Circular RNAs are a class of non-coding RNAs that are receiving extensive attention. Despite reports showing circular RNAs acting as microRNA sponges, the biological functions of circular RNAs remain largely unknown. We show that in patient tumor samples and in a panel of cancer cells, circ-Foxo3 was minimally expressed. Interestingly, during cancer cell apoptosis, the expression of circ-Foxo3 was found to be significantly increased. We found that silencing endogenous circ-Foxo3 enhanced cell viability, whereas ectopic expression of circ-Foxo3 triggered stress-induced apoptosis and inhibited the growth of tumor xenografts. Also, expression of circ-Foxo3 increased Foxo3 protein levels but repressed p53 levels. By binding to both, circ-Foxo3 promoted MDM2-induced p53 ubiquitination and subsequent degradation, resulting in an overall decrease of p53. With low binding affinity to Foxo3 protein, circ-Foxo3 prevented MDM2 from inducing Foxo3 ubiquitination and degradation, resulting in increased levels of Circular RNAs are a large class of non-coding RNAs that are circularized by joining free 3'-to 5'-ends, forming a circular structure. 1-4Although circular RNAs were initially characterized over 30 years ago, their functions in mammalian cells are still largely unknown. Most circular RNAs are predominantly found in the cytoplasm and contain exons, known as circRNAs.5 A relatively smaller group of circular RNAs that contain both exons and introns are known as EIciRNAs, and are predominantly found in the nucleus. 6 Recent studies have indicated that some circular RNAs contain miRNA binding sites and may function as sponges to arrest miRNA functions. 7,8 It has further been reported that EIciRNAs increase the transcription of their parental genes.9 Recently, we showed that the circular RNA circ-Foxo3 could function by binding to proteins in related signal pathways. 10,11 In the present study, we used computational approach to elucidate the interaction of circ-Foxo3 with MDM2 and p53. The RING-finger domain in the carboxyl terminal of the MDM2 is known to bind RNA specifically in a sequence-specific manner, 12 whereas p53 interacts with RNA via its C-terminal regulatory domain. 13 Our study comprised of computer-aided RNA structure modeling of circ-Foxo3 employing minimum free energy algorithm and machine translation system followed by its molecular interaction with MDM2 (RING-finger domain) and p53 (C-terminal regulatory domain) that includes docking, scoring, clustering, and refinement of the most promising models. The interaction was further confirmed by an approach of molecular experiments to explicate the biological functions of circ-Foxo3. ResultsDecreased expression of circ-Foxo3 in tumors and cancer cells. Downregulation of Foxo3 is often observed in cancer development.14,15 Both circ-Foxo3 and Foxo3 mRNA are encoded by the FOXO3 gene. 16 We found that the levels of circ-Foxo3 in tumor specimen were significantly lower than in the adjacent benign tissue (Figure 1a). We examined circ-Foxo3 expression and detected s...

Circular RNAs (circRNAs) are a subclass of noncoding RNAs widely expressed in mammalian cells. We report here the tumorigenic capacity of a circRNA derived from angiomotin-like1 (circ-Amotl1). Circ-Amotl1 is highly expressed in patient tumor samples and cancer cell lines. Single-cell inoculations using circ-Amotl1-transfected tumor cells showed a 30-fold increase in proliferative capacity relative to control. Agarose colony-formation assays similarly revealed a 142-fold increase. Tumor-take rate in nude mouse xenografts using 6-day (219 cells) and 3-day (9 cells) colonies were 100%, suggesting tumor-forming potential of every cell. Subcutaneous single-cell injections led to the formation of palpable tumors in 41% of mice, with tumor sizes >1 cm in 1 month. We further found that this potent tumorigenicity was triggered through interactions between circ-Amotl1 and c-myc. A putative binding site was identified in silico and tested experimentally. Ectopic expression of circ-Amotl1 increased retention of nuclear c-myc, appearing to promote c-myc stability and upregulate c-myc targets. Expression of circ-Amotl1 also increased the affinity of c-myc binding to a number of promoters. Our study therefore reveals a novel function of circRNAs in tumorigenesis, and this subclass of noncoding RNAs may represent a potential target in cancer therapy.