Significant progress has been made in circular RNA (circRNA) research in recent years. Increasing evidence suggests that circRNAs play important roles in many cellular processes, and their dysregulation is implicated in the pathogenesis of various diseases. CircRNAs are highly stable and usually expressed in a tissue- or cell type-specific manner. Therefore, they are currently being explored as potential therapeutic targets. Gain-of-function and loss-of-function approaches are typically performed using circRNA expression plasmids and RNA interference-based strategies, respectively. These strategies have limitations that can be mitigated using nanoparticle and exosome delivery systems. Furthermore, recent developments show that the cre-lox system can be used to knockdown circRNAs in a cell-specific manner. While still in the early stages of development, the CRISPR/Cas13 system has shown promise in knocking down circRNAs with high specificity and efficiency. In this review, we describe circRNA properties and functions and highlight their significance in disease. We summarize strategies that can be used to overexpress or knockdown circRNAs as a therapeutic approach. Lastly, we discuss major challenges and propose future directions for the development of circRNA-based therapeutics.

Yap is the key component of Hippo pathway which plays crucial roles in tumorigenesis. Inhibition of Yap activity could promote apoptosis, suppress proliferation, and restrain metastasis of cancer cells. However, how Yap is regulated is not fully understood. Here, we reported Yap being negatively regulated by its circular RNA (circYap) through the suppression of the assembly of Yap translation initiation machinery. Overexpression of circYap in cancer cells significantly decreased Yap protein but did not affect its mRNA levels. As a consequence, it remarkably suppressed proliferation, migration and colony formation of the cells. We found that circYap could bind with Yap mRNA and the translation initiation associated proteins, eIF4G and PABP. The complex containing overexpressed circYap abolished the interaction of PABP on the poly(A) tail with eIF4G on the 5′-cap of the Yap mRNA, which functionally led to the suppression of Yap translation initiation. Individually blocking the binding sites of circYap on Yap mRNA or respectively mutating the binding sites for PABP and eIF4G derepressed Yap translation. Significantly, breast cancer tissue from patients in the study manifested dysregulation of circYap expression. Collectively, our study uncovered a novel molecular mechanism in the regulation of Yap and implicated a new function of circular RNA, supporting the pursuit of circYap as a potential tool for future cancer intervention.

TP53 mutations occur in many different types of cancers that produce mutant p53 proteins. The mutant p53 proteins have lost wild-type p53 activity and gained new functions that contribute to malignant tumor progression. Different p53 mutations create distinct profiles in loss of wild-type p53 activity and gain of functions. Targeting the consequences generated by the great number of p53 mutations would be extremely complex. Therefore, in this study we used a workaround and took advantage of the fact that mutant p53 cannot bind H2AX. Using this, we developed a new approach to repress the acquisition of mutant p53 functions. We show here that the delivery of a circular RNA circ-Ccnb1 inhibited the function of three p53 mutations. By microarray analysis and real-time PCR, we detected decreased circ-Ccnb1 expression levels in patients bearing breast carcinoma. Ectopic delivery of circ-Ccnb1 inhibited tumor growth and extended mouse viability. Using proteomics, we found that circ-Ccnb1 precipitated p53 in p53 wild-type cells, but instead precipitated Bclaf1 in p53 mutant cells. Further experiments showed that H2AX serves as a bridge, linking the interaction of circ-Ccnb1 and wild-type p53, thus allowing Bclaf1 to bind Bcl2 resulting in cell survival. In the p53 mutant cells, circ-Ccnb1 formed a complex with H2AX and Bclaf1, resulting in the induction of cell death. We found that this occurred in three p53 mutations. These results shed light on the possible development of new approaches to inhibit the malignancy of p53 mutations.