: circRNA is important for development and physiology
Circular RNAs are believed to be essential to biological functions and development processes. To research that, scientists utilize CRISPR/Cas9 technology for generating loss-of-function alleles. In the study by Piwecka et al., the knockout of circRNA Cdr1 in the mouse genome resulted in neuronal malfunction. The synaptic transmission was interrupted and, interestingly, the nervous system, the brain, in particular, has abundant expression of circRNAs, which suggests their potential functionality. The proposed mechanism for this phenomenon is the interaction of Cdr1 with miRNAs (miR7, miR-671) in the wild type. When Cdr1 is lost, the miRNAs are deregulated. This finding suggests that circRNAs may play a critical role in neural development.
Another suggested role in maintaining fertility. The circRNAs are abundant and variable in human and animal testes. The research by Gao et al. focused on circRNAs from BOULE – a highly conserved gene with a known role in spermatogenesis. The study showed that circRNAs interact with heat shock proteins (HSPs) (most likely facilitating their ubiquitination) and this interaction is conserved. Lack of circBoule RNAs causes high HSP2 levels and results in lower fertility. Therefore, circular RNAs may become a focus for further studies on maintaining fertility.
Wang S, Zhang Y, Cai Q, et al.Circular RNA FOXP1 promotes tumor progression and Warburg effect in gallbladder cancer by regulating PKLR expres- sion[J].Molecular Cancer, 2019,18(1):145.
Mahmoudi E, Kiltschewskij D, Fitzsimmons C, et al. Depolarization-Associated CircRNA Regulate Neural Gene Expression and in Some Cases May Function as Templates for Translation[J]. Cell, 2020, 9(1), 25.
Ju HQ , Zhao Q , Wang F, et al. A circRNA signature predicts postoperative recurrence in stage II/III colon cancer[J]. EMBO Mol Med, 2019, 11: e10168.
Memczak, S., Jens, M., Elefsinioti, A. et al. Circular RNAs are a large class of animal RNAs with regulatory potency. Nature 495, 333–338 (2013).
Shi, L., Liu, B., Shen, D. et al. A tumor-suppressive circular RNA mediates uncanonical integrin degradation by the proteasome in liver cancer. Science Advances 7(13).