Expression Levels Of circRNAs Research Articles

Genetic variation of circHIBADH enhances prostate cancer risk through regulating HNRNPA1-related RNA splicing.

The current study aimed to investigate associations of circRNAs and related genetic variants with risk of prostate cancer (PCa) as well as to elucidate biological mechanisms underlying the associations. By using the MiOncoCirc database, we first compared expression levels of circRNAs between 25 paired PCa and adjacent normal tissues to identify risk-associated circRNAs. We then used logistic regression models to evaluate associations between genetic variants in candidate circRNAs and PCa risk among 4662 prostate cancer patients and 3114 healthy controls, and identified circHIBADH rs11973492 as a significant risk-associated variant (odds ratio = 1.20, 95% confidence interval: 1.08-1.34, P = 7.06 × 10 -4) in a dominant genetic model, which altered the secondary structure of the corresponding RNA chain. In the in silico analysis, we found circHIBADH to sponge and silence 21 RNA-binding proteins (RPBs) enriched in the RNA splicing pathway, among which HNRNPA1 was identified and validated as a hub RBP using an external RNA-sequencing data as well as the in-house (four tissue samples) and publicly available single-cell transcriptomes. Additionally, we demonstrated that HNRNPA1 might influence hallmarks including MYC, DNA repair, and E2F target signaling pathways, thereby promoting carcinogenesis. In conclusion, genetic variants in circHIBADH may act as a sponge and inhibitor of RNA splicing-associated RBPs including HNRNPA1, playing an oncogenic role in PCa.

Mechanism of circRNA_SMG6 mediating lung macrophage ECM degradation via miR-570-3p in microplastics-induced emphysema

Microplastics (MPs) are plastic particles < 5 mm in diameter, of which polystyrene microplastics (PS-MPs) are representative type. The extracellular matrix (ECM) degradation of macrophages is associated with the development of emphysema. Additionally, circular RNAs (circRNAs) have a regulatory role in epigenetic mechanisms related to lung disease. However, the mechanisms of the ECM degradation and circRNAs in MPs-induced emphysema are still unclear. In our study, Sprague-Dawley (SD) rats were treated with 0, 0.5, 1.0 and 2.0 mg/m3 100 nm PS-MPs for 90 days in an inhalation experiment. PS-MPs-exposed rats showed elevated airway resistance and pulmonary dysfunction. Lung histopathology exhibited inflammatory cell infiltration, septal thickening and alveolar dilatation. Exposure to PS-MPs was able to induce elevated levels of ECM degradation-related markers MMP9 and MMP12, as well as reduced levels of elastin in rat lung tissues. CircRNA_SMG6 is a non-coding RNA (ncRNA) with a homologous circular structure in human, rat and mouse. The expression level of circRNA_SMG6 was decreased in both rat lung tissues exposed to PS-MPs and PS-MPs-treated THP-1 cells. The luciferase reporter gene demonstrated that circRNA_SMG6 combined with miR-570-3p and co-regulated PTEN, the target gene of miR-570-3p. Moreover, overexpression of circRNA_SMG6 or inhibition of miR-570-3p attenuated PS-MPs-induced ECM degradation in THP-1 cells. Taken together, circRNA_SMG6 may have a significant function in the deterioration of emphysema caused by PS-MPs-induced macrophage ECM degradation by regulating miR-570-3p. Our findings reveal a novel mechanism of emphysema caused by PS-MPs and provide valuable information for assessing the health risks of MPs.

CircCOL1A1 promotes proliferation, migration, and invasion of colorectal cancer (CRC) cells and glutamine metabolism through GLS1 up-regulation by sponging miR-214-3p

BackgroundCircular ribose nucleic acids (circRNAs), an abundant type of noncoding RNAs, are widely expressed in eukaryotic cells and exert a significant impact on the initiation and progression of various disorders, including different types of cancer. However, the specific role of various circRNAs in colorectal cancer (CRC) pathology is still not fully understood.MethodsThe initial step involved the use of quantitative reverse transcription polymerase chain reaction (RT-qPCR) to assess the expression levels of circRNAs and messenger RNA (mRNA) in CRC cell lines and tissues. Subsequently, functional analyses of circCOL1A1 knockdown were conducted in vitro and in vivo through cell counting kit (CCK)-8, colony formation and transwell assays, as well as xenograft mouse model of tumor formation. Molecular expression and interactions were investigated using luciferase reporter assays, Western blot analysis, RNA immunoprecipitation (RIP), and immunohistochemical staining.ResultsThe RT-qPCR results revealed elevated levels of circCOL1A1 expressions in CRC tissues and cell lines as compared to the normal counterparts. In addition, circCOL1A1 expression level was found to be correlated with TNM stage, lymph node metastases, distant metastases, and invasion. Knockdown of circCOL1A1 resulted in impaired invasion, migration, and proliferation of CRC cells, and suppressed tumor generation in the animal model. We further demonstrated that circCOL1A1 could act as a sponge for miR-214-3p, suppressing miR-214-3p activity and leading to the upregulation of GLS1 protein to promote glutamine metabolism.ConclusionThese findings suggest that circCOL1A1 functions as an oncogenic molecule to promote CRC progression via miR-214-3p/GLS1 axis, hinting on the potential of circCOL1A1 as a therapeutic target for CRC.

Responses of keratinocytes to Trichophyton mentagrophyte infection based on whole transcriptome analysis.

Dermatophytosis is an intractable superficial mycosis in humans and animals mainly caused by Trichophyton mentagrophytes (T. mentagrophytes), with a global prevalence of about 20%. Keratinocytes are the most abundant participants in skin immunity, and they also play a role in the first-line defence against T. mentagrophytes. However, no studies of keratinocyte responses against T. mentagrophytes infection based on the whole transcriptome have been reported. Here, we systematically analysed changes in keratinocytes infected with T. mentagrophytes using whole transcriptome sequencing technology. The phenotypic changes in keratinocytes after infection with 1 × 105 conidia/mL T. mentagrophytes were observed by light microscopy, scanning electron microscopy, transmission electron microscopy and terminal deoxynucleotidyl transferase dUTP nick end labeling. RNA-sequencing (RNA-seq), small RNA-seq technology and related bioinformatics methods were used to systematically analyse the whole transcriptome changes in keratinocytes upon T. mentagrophytes stimulation. We found that T. mentagrophytes infection caused morphological changes, membrane damage, the formation of irregular organelles and keratinocyte apoptosis. A total of 204 differentially expressed (DE) circular RNAs (circRNAs), 868 DE long noncoding RNAs (lncRNAs), 2973 DE mRNAs and 209 DE micro RNAs (miRNAs) were identified between noninfected and T. mentagrophytes-infected keratinocytes. The expression level of selected RNAs was validated by quantitative real-time polymerase chain reaction (qRT-PCR). Functional enrichment analysis revealed that the parental genes of DE circRNAs were related to cell response, cell death and establishment of the skin barrier. Genes targeted by miRNA were involved in regulating the initiation of the immune response. Based on the expression level of circRNAs, lncRNAs, mRNAs and miRNAs, circRNA-miRNA-mRNA competing endogenous (ceRNA) networks comprised of 159 DE miRNAs, 141 DE circRNAs and 2307 DE mRNAs, and lncRNA-miRNA-mRNA ceRNA networks comprised of 790 DE lncRNAs, 190 DE miRNAs and 2663 DE mRNAs were constructed. The reliability of two selected ceRNA networks was verified using qRT-PCR. Further functional enrichment analysis revealed that the DE mRNAs interacting with circRNAs and lncRNAs in the ceRNA network mainly participated in fungal recognition, inflammation, the innate immune response and the death of keratinocytes. Our findings might provide new evidence on the pathogenesis of T. mentagrophytes-induced dermatophytosis, which is essential for identifying new therapeutic targets for dermatophytosis treatment.

Circular RNA Expression of Peripheral Blood Mononuclear Cells Associated with Risk of Acute Exacerbation in Smoking Chronic Obstructive Pulmonary Disease.

Circular RNAs (circRNAs) are newly identified endogenous non-coding RNAs that function as crucial gene modulators in the development of several diseases. By assessing the expression levels of circRNAs in peripheral blood mononuclear cells (PBMCs) from patients with chronic obstructive pulmonary disease (COPD), this study attempted to find new biomarkers for COPD screening. We confirmed altered circRNA expression in PBMCs of COPD (n=41) vs controls (n=29). Further analysis focused on the highest and lowest circRNA expression levels. The T-test is used to assess the statistical variances in circRNAs among COPD patients in the smoking and non-smoking cohorts. Additionally, among smokers, the Spearman correlation test assesses the association between circRNAs and clinical indicators. Two circRNAs, hsa_circ_0042590 and hsa_circ_0049875, that were highly upregulated and downregulated in PBMCs from COPD patients were identified and verified. Smokers with COPD had lower hsa_circ_0042590 and higher hsa_circ_0049875, in comparison to non-smokers. There was a significant correlation (r=0.52, P<0.01) between the number of acute exacerbations (AEs) that smokers with COPD experienced in the previous year and the following year (r=0.67, P<0.001). Moreover, hsa_circ_0049875 was connected to the quantity of AEs in the year prior (r=0.68, P<0.0001) as well as the year after (r=0.72, P<0.0001). AUC: 0.79, 95% CI: 0.1210-0.3209, P<0.0001) for hsa_circ_0049875 showed a strong diagnostic value for COPD, according to ROC curve analysis. Hsa_circ_0042590 showed a close second with an AUC of 0.83 and 95% CI: -0.1972--0.0739 (P <0.0001). This research identified a strong correlation between smoking and hsa_circ_0049875 and hsa_circ_0042590 in COPD PBMCs. The number of AEs in the preceding and succeeding years was substantially linked with the existence of hsa_circ_0042590 and hsa_circ_0049875 in COPD patients who smoke. Additionally, according to our research, hsa_circ_0049875 and hsa_circ_0042590 may be valuable biomarkers for COPD diagnosis.

A novel peptide PDHK1-241aa encoded by circPDHK1 promotes ccRCC progression via interacting with PPP1CA to inhibit AKT dephosphorylation and activate the AKT-mTOR signaling pathway

BackgroundClear cell renal cell carcinoma (ccRCC) is the most prevalent kidney cancer with high aggressive phenotype and poor prognosis. Accumulating evidence suggests that circRNAs have been identified as pivotal mediators in cancers. However, the role of circRNAs in ccRCC progression remains elusive.MethodsThe differentially expressed circRNAs in 4 paired human ccRCC and adjacent noncancerous tissues ccRCC were screened using circRNA microarrays and the candidate target was selected based on circRNA expression level using weighted gene correlation network analysis (WGCNA) and the gene expression omnibus (GEO) database. CircPDHK1 expression in ccRCC and adjacent noncancerous tissues (n = 148) were evaluated along with clinically relevant information. RT-qPCR, RNase R digestion, and actinomycin D (ActD) stability test were conducted to identify the characteristics of circPDHK1. The subcellular distribution of circPDHK1 was analyzed by subcellular fractionation assay and fluorescence in situ hybridization (FISH). Immunoprecipitation-mass spectrometry (IP-MS) and immunofluorescence (IF) were employed to evaluate the protein-coding ability of circPDHK1. ccRCC cells were transfected with siRNAs, plasmids or lentivirus approach, and cell proliferation, migration and invasion, as well as tumorigenesis and metastasis in nude mice were assessed to clarify the functional roles of circPDHK1 and its encoded peptide PDHK1-241aa. RNA-sequencing, western blot analysis, immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) assays were further employed to identify the underlying mechanisms regulated by PDHK1-241aa.ResultsCircPDHK1 was upregulated in ccRCC tissues and closely related to WHO/ISUP stage, T stage, distant metastasis, VHL mutation and Ki-67 levels. CircPDHK1 had a functional internal ribosome entry site (IRES) and encoded a novel peptide PDHK1-241aa. Functionally, we confirmed that PDHK1-241aa and not the circPDHK1 promoted the proliferation, migration and invasion of ccRCC. Mechanistically, circPDHK1 was activated by HIF-2A at the transcriptional level. PDHK1-241aa was upregulated and interacted with PPP1CA, causing the relocation of PPP1CA to the nucleus. This thereby inhibited AKT dephosphorylation and activated the AKT-mTOR signaling pathway.ConclusionsOur data indicated that circPDHK1-encoded PDHK1-241aa promotes ccRCC progression by interacting with PPP1CA to inhibit AKT dephosphorylation. This study provides novel insights into the multiplicity of circRNAs and highlights the potential use of circPDHK1 or PDHK1-241aa as a therapeutic target for ccRCC.

Exosomes derived from BMSCs enhance diabetic wound healing through circ-Snhg11 delivery

BackgroundExosomes (Exos) generated from bone mesenchymal stem cells (BMSCs) are elucidated to enhance cutaneous wound healing in mice models of diabetes mellitus (DM). While underlying mechanisms remain unknown.MethodsNext-generation sequencing (NGS) was used to examine changes in circRNA expression levels following Exo treatment. Luciferase assays were used to determine the interactions between RNAs. Immunofluorescence staining was used to examine reactive oxygen species (ROS) in endothelial progenitor cells (EPCs) cultured in high glucose (HG) conditions. Therapeutic effects regarding Exos were also examined by immunofluorescence.ResultsWe found that Exo treatment enhanced cutaneous wound healing significantly. NGS indicated that circ-Snhg11 was involved in Exo-mediated tissue repairing. Downregulation of circ-Snhg11 decreased Exo-mediated therapy responses during wound healing in diabetic mouse. Our luciferase reporter data confirmed that SLC7A11 and miR-144-3p were circ-Snhg11 downstream targets. miR-144-3p overexpression or SLC7A11 knockdown altered the protective effects of circ-Snhg11 upon EPCs exposed to HG conditions. Upregulation of circ-Snhg11 incremented therapy effects of Exo treatment during wound healing in DM mice through enhanced angiogenesis along with a reduction in GPX4-mediated ferroptosis.Conclusionscirc-Snhg11 in BMSC-Exos enhanced SLC7A11/GPX4-mediated anti-ferroptosis signals via miR-144-3p sponging resulting in enhanced diabetic wound healing and improved angiopoiesis.

The Function of Circular RNAs in Myocardial Ischemia-Reperfusion Injury: Underlying Mechanisms and Therapeutic Advancement.

Myocardial ischemia reperfusion injury (MIRI) represents a prevalent and severe cardiovascular condition that arises primarily after myocardial infarction recanalization, cardiopulmonary bypass surgery, and both stable and unstable angina pectoris. MIRI can induce malignant arrhythmias and heart failure, thereby increasing the morbidity and mortality rates associated with cardiovascular diseases. Hence, it is important to assess the potential pathological mechanisms of MIRI and develop effective treatments. The role of circular RNAs (circRNAs) in MIRI has increasingly become a topic of interest in recent years. Moreover, significant evidence suggests that circRNAs play a critical role in MIRI pathogenesis, thereby representing a promising therapeutic target. This review aimed to provide a comprehensive overview of the current understanding of the role of circRNAs in MIRI and discuss the mechanisms through which circRNAs contribute to MIRI development and progression, including their effects on apoptosis, inflammation, oxidative stress, and autophagy. Furthermore, the potential therapeutic applications of circRNAs in MIRI treatment, including the use of circRNA-based therapies and modulation of circRNA expression levels, have been explored. Overall, this paper highlights the importance of circRNAs in MIRI and underscores their potential as novel therapeutic targets.

Hsa_circ_0086414/transducer of ERBB2 (TOB2) axis-driven lipid elimination and tumor suppression in clear cell renal cell cancer via perilipin 3

BackgroundRenal cell cancer (RCC) is characterized by abnormal lipid accumulation. However, the specific mechanism by which such lipid deposition is eliminated remains unclear. Circular RNAs (circRNAs) widely regulate various biological processes, but the effect of circRNAs on lipid metabolism in cancers, especially clear cell renal cell carcinoma (ccRCC), remains poorly understood. MethodsThe downregulated circRNA, hsa_circ_0086414, was identified from high-throughput RNA-sequencing data of human ccRCC and pair-matched normal tissues. The target relationship between circRNA_0086414 and miR-661, and the transducer of ERBB2 (TOB2) was predicted using publicly available software programs and verified by luciferase reporter assays. The clinical prognostic value of TOB2 was evaluated by bioinformatic analysis. The expression levels of circRNA_0086414, miR-661, TOB2, and perilipin 3 (PLIN3) were measured by quantitative reverse-transcription polymerase chain reaction or western blot analysis. Cell Counting Kit-8, transwell assays, and xenograft models were employed to assess the biological behaviors of the hsa_circ_0086414/TOB2 axis. Oil Red staining and triglyceride assay was conducted to assess lipid deposition. ResultsHerein, we identified a downregulated circRNA, hsa_circ_0086414. Functionally, the restored hsa_circ_0086414 inhibited ccRCC proliferation, metastasis, and lipid accumulation in vitro and in vivo. Furthermore, the downregulated TOB2 predicted adverse prognosis and promoted cancer progression and lipid deposition in ccRCC. Mechanically, the binding of hsa_circ_0086414 to miR-661, as a miRNA sponge, upregulates the expression of TOB2, wielding an anti-oncogene effect. Importantly, the restored hsa_circ_0086414/TOB2 axis significantly contributed to the elimination of lipid deposition by inhibiting the lipid metabolism regulator PLIN3 in ccRCC cells. ConclusionsOur data highlight the importance of the hsa_circ_0086414/TOB2/PLIN3 axis as a tumor suppressor and lipid eliminator in ccRCC. The positive modulation of the hsa_circ_0086414/TOB2 axis might lead to the development of novel treatment strategies for ccRCC.

Droplet Digital PCR for the Detection and Quantification of Bona Fide CircRNAs.

CircRNAs are covalently closed RNA molecules gaining increasing attention over the years. Initially considered mere splicing errors, circRNAs are now recognized as a novel class of endogenous, conserved RNAs, expressed in many different species. The unique structure, the low levels of expression, and the almost complete sequence overlap with the cognate linear RNA make their detection and quantification challenging. Moreover, it has become crucial to prove the circular nature of the targeted transcript and unequivocally distinguish the circRNA from its linear counterpart. Nowadays, the most widely used technique to quantify circRNA expression is real-time quantitative PCR (qPCR). However, in the particular case of quantification of circles, it shows several technical shortcomings which affect the accuracy of the quantification. To precisely assess circRNA expression level, droplet digital PCR (ddPCR) is rapidly taking over for the more popular qPCR. In this chapter, we describe the detailed procedure based on droplets partitioning to quantify both linear and circRNA abundancy and demonstrate the circularity of the transcript under study with high precision, in a single experiment.

Qingjie Huagong decoction inhibits pancreatic acinar cell pyroptosis by regulating circHipk3/miR-193a-5p/NLRP3 pathway

BackgroundSafer and more effective drugs are needed for the treatment of acute pancreatitis (AP). Qingjie Huagong decoction (QJHGD) has been applied to treat AP for many years and has shown good clinical effects. However, the potential mechanism has not yet been determined. PurposeTo investigate the role and underlying mechanism of the effects of QJHGD on AP both in vitro and in vivo. MethodsQJHGD was characterized by UHPLC-Q-Orbitrap-MS. The protective effect of QJHDG and the underlying mechanism were investigated in MPC-83 cells in vitro. A caerulein-induced AP model was established to evaluate the protective effect of QJHGD in mice. CCK-8 assays were used to detect cell viability. The contents of inflammatory mediators were determined by ELISA. Expression levels of circRNA, miRNA and mRNA were determined by qRT-PCR. Protein expression was determined using Western blot. Pancreatic tissues were assessed by hematoxylin and eosin staining as well as immunohistochemical and immunofluorescence analyses. Pull-down and luciferase activity assays were performed to determine the regulatory relationships of circHipk3, miR-193a-5p and NLRP3. ResultsOur results confirmed that mmu-miR-193a-5p was sponged by mmu-circHipk3, and NLRP3 was a target of miR-193a-5p. In vitro experiments showed that QJHGD enhanced MPC-83 cell viability by regulating circHipk3 sponging mir-193a-5 targeting NLRP3 and inhibiting pyroptosis-related factors. Finally, we showed that QJHGD ameliorated pancreatic tissue injury in AP mice via this pathway. ConclusionThis study demonstrate that QJHDG exerted its anti-AP effects via the circHipk3/miR-193a-5p/NLRP3 pathway, revealing a novel mechanism for the therapeutic effect of QJHDG on AP.

Brief Report: Circular Runt-related transcription factor (circRUNX1) as potential biomarker for cancer recurrence in EGFR mutation-positive surgically resected NSCLC

IntroductionAs recently evidenced by the ADAURA trial, most patients with stages IB to IIIA of resected EGFR-mutant lung adenocarcinoma benefit from osimertinib as adjuvant therapy. Nevertheless, predictive markers of response and recurrence are still an unmet need for more than 10% of these patients. Some circular RNAs (circRNAs) have been reported to play a role in tumor growth and proliferation. In this project, we studied circRNA expression levels in formalin-fixed, paraffin-embedded lung tumor samples to explore their biomarker potential and develop a machine learning (ML)-based signature that could predict the benefit of adjuvant EGFR tyrosine kinase inhibitors in patients with EGFR-mutant NSCLC. MethodsPatients with surgically resected EGFR mutant-positive, stages I to IIIB NSCLC were recruited from February 2007 to December 2015. Formalin-fixed, paraffin-embedded tumor samples were retrospectively collected from those patients with a follow-up period of more than or equal to 36 months (N = 76). Clinicopathologic features were annotated. Total RNA was purified and quantified prior nCounter processing with our circRNA custom panel. Data analysis and ML were performed taking into consideration circRNA expression levels and recurrence-free survival (RFS). RFS was defined from the day of surgery to the day when recurrence was detected radiologically or the death owing to any cause. ResultsOf the 76 patients with EGFR mutation-positive NSCLC included in the study, 34 relapsed within 3 years after resection. The median age of the relapsing cohort was 71.5 (range: 49–89) years. Most patients were nonsmokers (n = 21; 61.8%) and of female sex (n = 21; 61.8%). Most cases (n = 17; 50%) presented an exon 21 mutation, whereas 15 and four patients had an exon 19 and exon 18 mutation, respectively. Differential expression analysis revealed that circRUNX1, along with circFUT8 and circAASDH, was up-regulated in relapsing patients (p < 0.05 and >2 fold-change). A ML-based circRNA signature predictive of recurrence in patients with EGFR mutation-positive NSCLC, comprising circRUNX1, was developed. Our final model including selected 6-circRNA signature with random forest algorithm was able to classify relapsing patients with an accuracy of 83% and an area under the receiver operating characteristic curve of 0.91.RFS was significantly shorter not only for the subgroup of patients with high versus low circRUNX1 expression but also for the group classified as recurrent by the ML circRNA signature when compared with those classified as nonrecurrent. ConclusionsOur findings suggest that circRUNX1 and the presented ML-developed signature could be novel tools to predict the benefit of adjuvant EGFR tyrosine kinase inhibitors with regard to RFS in patients with EGFR-mutant NSCLC. The training and validation phases of our ML signature will be conducted including bigger independent cohorts.

Circ_0004535/miR-1827/CASP8 network involved in type 2 diabetes mellitus with nonalcoholic fatty liver disease.

Diagnostic delay in type 2 diabetes mellitus (T2DM) with nonalcoholic fatty liver disease (NAFLD) patients often leads to a serious public health problem. Understanding the pathophysiological mechanisms of disease will help develop more effective treatments. High-throughput sequencing was used to determine the expression levels of circRNAs, and mRNAs in health controls, T2DM patients, and T2DM with NAFLD patients. Differentially expressed genes (DEcircRs, DEmRs) in T2DM with NAFLD were identified by differential analysis. The miRNAs with targeted relationship with the DEcircRs and DEmRs were respectively predicted to construct a ceRNA regulatory network. In addition, enrichment analysis of DEmRs in the ceRNA network was performed. The expression of important DEcircRs was further validated by quantitative real-time PCR (qRT-PCR). The steatosis was detected in glucose treated LO2 cells by overexpressing circ_0004535, and CASP8. There were 586 DEmRs, and 10 DEcircRs in both T2DM and T2DM with NAFLD patients. Combined with predicted results and differential analysis, the ceRNA networks were constructed. The DEmRs in the ceRNA networks were mainly enriched in Toll-like receptor signaling pathway, and apoptosis. Importantly, dual luciferase experiments validated the targeted binding of hsa_circ_0004535 and hsa-miR-1827 or hsa-miR-1827 and CASP8. qRT-PCR experiments validated that hsa_circ_0004535, and CASP8 was downregulated and hsa-miR-1827 was upregulated expression in peripheral blood of T2DM with NAFLD patients. Abnormal cell morphology, and increased lipid droplet fusion were observed in the glucose treated LO2 cells, overexpression of circ_0004535 and CASP8 ameliorated these changes. Our work provides a deeper understanding of ceRNA mediated pathogenesis of T2DM with NAFLD and provides a novel strategy for treatment.

Hsa_circ_0020134 promotes liver metastasis of colorectal cancer through the miR-183-5p-PFN2-TGF-β/Smad axis

Circular RNAs (circRNAs) are a distinct class of non-coding RNAs that play regulatory roles in the initiation and progression of tumors. With advancements in transcriptome sequencing technology, numerous circRNAs that play significant roles in tumor-related genes have been identified. In this study, we used transcriptome sequencing to analyze the expression levels of circRNAs in normal adjacent tissues, primary colorectal cancer (CRC) tissues, and CRC tissues with liver metastasis. We successfully identified the circRNA hsa_circ_0020134 (circ0020134), which exhibited significantly elevated expression specifically in CRC with liver metastasis. Importantly, high levels of circ0020134 were associated with a poor prognosis among patients. Functional experiments demonstrated that circ0020134 promotes the proliferation and metastasis of CRC cells both in vitro and in vivo. Mechanistically, upregulation of circ0020134 was induced by the transcription factor, PAX5, while miR-183-5p acted as a sponge for circ0020134, leading to partial upregulation of PFN2 mRNA and protein levels, thereby further activating the downstream TGF-β/Smad pathway. Additionally, downregulation of circ0020134 inhibited epithelial-mesenchymal transition (EMT) in CRC cells, which could be reversed by miR-183-5p inhibitor treatment. Collectively, our findings confirm that the circ0020134-miR-183-5p-PFN2-TGF-β/Smad axis induces EMT transformation within tumor cells, promoting CRC proliferation and metastasis, thus highlighting its potential as a therapeutic target for patients with CRC liver metastasis.

Comprehensive analysis of circRNA expression profile and circRNA-miRNA-mRNA network susceptibility to very early-onset schizophrenia

To explore the potential role of circular RNAs (circRNAs) in children developing very early-onset schizophrenia (VEOS). Total RNA was extracted from the plasma samples of 10 VEOS patients and eight healthy controls. Expression profiles of circRNAs, micro RNAs (miRNAs), and messenger RNAs (mRNAs) were analyzed using RNA-seq. The interaction networks between miRNAs and targets were predicted using the miRanda tool. A differentially expressed circRNA-miRNA-mRNA (ceRNA) network was further constructed. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses of the target mRNAs in the ceRNA network were performed to predict the potential functions of their host genes. The patient group and the control group were also compared on the regulatory patterns of circRNAs on mRNAs. 1934 circRNAs were identified from the samples and reported for the first time in schizophrenia. The circRNA expression levels were lower in the VEOS group than in the healthy control group, and 1889 circRNAs were expressed only in the control group. Differential expression analysis (i.e., log2fold change > 1.5, p 0.05) identified 235 circRNAs (1 up-regulated, 234 down-regulated), 11 miRNAs (7 up-regulated, 4 down-regulated), and 2,308 mRNAs (1906 up-regulated, 402 down-regulated) respectively. In VEOS, a ceRNA network with 10 down-regulated circRNA targets, 6 up-regulated miRNAs, and 47 down-regulated mRNAs was constructed. The target genes were involved in the membrane, the signal transduction, and the cytoskeleton and transport pathways. Finally, different expression correlation patterns of circRNA and mRNA in the network were observed between the patient group and the control group. The current research is the first to reveal the differentially expressed circRNAs in the plasma of VEOS patients. A circRNA-miRNA-mRNA network was also conducted in this study. It may be implied that the circRNAs in this network are potential diagnostic biomarkers for VEOS and they play an important role in the onset and development of VEOS symptoms.

Serum circRNA_100199 is a Prognostic Biomarker in Acute Myeloid Leukemia.

An aberrant level of serum microRNA expression has been demonstrated to be a prognostic marker for acute myeloid leukemia (AML). The therapeutic relevance of serum circRNA 100199 remained unknown, however. This research aimed to investigate the probable prognostic significance of serum circRNA_100199 for AML. This study included a total of 200 participants consisting of 114 AML-diagnosed patients and 86 healthy people. Blood samples were taken, and the level of circRNA_100199 in the serum was measured using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to explore its potential clinical significance. Our study demonstrated that circRNA_100199 expression in the serum was substantially higher in AML subjects than in healthy persons. This increase in serum circRNA_100199 levels was particularly noticeable in M4/M5 subtype AML patients, and those with poor cytogenetic risk or higher white blood cell counts. Using receiver operating characteristic (ROC) analysis, AML cases were effectively differentiated from healthy persons based on the level of serum circRNA_100199. Furthermore, it was found that high serum circRNA_100199 expression was strongly linked with shorter survival times and more severe clinical features. Our study also confirmed that high serum circRNA_100199 expression was an independent predictor of relapse-free survival (RFS) and overall survival (OS) in AML patients. Interestingly, the serum expression level of circRNA_100199 was significantly reduced following treatment, and its levels were substantially lower in AML patients who achieved complete remission (CR) than those who did not. Overall, these findings suggest that serum circRNA_100199 has the potential to be a favorable prognostic biomarker for AML.

Expression profiles of circular RNAs in spermatozoa from aging men.

Advanced paternal age (APA) is associated with decreased fertility, but the mechanism underlying APA remains unknown. CircRNAs have been reported to be ideal candidate biomarkers for diagnostic and therapeutic applications in many diseases and are also involved in spermatogenesis. Hence, we aimed to assess the circRNA expression profile of spermatozoa from aging men. We recruited 6 subjects, including 3 in the younger group (men age < 40) and 3 in the APA group (men age ≥ 40). RNA sequencing was exploited to identify the expression profiles of circRNAs between the two groups. The expression levels of circRNAs were validated using real-time quantitative polymerase chain reaction (RT-qPCR). Kyoto Encyclopedia of Genes and Genomes biological pathway analysis and Gene Ontology analysis were performed to evaluate the functions of differentially expressed circRNAs (DE-circRNAs) between the two groups. In total, 18,787 circRNAs were sequenced in the spermatozoa of two groups. Our analysis revealed that there were 1056 downregulated circRNAs and 1228 upregulated circRNAs between the two groups, and KEGG analysis showed they were mainly involved in pathways including the DNA repair signaling pathway, meiotic recombination signaling pathway, and PI3K/AKT signaling pathway. In conclusion, our study suggested that circRNAs play a vital role in spermatozoa from aging men and provided a fresh perspective on the specific regulatory mechanism of spermatozoa from aging men.

Bioinformatics and Experimental Identification of circ_0001535 Associated with Diagnosis and Development of Alzheimer's Disease.

Alzheimer's disease (AD) is a type of disease frequently occurs in the elderly population. Diagnosis and treatment methods for this disease are still lacking, and more research is required. In addition, little is known about the function of the circular RNAs (circRNAs) in AD. In this research, RNA expression data of AD from the Gene Expression Omnibus (GEO) database were downloaded. The expression levels of circRNAs in cerebrospinal fluid samples of healthy participants and AD patients were measured by reverse transcription‑quantitative PCR (RT-qPCR). The diagnosed value of differential expressed circRNAs was analyzed with the Receiver operating characteristic curve (ROC). Pathways related to circ_0001535 were found using gene set enrichment analysis (GSEA) and Metascape. The direct interactions between circ_0001535 and E2F transcription factor 1 (E2F1) or E2F1 and dihydrofolate reductase (DHFR) were verified using Chromatin immunoprecipitation (ChIP) and RNA Binding Protein Immunoprecipitation (RIP) assays. Cell Counting Kit-8 (CCK8) and flow cytometry were used to identify the function of circ_0001535/E2F1/DHFR axis on the proliferation and apoptosis of AD cells. In total, 12 circRNAs have been linked to AD diagnosis. The expression levels of 7 circRNAs differed between AD patients and control groups. Circ_0001535 had the most diagnose value among these circRNAs. Hence, circ_0001535 was regarded as a key circRNA in the present study. E2F1/DHFR axis was predicted to be regulated by circ_0001535. In addition, IP assays experiment results showed that E2F1 could bind to the promoter region of DHFR and be regulated by circ_0001535. In vitro results showed that circ_0001535 overexpression could promote DHFR expression, while E2F1 knock down could inhibit DHFR expression in SH-SY5Y cells. Finally, rescue experiments suggested that circ_0001535 could reduce Aβ25-35-induced SH-SY5Y cell proliferation and facilitate apoptosis through E2F1/DHFR axis. Our research in AD circRNA can offer important information regarding the role of specific circRNAs in the AD environment and point to specific future areas of therapeutic intervention in AD.

Circular RNA expression profiles and circRNA-miRNA-mRNA crosstalk in pre-eclamptic placenta

Objective: To identify the expression profile of circular RNA (circRNA) in placenta of pre-eclampsia (PE) pregnant women by high-throughput sequencing, and to construct the circRNA-microRNA (miRNA)-messenger RNA (mRNA) interaction network, so as to reveal the related pathways and regulatory mechanisms of PE. Methods: The clinical data and placentas of 42 women with PE (PE group) and 30 normal pregnant women (control group) who delivered in West China Second University Hospital from November 2019 to June 2021 were collected. (1) High-throughput sequencing was used to establish the differentially expressed circRNA profiles in placental tissues of 5 pairs of PE group and the control group. (2) Real-time quantitative PCR (qRT-PCR) was used to verify the expression levels of 6 differentially expressed circRNAs in placental tissues of PE group and control group. (3) Bioinformatics analysis was used to predict the target miRNA and analyze the co-expressed mRNA to construct a competitive endogenous RNA (ceRNA) network. The differentially expressed circRNAs were analyzed by Gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) pathways. (4) Logistic regression analysis, Pearson correlation and Kendall's tau-b correlation analysis were used to test the correlation between the three differentially expressed circRNAs and the risk of PE and clinical characteristics. (5) circRNA_05393 was selected for subsequent functional study. Small interfering RNA (siRNA) and overexpression plasmid were used to knock down or increase the expression level of circRNA_05393 in trophoblast cell line HTR-8/SVneo cells, respectively. Transwell assay was used to detect the migration and invasion ability of the trophoblasts in vitro. Cell counting kit-8 assay was used to detect the proliferation ability of the trophoblasts. Results: (1) Seventy-two differentially expressed circRNAs were identified by high-throughput sequencing, of which 35 were up-regulated and 37 were down-regulated. (2) qRT-PCR showed that compared with the control group, circRNA_00673 (1.306±0.168 vs 2.059±0.242; t=2.356, P=0.021) and circRNA_07796 (1.275±0.232 vs 1.954±0.230; t=2.018, P=0.047) were significantly increased, while circRNA_05393 (1.846±0.377 vs 0.790±0.094; t=3.138, P=0.002) was significantly decreased. (3) The circRNA-miRNA-mRNA interaction network contained 3 circRNAs, 8 miRNAs and 53 mRNAs. GO functional annotation analysis showed that the biological process was mainly enriched in iron ion homeostasis, membrane depolarization during action potential and neuronal action potential. In terms of cellular components, they were mainly enriched in cytoskeleton and membrane components. In terms of molecular function, they were mainly enriched in the activity of voltage-gated sodium channel and basic amino acid transmembrane transporter. KEGG pathway enrichment analysis showed that mRNAs in the interaction network were mainly enriched in complement and coagulation cascade, glycine, serine and threonine metabolism, p53 signaling pathway and peroxisome proliferators-activated receptors (PPAR) signaling pathway. (4) Logistic regression analysis showed that down-regulation of circRNA_05393 expression was a risk factor for PE (OR=0.044, 95%CI: 0.003-0.596; P=0.019). Correlation analysis showed that circRNA_05393 was significantly correlated with systolic blood pressure and diastolic blood pressure in PE pregnant women (both P<0.05). (5) Knock down or overexpression of circRNA_05393 significantly reduced or increased the migration and invasion abilities of HTR-8/SVneo cells (all P<0.05), but had no significant effect on the ability of tube formation and proliferation (all P>0.05). Conclusions: The construction of circRNA expression profile in placenta and the exploration of circRNA-miRNA-mRNA interaction network provide the possibility to reveal the regulatory mechanism of specific circRNA involved in PE. Inhibition of circRNA_05393 may induce the progression of PE by reducing the migration and invasion of trophoblasts.

Comprehensive Analysis of Circular RNAs in Endothelial Cells.

Non-coding RNAs constitute a heterogeneous group of molecules that lack the ability to encode proteins but retain the potential ability to influence cellular processes through a regulatory mechanism. Of these proteins, microRNAs, long non-coding RNAs, and more recently, circular RNAs have been the most extensively described. However, it is not entirely clear how these molecules interact with each other. For circular RNAs, the basics of their biogenesis and properties are also lacking. Therefore, in this study we performed a comprehensive analysis of circular RNAs in relation to endothelial cells. We identified the pool of circular RNAs present in the endothelium and showed their spectrum and expression across the genome. Using different computational strategies, we proposed approaches to search for potentially functional molecules. In addition, using data from an in vitro model that mimics conditions in the endothelium of an aortic aneurysm, we demonstrated altered expression levels of circRNAs mediated by microRNAs.