miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner.

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

CircRNA CDR1as affects functional repair after spinal cord injury and regulates fibrosis through the SMAD pathway.

Spinal cord injury (SCI) is a complex problem in modern medicine. Fibroblast activation and fibroscarring after SCI impede nerve recovery. Non-coding RNA plays an important role in the progression of many diseases, but the study of its role in the progression of spinal fibrosis is still emerging. Here, we investigated the function of circular RNAs, specifically antisense to the cerebellar degeneration-related protein 1 (CDR1as), in spinal fibrosis and characterized its molecular mechanism and pathophysiology. The presence of CDR1as in the spinal cord was verified by sequencing and RNA expression assays. The effects of inhibition of CDR1as on scar formation, inflammation and nerve regeneration after spinal cord injury were investigated in vivo and in vitro. Further, gene expression of miR-7a-5p and protein expression of transforming Growth Factor Beta Receptor II (TGF-ßR2) were measured to evaluate their predicted interactions with CDR1as. The regulatory effects and activation pathways were subsequently verified by miR-7a-5p inhibitor and siCDR1as. These results indicate that CDR1as/miR-7a-5p/TGF-ßR2 interactions may exert scars and nerves functions and suggest potential therapeutic targets for treating spinal fibrotic diseases.

TGF-ß signaling promotes cervical cancer metastasis via CDR1as.

BACKGROUND: Due to the lack of effective treatment, metastasis is the main cause of cancer related deaths. TGF-ß pathway has been reported related to cervical cancer metastasis. However, mechanism is still unclear. METHODS: After agonist of TGF-ß treatment, RNA sequencing revealed the expression profiles of circRNA in cervical cancer. In situ hybridization was used to analysis relationship between CDR1as and prognosis. Real-time PCR, Western blot, RNA interference, Transwell assay, Wound healing assay, RNA pulldown assay and RIP assays were performed in vitro. And in vivo cervical cancer model (including foot pad model and subcutaneous tumor formation) was also performed. RESULTS: CDR1as was found upregulated obviously following TGF-ß activation. In situ hybridization showed CDR1as was positively correlated with lymph node metastasis and shortened survival length. Simultaneously, overexpression of CDR1as promoted cervical cancer metastasis in vitro and in vivo. It was also found that CDR1as could facilitate the orchestration of IGF2BP1 on the mRNA of SLUG and stabilize it from degradation. Silencing IGF2BP1 hampers CDR1as related metastasis in cervical cancer. Additionally, effective CDR1as has been proven to activate TGF-ß signaling factors known to promote EMT, including P-Smad2 and P-Smad3. CONCLUSIONS: Our study proved TGF-ß signaling may promote cervical cancer metastasis via CDR1as.

Localised Cdr1as activity is required for fear extinction memory.

Circular RNAs (circRNAs) comprise a novel class of regulatory RNAs that are abundant in the brain, particularly within synapses. They are highly stable, dynamically regulated, and display a range of functions, including serving as decoys for microRNAs and proteins and, in some cases, circRNAs also undergo translation. Early work in animal models revealed an association between circRNAs and neurodegenerative and neuropsychiatric disorders; however, little is known about the link between circRNA function and memory. To address this, we examined circRNA in synaptosomes derived from the medial prefrontal cortex of fear extinction-trained male C57BL/6J mice and found 12,837 circRNAs that were enriched at the synapse, including cerebellar degeneration-related protein 1 antisense RNA (Cdr1as). Targeted knockdown of Cdr1as in the neural processes of the infralimbic cortex led to impaired fear extinction memory. These findings highlight the involvement of localised circRNA activity at the synapse in memory formation.

CircRNA CDR1as: a novel diagnostic and prognostic biomarker for gastric cancer.

BACKGROUND: Circular RNA (circRNA) CDR1as is emerging as a vital tumour regulator. This study aimed to investigate its diagnostic and prognostic value and molecular mechanisms for gastric cancer (GC). METHODS: CDR1as expression in GC and adjacent normal tissues (n = 82), paired plasma (n = 65) and plasma exosome samples (n = 68) from GC patients and healthy controls were determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Correlations between CDR1as level and clinicopathological factors of GC patients were analysed. Its diagnostic and prognostic value was evaluated by receiver operating characteristic (ROC) curves and Cox regression analysis combined with Kaplan-Meier plots. CDR1as-regulated proteins and signalling pathways were identified by quantitative proteomics and bioinformatic analysis. RESULTS: CDR1as was downregulated in GC tissues and associated with tumour size and neural invasion. Plasma- and exosome-derived CDR1as was upregulated in GC patients while plasma-derived CDR1as level was related to lymphatic metastasis. Area under ROC curve (AUC) of tissue-, plasma- and exosome-derived CDR1as was 0.782, 0.641, 0.536 while combination of plasma CDR1as, serum CEA and CA19-9 increased AUC to 0.786. Distal metastasis, TNM stage and tissue-derived CDR1as level were independent predictors for overall survival (OS) of patients. MiRNA signalling networks and glycine, serine and threonine metabolism were regulated by CDR1as and HSPE1 might be a key protein. CONCLUSIONS: CDR1as is a crucial regulator and promising biomarker for GC diagnosis and prognosis.

CDR1as level in tumour tissues and plasma of GC patients was associated with tumour progression. The findings indicate that CDR1as is involved in GC progression and is a potential diagnostic and prognostic biomarker.

miR-145-3p Inhibits MuSCs Proliferation and Mitochondria Mass via Targeting MYBL1 in Jianzhou Big-Eared Goats.

Muscle growth and injury-induced regeneration are controlled by skeletal muscle satellite cells (MuSCs) through myogenesis in postnatal animals. Meanwhile, myogenesis is accompanied by mitochondrial function and enzyme activity. Nevertheless, the underlying molecular mechanisms involving non-coding RNAs including circular RNAs (circRNAs) and microRNAs (miRNAs) remain largely unsolved. Here, we explored the myogenic roles of miR-145-3p and MYBL1 on muscle development and mitochondrial mass. We noticed that overexpression of miR-145-3p inhibited MuSCs proliferation and reduced the number of viable cells. Meanwhile, deficiency of miR-145-3p caused by LNAantimiR-145-3p or an inhibitor retarded the differentiation of MuSCs. miR-145-3p altered the mitochondrial mass in MuSCs. Moreover, miR-145-3p targeted and negatively regulated the expression of CDR1as and MYBL1. The knockdown of the MYBL1 using ASO-2'MOE modification simulated the inhibitory function of miR-145-3p on cell proliferation. Additionally, MYBL1 mediated the regulation of miR-145-3p on Vexin, VCPIP1, COX1, COX2, and Pax7. These imply that CDR1as/miR-145-3p/MYBL1/COX1, COX2, VCPIP1/Vexin expression at least partly results in a reduction in mitochondrial mass and MuSCs proliferation. These novel findings confirm the importance of mitochondrial mass during myogenesis and the boosting of muscle/meat development in mammals.

Circulating expression levels of CircHIPK3 and CDR1as circular-RNAs in type 2 diabetes patients.

BACKGROUND: Recent investigations suggested that deregulated levels of Circular RNAs (circRNAs) could be associated with type 2 diabetes mellitus (T2DM) pathogenesis. Accordingly, this study aimed to determine the expression levels of circulating CircHIPK3, CDR1as and their correlation with biochemical parameters in patients with T2DM, pre-diabetes and control subjects. METHODS AND RESULTS: The expression of circRNAs in peripheral blood was determined using QRT-PCR in 70 patients with T2DM, 60 pre-diabetes and in 69 age and sex matched healthy controls. Moreover, bioinformatics tools were applied to explore and predict the potential interactions between circRNAs and other non-coding RNAs (ncRNAs). Our analysis revealed that the expression level of CircHIPK3 was significantly elevated in T2DM patients compared to healthy participants (P < 0.001) and pre-diabetes subjects (P = 0.018). In addition, ROC analysis suggested that at the cutoff value of 0.24 and the sensitivity and specificity of 50% and 88.4%, respectively, CircHIPK3 could distinguish between T2DM patients and control subjects. Furthermore, it was observed that the expression level of CDR1as is higher in pre-diabetic individuals than healthy individuals (P = 0.004). Finally, Spearman correlation analysis showed that there was a significant correlation between CircHIPK3 and CDR1as expression levels and clinical and anthropometrical parameters such as BMI, systolic and diastolic blood pressure, HbA1c and fasting blood glucose (P < 0.005). CONCLUSIONS: The data of this study provided evidence that the expression levels of CircHIPK3, CDR1as increased in T2DM and pre-diabetes subjects, respectively.

Analyses of circRNA Expression throughout the Light-Dark Cycle Reveal a Strong Regulation of Cdr1as, Associated with Light Entrainment in the SCN.

Circular RNAs (circRNAs) are a large class of relatively stable RNA molecules that are highly expressed in animal brains. Many circRNAs have been associated with CNS disorders accompanied by an aberrant wake-sleep cycle. However, the regulation of circRNAs in brain homeostasis over daily light-dark (LD) cycles has not been characterized. Here, we aim to quantify the daily expression changes of circRNAs in physiological conditions in healthy adult animals. Using newly generated and public RNA-Seq data, we monitored circRNA expression throughout the 12:12 h LD cycle in various mouse brain regions. We identified that Cdr1as, a conserved circRNA that regulates synaptic transmission, is highly expressed in the suprachiasmatic nucleus (SCN), the master circadian pacemaker. Despite its high stability, Cdr1as has a very dynamic expression in the SCN throughout the LD cycle, as well as a significant regulation in the hippocampus following the entry into the dark phase. Computational integration of different public datasets predicted that Cdr1as is important for regulating light entrainment in the SCN. We hypothesize that the expression changes of Cdr1as in the SCN, particularly during the dark phase, are associated with light-induced phase shifts. Importantly, our work revises the current beliefs about natural circRNA stability and suggests that the time component must be considered when studying circRNA regulation.

CDR1as regulated by hnRNPM maintains stemness of periodontal ligament stem cells via miR-7/KLF4.

CDR1as is a well-identified circular RNA with regulatory roles in a variety of physiological processes. However, the effects of CDR1as on stemness of periodontal ligament stem cells (PDLSCs) and the underlying mechanisms remain unclear. In this study, we detect CDR1as in human PDLSCs, and subsequently demonstrate that CDR1as maintains PDLSC stemness. Knockdown of CDR1as decreases the expression levels of stemness-related genes and impairs the cell's multi-differentiation and cell migration abilities, while overexpression of CDR1as increases the expression levels of stemness-related genes and enhances these abilities. Furthermore, our results indicate that the RNA-binding protein hnRNPM directly interacts with CDR1as and regulates its expression in PDLSCs. In addition, we show that CDR1as promotes the expression of stemness-related genes in PDLSCs by inhibiting miR-7-mediated suppression of KLF4 expression. Collectively, our results demonstrate that CDR1as participates in the molecular circuitry that regulates PDLSC stemness.

The expanding role of CDR1-AS in the regulation and development of cancer and human diseases.

CircRNAs are a superabundant and highly conserved group of noncoding RNAs (ncRNAs) that are characterized by their high stability and integrity compared with linear forms of ncRNAs. Recently, their critical role in gene expression regulation has been shown; thus, it is not far-fetched to believe that their abnormal expression can be a cause of different kinds of diseases such as cancer, neurodegenerative, and autoimmune diseases. They can have a function in variety of biological processes such as microRNA (miRNA) sponging, interacting with RNA-binding proteins, or even an ability to translate to proteins. A huge challenge in finding diagnostic biomarkers is finding noninvasive biomarkers that can be detected in human fluids, especially blood samples. CircRNAs are becoming candidate biomarkers for diagnosis and prognosis of these diseases through their ability to transverse from the blood-brain barrier and their broad presence in circulating exosomes. The circRNA for miRNA-7 (ciRS-7) is newly recognized, and acknowledged to being related to human pathology and cancer progression. In this review, we first briefly summarize the latest studies about their characteristics, biogenesis, and their mechanisms of action in the regulation and development of human diseases. Finally, we provide a list of diseases that are linked to one member of this novel class of ncRNAs called ciRS-7.

Circular RNA-CDR1as is involved in lung injury induced by long-term formaldehyde inhalation.

OBJECTIVE: Formaldehyde (FA) is known to induce lung injury, but the underlying molecular mechanism remains largely unclear. CDR1as is an important member of the circular RNAs (circRNAs) family and functions as miRNA sponges with gene-regulatory potential. Our earlier circRNA microarray data showed CDR1as was highly expressed in lung tissue exposed to FA. However, the mechanism of circRNA-CDR1as mediates the FA-exposed lung injury is still unclear. This study aimed to explore the role of CDR1as in lung injury. MATERIALS AND METHODS: In this study, FA was inhaled at doses of 0.5, 2.46, and 5 mg/m3, respectively. After exposure 8 weeks, lung histopathological examination, lung injury score, and IL-1ß in bronchoalveolar lavage fluid (BALF) were determined. The expressions of CDR1as, rno-miR-7b and Atg7 were detected and the potential interaction of circRNA/miRNA/mRNA was predicted by bioinformatics analysis, including drawing circRNA/miRNA/mRNA interaction network, GO and KEGG analysis. RESULTS: Our results indicated FA inhalation upregulated the expression of CDR1as in lung tissues in a dose-dependent manner while the expression of rno-miR-7b decreased and Atg7 increased. Moreover, the alteration of CDR1as was positively correlated with lung injury. DISCUSSION AND CONCLUSIONS: CircRNA/miRNA/mRNA prediction further explained the possible effect mechanisms of CDR1as. These data implicated that CDR1as might be a critical regulator involved in lung injury induced by FA.

Diagnostic and prognostic value of circular RNA CDR1as/ciRS-7 for solid tumours: A systematic review and meta-analysis.

The circular RNA, CDR1as/ciRS-7, functions as a vital regulator in various cancers; however, the predictive value of CDR1as remains controversial. Therefore, a comprehensive analysis for clarifying the precise diagnostic and prognostic value of CDR1as in solid tumours is needed. A literature review of several databases was conducted for identifying potential studies. Pooled odds ratios (ORs) and hazard ratios (HRs) were used for evaluating the diagnostic accuracy variables and survival. Overall, 15 studies (1787 patients) and 11 studies (1578 patients) were included for diagnostic and prognostic outcome syntheses, respectively. Up-regulated CDR1as expression was found to be correlated with worse clinicopathological characteristics, including the T status, N status, histological grade, TNM stage and distant metastasis. The synthesized sensitivity was 0.72 (95% confidence interval [CI], 0.65-0.79), and the specificity was 0.80 (95% CI, 0.74-0.86). The positive likelihood ratio (LR), negative LR and diagnostic odds ratio (DOR) were 3.70, 0.34 and 10.80, respectively. The area under the receiver operator characteristic curve was 0.84 (95% CI, 0.80-0.87). In the pooled prognostic analysis, patients with high CDR1as expression had worse overall survival (HR = 2.40, P < 0.001) and disease-free survival (HR = 1.74, P < 0.001). These results suggest that CDR1as is a reliable diagnostic and prognostic biomarker with high accuracy and efficiency, which may potentially facilitate clinical decisions on solid tumours in the future.

Circular RNA CDR1as disrupts the p53/MDM2 complex to inhibit Gliomagenesis.

BACKGROUND: Inactivation of the tumor suppressor p53 is critical for pathogenesis of glioma, in particular glioblastoma multiforme (GBM). MDM2, the main negative regulator of p53, binds to and forms a stable complex with p53 to regulate its activity. Hitherto, it is unclear whether the stability of the p53/MDM2 complex is affected by lncRNAs, in particular circular RNAs that are usually abundant and conserved, and frequently implicated in different oncogenic processes. METHODS: RIP-seq and RIP-qPCR assays were performed to determine the most enriched lncRNAs (including circular RNAs) bound by p53, followed by bioinformatic assays to estimate the relevance of their expression with p53 signaling and gliomagenesis. Subsequently, the clinical significance of CDR1as was evaluated in the largest cohort of Chinese glioma patients from CGGA (n = 325), and its expression in human glioma tissues was further evaluated by RNA FISH and RT-qPCR, respectively. Assays combining RNA FISH with protein immunofluorescence were performed to determine co-localization of CDR1as and p53, followed by CHIRP assays to confirm RNA-protein interaction. Immunoblot assays were carried out to evaluate protein expression, p53/MDM2 interaction and p53 ubiquitination in cells in which CDR1as expression was manipulated. After AGO2 or Dicer was knocked-down to inhibit miRNA biogenesis, effects of CDR1as on p53 expression, stability and activity were determined by immunoblot, RT-qPCR and luciferase reporter assays. Meanwhile, impacts of CDR1as on DNA damage were evaluated by flow cytometric assays and immunohistochemistry. Tumorigenicity assays were performed to determine the effects of CDR1as on colony formation, cell proliferation, the cell cycle and apoptosis (in vitro), and on tumor volume/weight and survival of nude mice xenografted with GBM cells (in vivo). RESULTS: CDR1as is found to bind to p53 protein. CDR1as expression decreases with increasing glioma grade and it is a reliable independent predictor of overall survival in glioma, particularly in GBM. Through a mechanism independent of acting as a miRNA sponge, CDR1as stabilizes p53 protein by preventing it from ubiquitination. CDR1as directly interacts with the p53 DBD domain that is essential for MDM2 binding, thus disrupting the p53/MDM2 complex formation. Induced upon DNA damage, CDR1as may preserve p53 function and protect cells from DNA damage. Significantly, CDR1as inhibits tumor growth in vitro and in vivo, but has little impact in cells where p53 is absent or mutated. CONCLUSIONS: Rather than acting as a miRNA sponge, CDR1as functions as a tumor suppressor through binding directly to p53 at its DBD region to restrict MDM2 interaction. Thus, CDR1as binding disrupts the p53/MDM2 complex to prevent p53 from ubiquitination and degradation. CDR1as may also sense DNA damage signals and form a protective complex with p53 to preserve p53 function. Therefore, CDR1as depletion may play a potent role in promoting tumorigenesis through down-regulating p53 expression in glioma. Our results broaden further our understanding of the roles and mechanism of action of circular RNAs in general and CDR1as in particular, and can potentially open up novel therapeutic avenues for effective glioma treatment.

The circular RNA CDR1as regulate cell proliferation via TMED2 and TMED10.

BACKGROUND: Circular RNAs (CircRNAs) are biologically active RNAs. CDR1as is one such circRNA previously reported to be a microRNA-7 (miR-7) sponge, thereby regulating associated gene expression. The specific underlying molecular mechanisms of CDR1as biology, however, remain largely unknown. METHODS: We performed CDR1as knockdown in order to explore its function in cell proliferation, migration, the cell cycle, and tumorigenesis. We further employed quantitative proteomic analyses and associated bioinformatics strategies to globally assess CDR1as-regulated proteins (CRPs). Western blotting and immunofluorescence staining were used to validate the proteomic results. We additionally investigated a specific link between TMED2, TMED10, and miR-7 via a dual-luciferase reporter system, and generated CDR1as knockout cell lines via CRISPR/Cas9 editing. RESULTS: We identified 353 proteins dysregulated upon CDR1as knockdown in 293 T cells. These CRPs were found to interact with one another and to play key roles in certain cellular pathways. Two such proteins, TMED2 and TMED10, were found to specifically contribute to the influence of CDR1as on cell proliferation. CDR1as may regulate these two TMED proteins through miR-7 sponging. We were able to further confirm these results using both CRISPRi cell lines and nude mouse models. CONCLUSION: This study suggested that CDR1as may regulate cell proliferation via serving as a miR-7 sponge, thereby regulating TMED2 and TMED10 expression. These results are an invaluable template for future streamlined studies of circRNAs.

Silencing CDR1as enhances the sensitivity of breast cancer cells to drug resistance by acting as a miR-7 sponge to down-regulate REGγ.

In our study, we aimed to investigate the role of CDR1as during competitive inhibition of miR-7 in the regulation of cisplatin chemosensitivity in breast cancer via regulating REGγ. RT-qPCR was applied to detect the expression of CDR1as and miR-7 in breast cancer tissues, breast cancer cell lines and corresponding drug-resistant cell lines. The correlation between CDR1as and miR-7 and between miR-7 and REGγ was evaluated. MCF-7-R and MDA-MB-231-R cells were selected followed by transfection of a series of mimics, inhibitors or siRNA. The effect of CDR1as on the half maximal inhibitor concentration (IC50), cisplatin sensitivity and cell apoptosis was also analysed. Furthermore, a subcutaneous xenograft nude mouse model was established to further confirm the effect of CDR1as on the chemosensitivity of breast cancer to cisplatin in vivo. Immunohistochemical staining was conducted to test the Ki-67 expression in nude mice. A positive correlation was found between the drug resistance and CDR1as expression in breast cancer. CDR1as could increase the resistance of breast cancer cells to cisplatin. miR-7 expression was low, while REGγ was highly expressed in MCF-7-R and MDA-MB-231-R cells. CDR1as competitively inhibited miR-7 and up-regulated REGγ. Overexpression of miR-7 could reverse the enhanced sensitivity of silenced CDR1as to drug-resistant breast cancer cells. Additionally, in vivo experiments demonstrated that CDR1as mediated breast cancer occurrence and its sensitivity to cisplatin. Silencing CDR1as decreased Ki-67 expression. Silencing CDR1as may inhibit the expression of REGγ by removing the competitive inhibitory effect on miR-7 and thus enhancing the sensitivity of drug-resistant breast cancer cells.

Inhibition of circular RNA CDR1as increases chemosensitivity of 5-FU-resistant BC cells through up-regulating miR-7.

This study aims to explore the mechanism of Circular RNA CDR1as implicating in regulating 5-fluorouracil (5-FU) chemosensitivity in breast cancer (BC) by competitively inhibiting miR-7 to regulate CCNE1. Expressions of CDR1as and miR-7 in 5-FU-resistant BC cells were determined by RT-PCR. CCK-8, colony formation assay and flow cytometry were applied to measure half maximal inhibitory concentration (IC50), 5-Fu chemosensitivity and cell apoptosis. Western blot was used to detect the expressions of apoptosis-related factors. CDR1as was elevated while miR-7 was inhibited in 5-FU-resistant BC cells. Cells transfected with si-CDR1as or miR-7 mimic had decreased IC50 and colony formation rate, increased expressions of Bax/Bcl2 and cleaved-Caspase-3/Caspase-3, indicating inhibition of CDR1as and overexpression of miR-7 enhances the chemosensitity of 5-FU-resistant BC cells. Targetscan software indicates a binding site of CDR1as and miR-7 and that CCNE1 is a target gene of miR-7. miR-7 can gather CDR1as in BC cells and can inhibit CCNE1. In comparison to si-CDR1as group, CCNE1 was increased and chemosensitivity to 5-Fu was suppressed in si-CDR1as + miR-7 inhibitor group. When compared with miR-7 mimic group, CDR1as + miR-7 mimic group had increased CCNE1 and decreased chemosensitivity to 5-Fu. Nude mouse model of BC demonstrated that the growth of xenotransplanted tumour in si-CDR1as + miR-7 inhibitor group was faster than that in si-CDR1as group. The tumour growth in CDR1as + miR-7 mimic group was faster than that in miR-7 mimic group. CDR1as may regulate chemosensitivity of 5-FU-resistant BC cells by inhibiting miR-7 to regulate CCNE1.

Circular RNA CDR1as sponges miR-7-5p to enhance E2F3 stability and promote the growth of nasopharyngeal carcinoma.

BACKGROUND: Circular RNA (circRNA) CDR1as plays an important role in the occurrence and development of human tumors. The purpose of this study is to investigate the molecular mechanism of circRNA CDR1as in the development of nasopharyngeal carcinoma (NPC). METHODS: The mRNA expressions of circRNA CDR1as, miR-7-5p, and E2F3 were detected by qRT-PCR. The effects of circRNA CDR1as, miR-7-5p, and E2F3 on NPC cells were investigated using cell counting kit-8 (CCK8) method, colony formation assay, and representative metabolite assay. The molecular mechanism of circRNA CDR1 in NPC was studied by bioinformatics and luciferase reporter assay. In addition, the biological activity of circRNA CDR1as was also investigated in NPC xenograft tumor mice model. RESULTS: The results showed that the circRNA CDR1as expression was significantly up-regulated in NPC tissues by comparison with non-tumor NPE tissues (p < 0.01), suggesting that circRNA CDR1as was associated with poor prognosis in NPC patients. Moreover, circRNA CDR1as could up-regulate E2F3 expression by binding miR-7-5p, and promote the growth and glucose metabolism of NPC cells. Meanwhile, circRNA CDR1as could promote NPC progression through the negative regulation of miR-7-5p in the xenograft tumor model. CONCLUSION: CircRNA CDR1as promoted the occurrence and development of NPCs by successively up-regulating the expression of miR-7-5p and E2F3, suggesting CircRNA CDR1as as a potential target for the treatment of NPC patients.Trial registration The study was approved by the cancer center's institutional research ethics committee on Oct 18, 2008 (2008GZ2847462).

CircRNA-Cdr1as Exerts Anti-Oncogenic Functions in Bladder Cancer by Sponging MicroRNA-135a.

BACKGROUND/AIMS: CircRNAs regulate gene expression in different malignancies. However, the role of Cdr1as in the tumourigenesis of bladder cancer and its potential mechanisms remain unknown. METHODS: qRT-PCR was used to detect Cdr1as and target miRNA expression in bladder cancer tissues and cell lines. Biological functional experiments were performed to detect the effects of Cdr1as on the biological behaviour of bladder cancer cells in vivo and in vitro. Bioinformatic analysis was utilised to predict potential miRNA target sites on Cdr1as. Ago2 RNA binding protein immunoprecipitation assay, RNA antisense purification assay, biotin pull down assay and RNA FISH were performed to detect the interaction between Cdr1as and target miRNAs. Western blot was used to determine the expression level of p21 in bladder cancer cells. RESULTS: Cdr1as was significantly down-regulated in bladder cancer tissues compared with adjacent normal tissues. Overexpression of Cdr1as inhibited the proliferation, invasion and migration of bladder cancer cells in vitro and slowed down tumour growth in vivo. Cdr1as sponged multiple miRNAs in bladder cancer. Moreover, Cdr1as directly bound to miR-135a and inhibited its activity in bladder cancer. CONCLUSION: Cdr1as is down-regulated and sponges multiple miRNAs in bladder cancer. It exerts anti-oncogenic functions by sponging microRNA-135a.

Circular RNA in Exosomes.

Circular RNAs (circRNAs) are a novel family of non-coding endogenous RNAs discovered in all eukaryotic cells and generated through a particular mechanism of alternative splicing called "back-splicing". These molecules show multiple functions, by acting as modulators of gene and miRNA expression, and may have a role in several biological processes, such as cell proliferation and invasion with, tumour development and progression, and in several mechanisms underlying other diseases. Their presence has been shown to be abundant in several body fluids such as blood and saliva. Based on their biogenesis mechanism, circRNAs may be categorized into five classes: exonic circRNAs, intronic circRNAs, antisense circRNAs, sense overlapping circRNAs and intergenic circRNAs. Recently, the presence of circRNAs, in addition to that of miRNAs and long non-coding RNAs, has been detected also in small extracellular vesicles called exosomes. Investigating the presence and expression levels of serum exosomal circRNAs could allow us, in future, to discriminate cancer patients from healthy individuals, identifying new potential exosome-based cancer biomarkers.In this chapter, we briefly will describe the major features and functions of exosomal circRNAs, discussing their potential role as molecular biomarkers for diagnosis, prognosis and monitoring of complex diseases, including cancer.

Circular RNA and Alzheimer's Disease.

Circular RNAs (circRNAs) represent a special group of noncoding single-stranded highly stable ribonucleic acid entities abundant in the eukaryotic transcriptome. These circular forms of RNAs are significantly enriched in human brain and retinal tissues. However, the biological evolution and function of these circRNAs are poorly understood. Recent reports showed circRNA to be an important player in the development of neurodegenerative diseases like Alzheimer's disease. With the progression of age, circRNA level increases in the brain and also in age-associated neurological disorder like Alzheimer's disease (AD), Parkinson's disease, inflammatory neuropathy, nervous system neoplasms, and prion diseases. One highly represented circRNA in the human brain and retina is a ciRS-7 (CDR1as) which acts as an endogenous, anticomplementary miRNA inhibitor or "sponge" to quench the normal functioning of miRNA-7. Low CDR1as level can lead to increase in miR-7 expression which downregulates the activity of ubiquitin protein ligase A (UBE2A), an important AD target, functionally involved in clearing toxic amyloid peptides from AD brain. This chapter focuses on the functional relationship of circRNA with AD and interplay of miRNA-mRNA-mediated genetic regulatory networks. Our conceptual understanding also suggests that circRNA can be considered as a potential biomarker and therapeutic target in AD diagnosis and treatment.