CircFOXO3 mediates hypoxia-induced autophagy of endometrial stromal cells in endometriosis.

Endometriosis is a benign gynecological disease that shares some common features of malignancy. Autophagy plays vital roles in endometriosis and influences endometrial cell metastasis, and hypoxia was identified as the initiator of this pathological process through hypoxia inducible factor 1 alpha (HIF-1α). A newly discovered circular RNA FOXO3 (circFOXO3) is critical in cell autophagy, migration, and invasion of various diseases and is reported to be related to hypoxia, although its role in endometriosis remains to be elucidated up to now. In this study, a lower circFOXO3 expression in ectopic endometrium was investigated. Furthermore, we verified that circFOXO3 could regulate autophagy by downregulating the level of p53 protein to mediate the migration and invasion of human endometrial stromal cells (T HESCs). Additionally, the effects of HIF-1α on circFOXO3 and autophagy were examined in T HESCs. Notably, overexpression of HIF-1α could induce autophagy and inhibit circFOXO3 expression, whereas overexpressing of circFOXO3 under hypoxia significantly inhibited hypoxia-induced autophagy. Mechanistically, the direct combination between HIF-1α and HIF-1α-binding site on adenosine deaminase 1 acting on RNA (ADAR1) promoter increased the level of ADAR1 protein, which bind directly with circFOXO3 pre-mRNA to block the cyclization of circFOXO3. All these results support that hypoxia-mediated ADAR1 elevation inhibited the expression of circFOXO3, and then autophagy was induced upon loss of circFOXO3 via inhibition of p53 degradation, participating in the development of endometriosis.

Circular RNA circFoxo3 Promotes Granulosa Cell Apoptosis Under Oxidative Stress Through Regulation of FOXO3 Protein.

Oxidative stress leads to ovarian functional decline by inducing granulosa cell (GC) apoptosis. Circular RNA circFoxo3 acts as a critical factor in regulating cell cycle and apoptosis, and cellular senescence in tumor cells. However, function of circFoxo3 is little understood in oxidative stress-induced injury of follicular GCs. In this study, we aimed to illustrate the regulation pattern of circFoxo3 in GCs under oxidative stress. CircFoxo3 was confirmed to be expressed in both human and mouse GCs by amplification with divergent primers and sequencing. In vitro and in vivo ovarian oxidative stress model, the expression of circFoxo3, FOXO3 protein, and its downstream targets were examined by quantitative real-time PCR and Western blotting, respectively. Knockdown of circFoxo3 was performed to evaluate the effects of circFoxo3-mediated GC apoptosis in vitro. RNA pull-down was used to discover the protein that interacted with circFoxo3 so as to illustrate the mechanism of circFoxo3 in GCs. Our results demonstrated that circFoxo3 was significantly upregulated in hydrogen peroxide (H2O2)-treated GCs and a 3-nitropropionic acid (3-NP)-induced mouse model of ovarian oxidative stress. Protein level of transcriptional factor FOXO3 was also remarkably increased in both in vitro and in vivo oxidative stress model, but FOXO3 mRNA expression revealed no significant difference. Knockdown of endogenous circFoxo3 downregulated FOXO3 protein level and blocked H2O2-induced cell apoptosis. CircFoxo3 could pull down high levels of MDM2 protein that induced FOXO3 ubiquitination and degradation. Furthermore, knockdown of MDM2 and circFoxo3 showed remarkably higher level of apoptosis when compared with the knockdown of circFoxo3 alone. Our study suggested that circFoxo3 regulated FOXO3 protein level in GCs by reducing interactions between FOXO3 and MDM2. In conclusion, circFoxo3 was positively associated with FOXO3 protein and jointly played crucial roles in mediating GC apoptosis induced by oxidative stress.

Circular RNA circFOXO3 facilitate non-small cell lung cancer progression through upregulating HMGB3 via sponging miR-545-3p/miR-506-3p.

PURPOSE: Circular RNAs (circRNAs) have emerged as a pivotal regulatory element in the progression of human cancers. Being an important member of circRNAs, circFOXO3 has been implicated in tumor invasion or metastasis of non-small cell lung cancer (NSCLC); however, the molecular mechanism underlying this promoting effect remains an enigma. The present study aims to study the function of circFOXO3 and dissect the relevant intracellular network in the progression and metastasis of NSCLC. METHODS: Quantitative real time PCR (RT-qPCR) assay and Western blotting were used to quantify the levels of RNAs and proteins respectively. starBase v2.0 and luciferase assay were used to validate the target of circRNAs or miRNAs. Cell Counting Kit-8 (CCK-8) assay was adopted to examine cell viability. Transwell was used to determine cell invasion and migration. Xenograft model was established to detect tumor growth. RESULTS: RT-qPCR showed that circFOXO3 was overexpressed in NSCLC cells and tissues. Knockdown of circFOXO3 not only inhibited NSCLC cell proliferation, migration and invasion in vitro but also suppressed tumor growth in vivo. starBase v2.0 and luciferase assay results collectively suggested that circFOXO3 sponged miR-545-3p and miR-506-3p. Dual-inhibition of circFOXO3 and its target miRNAs suppressed the reduction of cell proliferation, migration and invasion induced by siRNA of circFOXO3 (si-circFOXO3), demonstrating that the effect of circFOXO3 on NSCLC was dependent on sponging miR-545-3p and miR-506-3p. Further bioinformatic analysis and biochemistry experiments revealed that miR-545-3p and miR-506-3p regulated the expression of a family member of high-mobility group box, HMGB3. CONCLUSION: Here, we show thatcircFOXO3 in NSCLC promotes the proliferation, migration and invasion of NSCLC cells, thereby promoting tumor growth. We further find that circFOXO3 sponges miR-545-3p/miR-506-3p that bind to 3'-UTR of HMGB3 mRNA, which constitutes the major network fulfilling the circFOXO3's promoting effect. Therefore, we proposed that circFOXO3 could be a potential therapeutic target for NSCLC.

Circular RNA circFOXO3 regulates KDM2A by targeting miR-214 to promote tumor growth and metastasis in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a pathological type of oral cancer, which accounts for over 90% of oral cancers. It has been widely shown that circRNA is involved in the regulation of multiple malignant oral diseases including OSCC. However, the mechanism underlying how circRNA regulates OSCC is still not clearly elucidated. In this article, we report circFOXO3 promotes tumor growth and invasion of OSCC by targeting miR-214 which specifically degrades the lysine demethylase 2A (KDM2A). CircRNA sequencing was conducted in OSCC tumor and tumor-side tissues, and the expression of circFOXO3 is found to be markedly increased in tumor tissues. CircFOXO3 is also highly expressed in several OSCC cell lines compared with human oral keratinocytes. Transwell assay and colony formation showed that knockdown of circFOXO3 prevents the invasion and proliferation of oral cancer cells. Via bioinformatic research, miR-214 was found to be the target of circFOXO3 and correlate well with circFOXO3 both in vitro and in vivo. KDM2A was then validated by database analysis and luciferase assay to be the direct target of miR-214. KDM2A helps to promote tumor invasiveness and proliferation of OSCC. Collectively, our results proved that circFOXO3 sponges miR-214 to up-regulate the expression of KDM2A, thus promotes tumor progression in OSCC.

Circular RNA Foxo3 Relieves Myocardial Ischemia/Reperfusion Injury by Suppressing Autophagy via Inhibiting HMGB1 by Repressing KAT7 in Myocardial Infarction.

INTRODUCTION: Myocardial infarction is coronary artery-related heart disease, and the leading cause of mortality globally. Circular RNAs (circRNAs) are a new type of regulatory RNAs and participate in multiple pathological cardiac progression. METHODS: However, the function of circFoxo3 in MI-induced myocardial injury remains obscure. RESULTS: Significantly, we identified that circFoxo3 was downregulated in the MI rat model and the overexpression of circFoxo3 ameliorated MI-induced cardiac dysfunction and attenuated MI-induced autophagy in rat model. Meanwhile, the overexpression of circFoxo3 repressed oxygen-glucose deprivation (OGD)-induced autophagy, apoptosis, inflammation, and injury of cardiomyocyte in vitro. Mechanically, we identified that the expression of KAT7 was reduced by circFoxo3 overexpression in cardiomyocytes. Meanwhile, the expression of HMGB1 was repressed by the depletion of KAT7 in cardiomyocytes. The enrichment of histone H3 lysine 14 acetylation (H3K14ac) and RNA polymerase II (RNA pol II) on HMGB1 promoter was inhibited by the knockdown of KAT7. Moreover, the overexpression of circFoxo3 suppressed HMGB1 expression and KAT7 overexpression rescued the expression of HMGB1 in cardiomyocytes. The enrichment of KAT7, H3K14ac, and RNA poly II on HMGB1 promoter was decreased by circFoxo3 overexpression, while the overexpression of KAT7 could reverse the effect. The overexpression of KAT7 or HMGB1 could reverse circFoxo3-attenuated cardiomyocyte injury and autophagy in vitro. Thus, we conclude that circular RNA circFoxo3 relieved myocardial ischemia/reperfusion injury by suppressing autophagy via inhibiting HMGB1 by repressing KAT7 in MI. DISCUSSION: Our finding provides new insight into the mechanism by which circFoxo3 regulates MI-related cardiac dysfunction by targeting KAT7/HMGB1 axis.

The Emerging Roles of circFOXO3 in Cancer.

Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs which are mainly formed by reverse splicing of precursor mRNAs. They are relatively stable and resistant to RNase R because of their covalently closed structure without 5' caps or 3' poly-adenylated tails. CircRNAs are widely expressed in eukaryotic cells and show tissue, timing, and disease specificity. Recent studies have found that circRNAs play an important role in many diseases. In particular, they affect the proliferation, invasion and prognosis of cancer by regulating gene expression. CircRNA Forkhead box O3 (circFOXO3) is a circRNA confirmed to be abnormally expressed in a variety of cancers, including prostate cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, and breast cancer, etc. At present, the feature of circFOXO3 as a molecular sponge is widely studied to promote or inhibit the development of cancers. However, the diverse functions of circFOXO3 have not been fully understood. Hence, it is important to review the roles of circFOXO3 in cancers. This review has summarized and discussed the roles and molecular mechanism of circFOXO3 and its target genes in these cancers, which can help to enrich our understanding to the functions of circRNAs and carry out subsequent researches on circFOXO3.

Pathogenic mechanisms and the potential clinical value of circFoxo3 in cancers.

Circular RNAs (circRNAs) are covalently closed circular structures that can function in various physiological and pathological processes by acting as microRNA (miRNA) sponges, RNA-binding protein (RBP) sponges, mRNA transcriptional regulators, and protein translational templates. circFoxo3 is one of the most studied circRNAs and is generated from the tumor suppressor gene Foxo3. Increasing studies have demonstrated the multiple functions of circFoxo3 in the pathogenesis of different cancer types. circFoxo3 plays important roles in cancer development mainly by binding to various miRNAs. The diagnostic potential of circFoxo3 has been revealed in several cancers. Some research results have been found to contradict the results of other studies, and this may be due to insufficient sample sizes and inconsistencies in the experimental and nomenclature methods. In this review, we systematically summarize current knowledge about the biogenesis and functions of circRNAs, elucidate the roles of circFoxo3 in different cancers, and explore the clinical applications of circFoxo3.

circFOXO3: Going around the mechanistic networks in cancer by interfering with miRNAs regulatory networks.

Circular RNAs (circRNA) have gained recent interest due to their functional versatility due to their interactions with other RNA species and proteins, all of which underline complex regulatory networks involved in pathogenic mechanisms. As a result, recent insights in circRNA biology are investigating their biomarker and therapeutic potential. One such circRNA is CircFOXO3, which consists of the circularized second exon of the FOXO3 mRNA, a member of the forkhead box transcription factor family involved in the regulation of developmental programs. Recent research focused on the role of circFOXO3 in the context of cancer has highlighted several implications in key tumorigenesis mechanisms, thus consolidating its relevance among other identified circRNAs. In this paper, we will focus on the currently identified case-specific implications of circFOXO3 in cancer, with a focus on the circFOXO3-miRNA-mRNA regulatory networks, its interactions with different proteins, and their cumulated biological effects upon tumor development. Therefore, we aim to provide an integrated perspective of the mechanistic implications of circFOXO3 in different cancers while also highlighting its biomarker or therapeutic potential based on the current evidence.

circFOXO3 protects cardiomyocytes against radiation­induced cardiotoxicity.

Radiation therapy, one of the major treatment options for cancer, can cause delayed heart damage. The circular RNA (circRNA) circFOXO3 (hsa_circ_0006404) is associated with cancer progression. However, the functions of circFOXO3 in radiation­induced cardiotoxicity remains unknown. The present study aimed to identify the functions of cirFOXO3 in radiation­induced cardiotoxicity. The present study established circFOXO3­knockdown (KD) or ­overexpressing (OE) cardiomyocytes. Functional assay results showed that KD of circFOXO3 in cardiomyocytes significantly increased DNA damage and apoptosis after radiation. By contrast, OE of circFOXO3 reduced DNA damage and apoptosis rates in response to radiation. Mechanistically, KD of circFOXO3 elevated the levels of Bax, caspase 3 and caspase 7, and decreased Bcl­2 expression, whereas OE of circFOXO3 decreased Bax, caspase 3 and caspase 7 expression, and increased Bcl­2 expression. Thus, the present study indicated that circFOXO3 protected cardiomyocytes from radiation­induced cardiotoxicity by reducing DNA damage and apoptosis. circFOXO3 may be a potential therapeutic target against radiation­induced cardiotoxicity.

CircFOXO3 rs12196996, a polymorphism at the gene flanking intron, is associated with circFOXO3 levels and the risk of coronary artery disease.

CircFOXO3 plays an important role in the pathogenesis of coronary artery disease (CAD). Single nucleotide polymorphisms (SNPs) at circRNA flanking introns may change its back-splicing and influence circRNA formation. Here, we aimed to investigate the influence of the polymorphisms at the circFOXO3 flanking introns on individual susceptibility to CAD. A total of 1185 individuals were included in the case-control study. In a multivariate logistic regression analysis, we determined that the rs12196996 G variant was significantly associated with increased CAD risk (OR = 1.36, P = 0.014). A similar trend of the association was observed in the recessive model (OR = 2.57, P = 0.003). Stratified analysis revealed a more significant association with CAD risk among younger subjects and non-smokers. Consistent with these results, the haplotype rs12196996G-rs9398171C containing rs12196996G allele was also associated with increased CAD risk (OR = 1.31, P = 0.013). Further investigation revealed that the rs12196996 GG genotype was associated with decreased circFOXO3 expression, but not linear FOXO3 levels. Taken together, our data provide the first evidence that the rs12196996 polymorphism at the circFOXO3 gene flanking intron is associated with CAD risk in the Chinese Han population, which is probably due to influence circFOXO3 levels.

CircFoxo3 Promotes Adriamycin Resistance Through Regulation of miR-199a-5p/ATP Binding Cassette Subfamily C Member 1 Axis in Hepatocellular Carcinoma.

INTRODUCTION: Chemotherapy resistance is the main cause of poor prognosis in patients with hepatocellular carcinoma (HCC). Therefore, it is important to understand the molecular mechanism of adriamycin (ADM) resistance in HCC. Increasing evidence indicates that circular RNAs (circRNAs) play a crucial regulatory role in different pathological processes. In the current study, we aimed to investigate the roles and the underlying molecular mechanism of circFoxo3 in ADM-resistant HCC. MATERIALS AND METHODS: Twenty-five pairs of clinical tumors samples and matched normal tissues were collected from patients with HCC. Gain- and loss-function experiments were performed to investigate the role of circFoxo3 in ADM-resistant cells. RESULTS: CircFoxo3 expression was increased in ADM-resistant HCC tissues and HCC cell lines and in metastatic tissues compared with non-metastatic tissues. CircFoxo3 knockdown reduces and circFoxo3 overexpression enhances HCC cell invasion and tumor growth. In addition, circFoxo3 interacted with miR-199a-5p and regulated miR-199a-5p expression. Furthermore, ATP Binding Cassette Subfamily C Member 1 (ABCC1) was identified as a new target of miR-199a-5p. CircFoxo3 interacted with miR-199a-5p to positively regulate ABCC1 expression, contributing to epithelial-mesenchymal transition progression. CONCLUSION: CircFoxo3 knockdown reduces and circFoxo3 overexpression enhances HCC cell invasion and tumor growth through regulation of miR-199a-5p/ABCC1 axis. Our findings reveal that circFoxo3 may be novel biomarkers and therapeutic target for HCC treatment.

CircFOXO3 functions as a molecular sponge for miR-143-3p to promote the progression of gastric carcinoma via upregulating USP44.

Gastric carcinoma (GC) ranks fifth in terms of cancer morbidity and third in cancer-related death worldwide and imposes enormous health and economic burdens. The molecular mechanisms underlying GC formation and progression remain unclear. Our aim was to identify the involvement of circular RNA circFOXO3 in GC, and to determine the underlying mechanisms. In this study, we revealed a stimulatory role of circular RNA circFOXO3 in tumor growth in vivo. CircFOXO3 enhanced GC cell proliferation and migration in vitro and promoted tumor growth of GC cells in vivo. Bioinformatic analysis revealed that circFOXO3 might regulate USP44 expression by specifically binding to microRNA (miR)-143-3p. Existence of circFOXO3-miR-143-3p-USP44 axis in GC cells was confirmed by RNA-binding protein immunoprecipitation, luciferase reporter assay, and an RNA pull-down experiments. All the data indicate that circFOXO3 promotes GC cell proliferation and migration by upregulating USP44 expression via targeting of miR-143-3p.

Circular RNA circFOXO3 promotes prostate cancer progression through sponging miR-29a-3p.

Circular RNA FOXO3 (CircFOXO3, also termed as Hsa_circ_0006404) is derived from exon 2 of forkhead box O3 (FOXO3) gene, and abnormal expression is shown in different diseases. However, whether circFOXO3 plays important roles in tumorigenesis and progression of prostate cancer (PCa) remains unclear. In this study, we found that circFOXO3 was up-regulated in both PCa tissues and serum samples. Moreover, circFOXO3 was positively correlated with the Gleason score in PCa samples. CircFOXO3 was observed to be up-regulated in Gleason score > 6 PCa samples compared with Gleason score = 6 PCa samples. Knock-down circFOXO3 could remarkably inhibit PCa cell cycle, proliferation and promote cell apoptosis in vitro. Furthermore, we demonstrated circFOXO3 could act as miR-29a-3p sponge to up-regulate SLC25A15 expression by bioinformatics analysis, dual-luciferase reporter assays and biotinylated RNA pull-down assays. SLC25A15 could reverse the tumour suppressing roles of knock-down circFOXO3 in PCa. Of note, we found that miR-29a-3p was down-regulated; however, SLC25A15 was overexpressed in PCa samples compared with normal tissues. In conclusion, circFOXO3 acts as a miR-29a-3p sponge to exhibit oncogenic activity that affects the cell cycle and cell apoptosis in PCa through transcriptional up-regulation of SLC25A15. Our analysis suggests circFOXO3 could act as promising prostate cancer biomarkers.

Reduction of circular RNA Foxo3 promotes prostate cancer progression and chemoresistance to docetaxel.

Dysregulation of circular RNA Foxo3 (circFoxo3) has been reported to be involved in breast cancer and non-small lung cancer progression. However, little is known about the role of circFoxo3 in prostate cancer, which the present study seeks to investigate. CircFoxo3 expression was analyzed in 22 low-grade prostate cancer samples, 24 high-graded prostate cancer samples, and 18 normal prostate tissues, finding that its quantity was significantly decreased in high-graded compared to low-grade prostate cancer and normal prostate tissues. CircFoxo3 inhibited prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, which was related to circFoxo3's repression of Foxo3 and EMT. Silencing circFoxo3 expression promoted prostate cancer cell survival, migration, invasion and chemoresistance to docetaxel, as well as the positive effects of androgen on prostate cancer viability. Delivery of circfoxo3 enhanced chemosensitivity to docetaxel of prostate tumor-bearing mice and prolonged the life span of mice, while reduction with siRNAs promoted chemoresistance to docetaxel and shorted the life span of the tumor-bearing mice. Targeting circFoxo3/Foxo3/EMT may provide an applicable strategy for exploring potential prognostic and therapeutic approaches for prostate cancer.

CircFOXO3 promotes glioblastoma progression by acting as a competing endogenous RNA for NFAT5.

BACKGROUND: Circular RNAs (circRNAs), a newly discovered type of endogenous noncoding RNA, have been proposed to mediate the progression of diverse types of tumors. Systematic studies of circRNAs have just begun, and the physiological roles of circRNAs remain largely unknown. Here, we focused on elucidating the potential role and molecular mechanism of circular forkhead box O3 (circFOXO3) in glioblastoma (GBM) progression. METHODS: First, we analyzed circFOXO3 alterations in GBM and noncancerous tissues through real-time quantitative reverse transcription PCR (qRT-PCR). Next, we used loss- and gain-of-function approaches to evaluate the effect of circFOXO3 on GBM cell proliferation and invasion. Mechanistically, fluorescent in situ hybridization, RNA pull-down, dual luciferase reporter, and RNA immunoprecipitation assays were performed to confirm the interaction between circFOXO3 and miR-138-5p/miR-432-5p in GBM. An animal model was used to verify the in vitro experimental findings. RESULTS: CircFOXO3 expression was significantly higher in GBM tissues than in noncancerous tissues. GBM cell proliferation and invasion were reduced by circFOXO3 knockdown and enhanced by circFOXO3 overexpression. Further biochemical analysis showed that circFOXO3 exerted its pro-tumorigenic activity by acting as a competing endogenous RNA (ceRNA) to increase expression of nuclear factor of activated T cells 5 (NFAT5) via sponging both miR-138-5p and miR-432-5p. Notably, tumor inhibition by circFOXO3 downregulation could be reversed by miR-138-5p/miR-432-5p inhibitors in GBM cells. Moreover, GBM cells with lower circFOXO3 expression developed less aggressive tumors in vivo. CONCLUSIONS: Our data demonstrate that circFOXO3 can exert regulatory functions in GBM and that ceRNA-mediated microRNA sequestration might be a potential strategy for GBM therapy.