Most RNAs generated by the human genome have no protein-coding ability and are termed non-coding RNAs. Among these include circular RNAs, which include exonic circular RNAs (circRNA), mainly found in the cytoplasm, and intronic RNAs (ciRNA), predominantly detected in the nucleus. The biological functions of circular RNAs remain largely unknown, although ciRNAs have been reported to promote gene transcription, while circRNAs may function as microRNA sponges. We demonstrate that the circular RNA circ-Foxo3 was highly expressed in non-cancer cells and were associated with cell cycle progression. Silencing endogenous circ-Foxo3 promoted cell proliferation. Ectopic expression of circ-Foxo3 repressed cell cycle progression by binding to the cell cycle proteins cyclin-dependent kinase 2 (also known as cell division protein kinase 2 or CDK2) and cyclin-dependent kinase inhibitor 1 (or p21), resulting in the formation of a ternary complex. Normally, CDK2 interacts with cyclin A and cyclin E to facilitate cell cycle entry, while p21works to inhibit these interactions and arrest cell cycle progression. The formation of this circ-Foxo3-p21-CDK2 ternary complex arrested the function of CDK2 and blocked cell cycle progression.

We conclude that ID-1, E2F1, FAK, and HIF1α interact with circ-Foxo3 and are retained in the cytoplasm and could no longer exert their anti-senescent and anti-stress roles, resulting in increased cellular senescence.

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 have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, circRNA functions remain largely unknown. CircRNAs can function as miRNA sponges, thereby reducing their ability to target mRNAs. We hypothesize that circRNAs may bind, store, sort, and sequester proteins to particular subcellular locations, and act as dynamic scaffolding molecules that modulate protein-protein interactions. Here, we review the biological implication and function of circRNA-protein interaction, and reveal a dynamic model of the interaction in various tissues, development stages and physiological conditions. Improved techniques to identify and characterize the dynamic RNA-protein interactions may elucidate the molecular mechanisms associated with the expression and functional diversity of circRNAs.

As central nodes in cardiomyocyte signaling, nuclear AKT appears to play a cardio-protective role in cardiovascular disease. Here we describe a circular RNA, circ-Amotl1 that is highly expressed in neonatal human cardiac tissue, and potentiates AKT-enhanced cardiomyocyte survival. We hypothesize that circ-Amotl1 binds to PDK1 and AKT1, leading to AKT1 phosphorylation and nuclear translocation. In primary cardiomyocytes, epithelial cells, and endothelial cells, we found that forced circ-Amotl1 expression increased the nuclear fraction of pAKT. We further detected increased nuclear pAKT in circ-Amotl1-treated hearts. In vivo, circ-Amotl1 expression was also found to be protective against Doxorubicin (Dox)-induced cardiomyopathy. Putative PDK1- and AKT1-binding sites were then identified in silico. Blocking oligonucleotides could reverse the effects of exogenous circ-Amotl1. We conclude that circ-Amotl1 physically binds to both PDK1 and AKT1, facilitating the cardio-protective nuclear translocation of pAKT.

It has been reported that the miR-106bB25 cluster, a paralog of the miR-17B92 cluster, possesses oncogenic activities. However, the precise role of each microRNA (miRNA) in the miR-106bB25 cluster is not yet known. In this study, we examined the function of miR-93, one of the microRNAs within the miR-106bB25 cluster, in angiogenesis and tumor formation. We found that miR-93 enhanced cell survival, promoted sphere formation and augmented tumor growth. Most strikingly, when miR-93-overexpressing U87 cells were co-cultured with endothelial cells, they supported endothelial cell spreading, growth, migration and tube formation. In vivo studies revealed that miR-93-expressing cells induced blood vessel formation, allowing blood vessels to extend to tumor tissues in high densities. Angiogenesis promoted by miR-93 in return facilitated cell survival, resulting in enhanced tumor growth. We further showed that integrinb8 is a target of miR-93. Higher levels of integrin-b8 are associated with cell death in tumor mass and in human glioblastoma. Silencing of integrin-b8 expression using small interfering RNA promoted cell proliferation, whereas ectopic expression of integrin-b8 decreased cell growth. These findings showed that miR-93 promotes tumor growth and angiogenesis by suppressing, at least in part, integrin-b8 expression. Our results suggest that inhibition of miR-93 function may be a feasible approach to suppress angiogenesis and tumor growth.

Clear cell renal cell carcinoma (ccRCC) is histologically defined by its lipid and glycogen-rich cytoplasmic deposits. Alterations in the VHL tumor suppressor stabilizing the hypoxia-inducible factors (HIFs) are the most prevalent molecular features of clear cell tumors. The significance of lipid deposition remains undefined. We describe the mechanism of lipid deposition in ccRCC by identifying the rate-limiting component of mitochondrial fatty acid transport, carnitine palmitoyltransferase 1A (CPT1A), as a direct HIF target gene. CPT1A is repressed by HIF1 and HIF2, reducing fatty acid transport into the mitochondria, and forcing fatty acids to lipid droplets for storage. Droplet formation occurs independent of lipid source, but only when CPT1A is repressed. Functionally, repression of CPT1A is critical for tumor formation, as elevated CPT1A expression limits tumor growth. In human tumors, CPT1A expression and activity are decreased versus normal kidney; and poor patient outcome associates with lower expression of CPT1A in tumors in TCGA. Together, our studies identify HIF control of fatty acid metabolism as essential for ccRCC tumorigenesis.

It has recently been shown that the upregulation of a pseudogene specific to a protein-coding gene could function as a sponge to bind multiple potential targeting microRNAs (miRNAs), resulting in increased gene expression. Similarly, it was recently demonstrated that circular RNAs can function as sponges for miRNAs, and could upregulate expression of mRNAs containing an identical sequence. Furthermore, some mRNAs are now known to not only translate protein, but also function to sponge miRNA binding, facilitating gene expression. Collectively, these appear to be effective mechanisms to ensure gene expression and protein activity. Here we show that expression of a member of the forkhead family of transcription factors, Foxo3, is regulated by the Foxo3 pseudogene (Foxo3P), and Foxo3 circular RNA, both of which bind to eight miRNAs. We found that the ectopic expression of the Foxo3P, Foxo3 circular RNA and Foxo3 mRNA could all suppress tumor growth and cancer cell proliferation and survival. Our results showed that at least three mechanisms are used to ensure protein translation of Foxo3, which reflects an essential role of Foxo3 and its corresponding non-coding RNAs.