CircDLGAP4 overexpression ameliorates neuronal injury in Parkinson's disease by binding to EIF4A3 and increasing HMGA2 expression.

Parkinson's disease (PD) is a prevalent neurodegenerative disease, and its prevalence increases steadily with age. Circular RNAs (circRNAs) are involved in various neurodegenerative diseases. Here, we aimed to explore the role of circRNA DLG-associated protein 4 (circDLGAP4) in 1-methyl-4-phenylpyridinium ion (MPP+ )-induced neuronal injury in PD. SH-SY5Y cells were treated with MPP+ to establish PD cell models. The levels of circDLGAP4 and high mobility group AT-hook 2 (HMGA2) in SH-SY5Y cells were detected. SH-SY5Y cell viability and apoptosis were detected. The levels of inflammatory damage (IL-1ß, IL-6, TNF-α) and oxidative stress (reactive oxygen species, lactate dehydrogenase, superoxide dismutase, and malondialdehyde)-related factors were measured. The binding of eukaryotic initiation factor 4A3 (EIF4A3) to circDLGAP4 and HMGA2 was analyzed using RNA pull-down or RNA immunoprecipitation. The stability of HMGA2 was detected after actinomycin D treatment, and its effects on neuronal injury were tested. CircDLGAP4 expression was decreased in MPP+ -induced SH-SY5Y cells. CircDLGAP4 upregulation restored cell activity, decreased apoptosis, and reduced inflammatory damage and oxidative stress in PD cell models. CircDLGAP4 bound to EIF4A3 to increase HMGA2 expression and stability. Silencing HMGA2 attenuated the protective effect of circDLGAP4 overexpression. Overall, circDLGAP4 upregulated HMGA2 by recruiting EIF4A3, thus increasing the mRNA stability of HMGA2 and alleviating neuronal injury in PD.

CircDLGAP4 induces autophagy and improves endothelial cell dysfunction in atherosclerosis by targeting PTPN4 with miR-134-5p.

OBJECTIVE: Circular RNAs (circRNAs), a new subgroup of non-coding RNAs in the human transcriptome, are crucial in atherosclerosis (AS). Here, a newly identified circRNA circDLGAP4 was demonstrated to be downregulated in oxidized forms of low-density lipoprotein (ox-LDL)-induced HUVECs. METHODS: This research adopted ox-LDL to stimulate human umbilical vein endothelial cells (HUVECs) to mimic AS in vitro. To further validate the protective action of circDLGAP4 in AS, a mouse model of AS was constructed with a high-fat diet. Functional assays evaluated circDLGAP4 role in AS in vitro and in vivo. Moreover, mechanism assays evaluated association of circDLGAP4/miR-134-5p/PTPN4. RESULTS: CircDLGAP4 was induced to promote cell proliferative behavior and autophagy, inhibit apoptotic and inflammatory activities in ox-LDL-treated HUVECs, and attenuated endothelial barrier function. CircDLGAP4 regulated PTPN4 by directly targeting miR-134-5p. Meanwhile, inhibiting miR-134-5p reduced ox-LDL-induced cell dysfunction. Knockout of PTPN4 reversed circDLGAP4 overexpression or miR-134-5p downregulation in vitro. In addition, reducing circDLGAP4 or overexpressing miR-134-5p increased the red atherosclerotic plaque and lesion area of AS mice, reduced autophagy level, and promoted the release of inflammatory cytokines. CONCLUSION: This study extends the role of circRNA in AS by inducing autophagy and improving endothelial dysfunction in AS via the circDLGAP4/miR-134-5p/PTPN4 axis.

Celastrol assuages oxygen-glucose deprivation and reoxygenation-induced damage in human brain microvascular endothelial cells through the circDLGAP4/miR-6085/GDF11 pathway.

The effect of Celastrol on cerebral ischemia-reperfusion remains unknown. The study aims to explore the role of circular RNA DLGAP4 (circDLGAP4) in cerebral ischemia-reperfusion and the underlying mechanism. Ischemia-reperfusion (I/R) injury of human brain microvascular endothelial cells (HBMECs) was induced by oxygen-glucose deprivation and reoxygenation (OGD/R). Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting analysis were performed to detect the expression of circDLGAP4, microRNA-6085 (miR-6085), growth differentiation factor 11 (GDF11), B-cell lymphoma-2 (BCL2) and BCL2-associated x protein (BAX). Cell viability, proliferation, and apoptosis were analyzed by cell counting kit-8, 5-Ethynyl-2'-deoxyuridine and flow cytometry analysis. Oxidative stress was analyzed by evaluating the levels of Malondialdehyde (MDA) and Reactive Oxygen Species (ROS) and the activity of Superoxide Dismutase (SOD). The associations among circDLGAP4, miR-6085 and GDF11 were identified by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. Celastrol reduced OGD/R-induced inhibition of circDLGAP4 expression in HBMECs. Celastrol treatment protected HBMECs from OGD/R-induced cell proliferation inhibition and apoptosis and oxidative stress promotion; however, circDLGAP4 depletion attenuated these effects. CircDLGAP4 acted as a sponge for miR-6085, and miR-6085 mimics restored circDLGAP4-mediated effects in OGD/R-stimulated HBMECs. In addition, GDF11 was identified as a targte of miR-6085, and participated in the regulation of miR-6085 to OGD/R-induced HBMEC damage. Further, circDLGAP4 absence inhibited GDF11 expression by interacting with miR-6085 under Celastrol treatment. Celastrol ameliorated OGD/R-induced HBMEC apoptosis and oxidative stress by circDLGAP4/miR-6085/GDF11 pathway, supporting the use of Celastrol as a therapeutic agent for cerebral infarction.

Diagnosis and prognostic value of circDLGAP4 in acute ischemic stroke and its correlation with outcomes.

Rationale and aims: Circular RNAs are a subclass of noncoding RNAs in mammalian cells and highly expressed in the central nervous system. Although their physiological functions are not yet completely defined, they are thought to promise as stroke biomarkers because of their stability in peripheral blood.Sample Size Estimate: 222 participants. Methods and design: The plasma of patients with acute ischemic stroke (AIS) (n = 111) and non-stroke controls (n = 111) from November 2017 to February 2019 were enrolled in our research. The expression of circDLGAP4 in plasma was evaluated using real-time PCR. Study outcomes: In patients with AIS, circDLGAP4 was significantly decreased in comparison with non-stroke controls. The CircDLGAP4 level had a significant AUC of 0.7896 with 91.72% sensitivity and 64.83% specificity in diagnosing AIS. Furthermore, the circDLGAP4 level was related to smoking history and previous transient ischemic attack/stroke/myocardial infarction in all samples. The change rate in circDLGAP4 within the first 7 days showed an AUC curve of 0.960 in predicting an stroke outcome. Conclusion: circDLGAP4 could serve as biomarker for AIS diagnosis and prediction of stroke outcomes.

circDlgap4 Alleviates Cerebral Ischaemic Injury by Binding to AUF1 to Suppress Oxidative Stress and Neuroinflammation.

Ischaemic stroke is one of the most common causes of mortality and morbidity.circDlgap4 has been implicated in ischemia/reperfusion injury through an unknown mechanism. Here, we studied the function of circDlgap4/AUF1 in ischaemic stroke and its underlying molecular mechanism. N2a cells and primary mouse cortical neurons were subjected to OGD to mimic neuronal injury during ischemia. BV-2 cells were treated with LPS to mimic neuroinflammation. The MTT assay was used to assess cell viability, while flow cytometry was used to measure cell apoptosis. qRT-PCR, western blotting, immunohistochemistry, and immunostaining were employed to determine the levels of circDlgap4, AUF1, NRF2/HO-1, proinflammatory cytokines, NF-κB pathway-related proteins, and IBA-1. RIP and RNA pulldown assays were employed to validate the interactions of circDlgap4/AUF1, AUF1/NRF2, and AUF1/cytokine mRNAs. mRNA degradation was used to determine the effects on mRNA stability. The tMCAO model was used as an in vivo model of ischaemic stroke. TCC staining and neurological scoring were performed to evaluate ischaemic injury. circDlgap4 was decreased following OGD and during tMCAO. circDlgap4 overexpression inhibited OGD-induced cell death and oxidative stress and LPS-induced increases in proinflammatory cytokines by increasing NRF2/HO-1. Knockdown of AUF1 blocked the effects of circDlgap4 overexpression. Mechanistically, RIP, RNA pulldown, and mRNA degradation assay results showed circDlgap4/AUF1/NRF2 mRNA formed a complex to stabilize NRF2 mRNA. Furthermore, AUF1 directly interacted with TNF-α, IL-1ß, and COX-2 mRNAs, and circDlgap4/AUF1 binding promoted the degradation of these mRNAs. Finally, circDlgap4 ameliorated ischaemic injury in vivo. circDlgap4 alleviates ischaemic stroke injury by suppressing oxidative stress and neuroinflammation by binding to AUF1.

Exosome-delivered circular RNA DLGAP4 induces chemoresistance via miR-143-HK2 axis in neuroblastoma.

Accumulating evidence validates that aerobic glycolysis is involved in chemotherapy resistance in human malignant tumors. In the present study, we explored the role of exosome-delivered circular RNA DLGAP4 (circDLGAP4), a novel identified circRNAs, in the chemoresistance of neuroblastoma (NB) cells. Our study demonstrated that doxorubicin-resistant cells expressed higher HK2, accompanied with enhanced glycolysis. In addition, circDLGAP4 was validated to act as a sponge for the HK2-targeting miR-143. As a molecular cargo, exosomes were found to deliver circDLGAP4 from doxorubicin-resistant cells to the sensitive cells. Functionally, exosomal circDLGAP4 enhanced glycolysis and drug resistance via regulating miR-143 and HK2 in NB cells. Consistently, upregulation of HK2 induced by circDLGAP4 or miR-143 inhibitors produced the similar malignant transformation in NB cells. However, knockdown of circDLGA P4 could reversed the drug resistance in the recipient cells. In conclusion, these findings demonstrate that exosome-delivered circDLGAP4 promotes the glycolysis, proliferation, and invasion of sensitive NB cells by regulating miR-143 and HK2, providing a novel link between drug resistance and circDLGAP4/miR-143/HK2 axis in drug-resistant NB.

CircDLGAP4 overexpression relieves oxygen-glucose deprivation-induced neuronal injury by elevating NEGR1 through sponging miR-503-3p.

Circular RNAs (circRNAs) have been reported to play vital regulatory roles in human diseases. However, the functions of circRNAs in ischemic stroke (IS) are limited. In this study, we aimed to explore the functions and mechanisms of circRNA DLG associated protein 4 (circDLGAP4) in IS development. Oxygen-glucose deprivation (OGD)-treated HCN-2 cells were used to mimic IS environment in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was used to detect the levels of circDLGAP4, microRNA-503-3p (miR-503-3p) and neuronal growth regulator 1 (NEGR1) mRNA. RNase R assay was conducted to analyze the stability of circDLGAP4. Cell Counting Kit-8 (CCK-8) assay and flow cytometry analysis were adopted for cell viability and death, respectively. Western blot assay was performed for protein levels. Enzyme-linked immunosorbent assay (ELISA) kits were used to examine the concentrations of inflammatory cytokines. Dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull-down assay were employed to analyze the relationships among circDLGAP4, miR-503-3p and NEGR1. CircDLGAP4 level was declined in HCN-2 cells after OGD treatment. CircDLGAP4 overexpression promoted cell viability and suppressed cell death and inflammatory cytokine concentrations in OGD-treated HCN-2 cells. CircDLGAP4 acted as the sponge for miR-503-3p and the impacts of circDLGAP4 overexpression on cell viability, death and inflammation in OGD-treated HCN-2 cells were reversed by miR-503-3p elevation. Furthermore, NEGR1 was the target gene of miR-503-3p. MiR-503-3p inhibition ameliorated OGD-induced HCN-2 cell impairments, but NEGR1 knockdown abolished the effects. CircDLGAP4 alleviated OGD-induced HCN-2 cell damage by regulating miR-503-3p/NEGR1 axis.

Circular RNA circDLGAP4 is involved in lung cancer development through modulating microRNA-143/CDK1 axis.

To investigate the role of circular RNA DLGAP4 (circDLGAP4) in lung cancer. circDLGAP4 expression was detected in lung cancer tissues and cell lines by PCR. The correlation between circDLGAP4 and clinicopathological characteristics of lung cancer patients was investigated. Moreover, the influences of depression of circDLGAP4 on the biological processes biological processes of lung cancer cells were explored in vitro. In addition, whether circDLGAP4 regulated lung cancer cell biological processes by sponging microRNA-143 (miR-143) to regulate cyclin-dependent kinase 1 (CDK1) expression was explored and verified in another lung cell line. CircDLGAP4 expression was remarkably elevated in lung cancer tissues and was significantly corrected with TNM stage and tumor metastasis. Suppression of circDLGAP4 inhibited the biological performances of lung cancer cells. Also, there was a negative regulatory relationship between circDLGAP4 and miR-143. Inhibition of miR-143 alleviated the influences of circDLGAP4 depression on lung cancer cell biological processes. Moreover, CDK1 was discovered as a target of miR-143, and miR-143 was involved in the process of lung cancer cell biological processes through targeting CDK1. Our findings reveal that circular RNA circDLGAP4 is involved in lung cancer development through modulating microRNA-143/CDK1 axis. circDLGAP4 may serve as a potential biomarker for the diagnosis or treatment of lung cancer.

The possible protective role of circDLGAP4 from peripheral blood in coronary heart disease / 临床检验杂志

Objective@#To investigate the expression and clinical significance of circDLGAP4 from peripheral blood in coronary heart disease (CAD). @*Methods@#The relative expression level of circDLGAP4 in peripheral blood leukocytes (PBLs) from 142 CAD patients and 169 healthy controls were detected by real-time PCR. Logistic regression, Spearman correlation and multivariate regression analysis were used to investigate the correlation of circDLGAP4 with CAD. THP-1 macrophages were treated with oxidized low density lipoprotein (ox-LDL) to construct an atherosclerotic foam cell model. The levels of circDLGAP4 mRNA were detected at different time points. @*Results@#The mRNA expression of circDLGAP4 in PBLs of CAD patients was significantly decreased compared with controls (P=0.019). With increased unit (2 -ΔCt ) of circDLGAP4 expression, the risk of CAD occurrence reduced by 41.6% (adjusted OR=0.584, 95% CI: 0.394-0.866, P=0.007). The expression of circDLGAP4 was negatively correlated with T2DM history (β=-0.182，P=0.030). The level of circDLGAP4 in ox-LDL-treated THP-1 macrophages was decreased in a time-dependent manner. @*Conclusion@#The expression of circDLGAP4 was significantly decreased in PBLs of CAD patients and THP-1 macrophages-derived foam cells, and might be a protective factor in the pathophysiology of CAD.