Dorsal root ganglion-derived exosomes deteriorate neuropathic pain by activating microglia via the microRNA-16-5p/HECTD1/HSP90 axis.

BACKGROUND: The activated microglia have been reported as pillar factors in neuropathic pain (NP) pathology, but the molecules driving pain-inducible microglial activation require further exploration. In this study, we investigated the effect of dorsal root ganglion (DRG)-derived exosomes (Exo) on microglial activation and the related mechanism. METHODS: A mouse model of NP was generated by spinal nerve ligation (SNL), and DRG-derived Exo were extracted. The effects of DRG-Exo on NP and microglial activation in SNL mice were evaluated using behavioral tests, HE staining, immunofluorescence, and western blot. Next, the differentially enriched microRNAs (miRNAs) in DRG-Exo-treated microglia were analyzed using microarrays. RT-qPCR, RNA pull-down, dual-luciferase reporter assay, and immunofluorescence were conducted to verify the binding relation between miR-16-5p and HECTD1. Finally, the effects of ubiquitination modification of HSP90 by HECTD1 on NP progression and microglial activation were investigated by Co-IP, western blot, immunofluorescence assays, and rescue experiments. RESULTS: DRG-Exo aggravated NP resulting from SNL in mice, promoted the activation of microglia in DRG, and increased neuroinflammation. miR-16-5p knockdown in DRG-Exo alleviated the stimulating effects of DRG-Exo on NP and microglial activation. DRG-Exo regulated the ubiquitination of HSP90 through the interaction between miR-16-5p and HECTD1. Ubiquitination alteration of HSP90 was involved in microglial activation during NP. CONCLUSIONS: miR-16-5p shuttled by DRG-Exo regulated the ubiquitination of HSP90 by interacting with HECTD1, thereby contributing to the microglial activation in NP.

Identification and Functional Analysis of Rare HECTD1 Missense Variants in Human Neural Tube Defects.

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased secretion of extracellular heat shock protein 90 (eHSP90) to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with human disease and suggest that sequence variation in HECTD1 may play a role in the etiology of human NTDs.

Nucleoporin 93, a new substrate of the E3 ubiquitin protein ligase HECTD1, promotes esophageal squamous cell carcinoma progression.

Nucleoporin 93 (NUP93) is an important component of the nuclear pore complex, exhibiting pro-tumorigenic properties in some cancers. However, its function in esophageal squamous cell carcinoma (ESCC) has not been elucidated. This study aimed to investigate the effects of NUP93 in ESCC and the underlying mechanisms involved. Through analysis of public human cancer datasets, we observed higher expression of NUP93 in esophageal cancer tissues than in normal tissues. Stable ESCC cell lines with NUP93 overexpression or knockdown were established by lentiviral vector transduction and puromycin selection. NUP93 knockdown suppressed the proliferation, colony formation, cell cycle transition, migration, and invasion of ESCC cells, while the overexpression of NUP93 displayed opposite effects. NUP93 positively regulated epithelial-mesenchymal transition and AKT signaling transduction in ESCC cells. In addition, NUP93 increased the expression of programmed death ligand 1 (PD-L1) in ESCC cells and attenuated NK cell-mediated lysis of ESCC cells. In vivo experiments demonstrated that NUP93 promotes the growth of ESCC in nude mice, enhances Ki67 and PD-L1 expression, and promotes AKT signaling transduction in xenografts. Mechanistically, we demonstrated that the HECT domain E3 ubiquitin protein ligase 1 (HECTD1) contributes to the ubiquitination and degradation of NUP93 and acts as a tumor suppressor in ESCC. To conclude, this study has shown that NUP93 has pro-tumor properties in ESCC and that HECTD1 functions as an upstream regulator of NUP93 in ESCC. These findings may contribute to the investigation of potential therapeutic targets in ESCC.

Centriolin interacts with HectD1 in a cell cycle dependent manner.

OBJECTIVE: The centrosome is universally recognized as the microtubule organizing center of animal cells, but emerging evidence suggests that it has other important functions including primary cilia formation, DNA damage checkpoints, and cell cycle progression. Despite this, the role of individual components of the centrosome remains unclear. Previous studies suggest that one component, centriolin, has an important function in cytokinesis and cell cycle progression, although its exact role in these processes is not known. To determine how centriolin influences the progression through the cell cycle, we sought to identify interacting partners that may be involved in regulating its function. RESULTS: This study provides evidence that the ubiquitin E3 ligase HectD1 binds to centriolin and that this association likely accounts for our observation that HectD1 co-localizes with centriolin at the centrosome during mitosis. In addition to its centrosomal localization, we also show that the expression of HectD1 fluctuates throughout the cell cycle, with the highest levels during mitosis, coinciding with a marked reduction in centriolin expression. We propose that the interaction between HectD1 and centriolin may be necessary for normal cell cycle progression and we speculate that this function may involve HectD1-mediated degradation of centriolin.

Circ-Bnc2 alleviates neuroinflammation in LPS-stimulated microglial cells to inhibit neuron cell apoptosis through regulating miR-497a-5p/HECTD1 axis.

BACKGROUND: Neuroinflammation caused by microglia cells activation and the apoptosis of neuron cells are associated with the occurrence of depression. Circ-Bnc2 has been shown to be significantly downregulated in depression mice, but its role in the progression of depression remains unclear. METHODS: Lipopolysaccharide (LPS) was used to treat BV2 microglial cells to induce neuroinflammation. The expression of circ-Bnc2, microRNA (miR)-497a-5p, and HECT domain E3 ubiquitin protein ligase 1 (HECTD1) was measured by quantitative real-time PCR. The protein levels of neuroinflammation markers, apoptosis markers, and HECTD1 were determined by western blot analysis. ELISA assay was used to examine the concentrations of inflammatory factors. After HT22 cells were cultured with the conditioned medium of LPS-induced BV2 cells, the proliferation and apoptosis of HT22 cells were assessed by cell counting kit 8 assay, EdU assay, and flow cytometry. In addition, the interaction between miR-497a-5p and circ-Bnc2 or HECTD1 was confirmed by dual-luciferase reporter assay, RIP assay, and RNA pull-down assay. RESULTS: Our data showed that circ-Bnc2 was lowly expressed in LPS-induced BV2 cells. Function experiments suggested that circ-Bnc2 could inhibit LPS-induced neuroinflammation in BV2 cells to repress HT22 cell apoptosis and promote proliferation. Circ-Bnc2 could sponge miR-497a-5p, and the neuroprotective function of circ-Bnc2 could be reversed by miR-497a-5p overexpression. Additionally, miR-497a-5p could target HECTD1. miR-497a-5p inhibitor could alleviate LPS-induced neuroinflammation in BV2 cells and reduce HT22 cell apoptosis, which also could be reversed by HECTD1 knockdown. Moreover, circ-Bnc2 had a positive regulation on HECTD1 expression by sponging miR-497a-5p. CONCLUSION: In summary, our results confirmed that circ-Bnc2 could inhibit neuroinflammation and neuron cell apoptosis by regulating miR-497a-5p/HECTD1 axis, suggesting that circ-Bnc2 might be a potential target for depression treatment.

CircUCK2 regulates HECTD1-mediated endothelial-mesenchymal transition inhibition by interacting with FUS and protects the blood-brain barrier in ischemic stroke.

Ischemic stroke (IS) has become a cerebrovascular disease of widespread concern. Overexpression of circUCK2 alleviates neuronal damage in IS. However, the specific regulatory mechanisms of circUCK2 are not fully understood. In this study, we found that circUCK2 and HECT domain E3 ubiquitin ligase 1 (HECTD1) were downregulated in IS models in vitro and in vivo. Overexpression of circUCK2 or HECTD1 inhibited endothelial-mesenchymal transition (EndoMT) and protected the blood-brain barrier (BBB) in transient middle cerebral artery occlusion mice from damage. It was further discovered that circUCK2 regulated HECTD1 expressions by interacting with fused in sarcoma (FUS). Moreover, FUS overexpression partially restored the effect of circUCK2 on EndoMT, and overexpression of HECTD1 weakened the effect of FUS on EndoMT. Collectively, circUCK2 upregulates the expression of HECTD1 by combining with FUS and inhibits EndoMT to alleviate BBB damage in IS both in vivo and in vitro.

Low HECTD1 mRNA expression is associated with poor prognosis and may be correlated with increased mitochondrial respiratory function in breast cancer.

HECT domain E3 ubiquitin ligase 1 (HECTD1) has been reported to be a negative regulator of epithelial-mesenchymal transition and to decrease breast cancer invasion and metastasis. However, the clinical significance and detailed role of HECTD1 in breast cancer remain elusive. We investigated the role of HECTD1 in two large breast cancer cohorts at our institution and The Cancer Genome Atlas using mRNA expression and bioinformatics analysis. We also examined the prognostic significance of HECTD1 mRNA expression by multivariate analysis and HECTD1 protein expression by immunohistochemistry using our cohort. HECTD1 mRNA expression was significantly lower in breast cancer tissues compared with those in adjacent normal tissues (P<0.001). HECTD1 mRNA expression levels also differed among breast cancer subtypes. Decreased HECTD1 mRNA expression was significantly associated with aggressive tumor characteristics, including large tumor size and high histological grade. HECTD1 mRNA expression was inversely associated with mitochondrial cellular respiratory function (oxidative phosphorylation (P<0.001, FDR q-value <0.001) the respiratory chain complex (P<0.001, FDR q-value <0.001) and reactive oxygen species (P<0.001, FDR q-value <0.001), but not with epithelial-mesenchymal transition, in breast cancer tissues. Low expression of HECTD1 mRNA was associated with shorter disease-free survival (log-rank: P=0.013) and overall survival (log-rank: P=0.038) in breast cancer patients. Multivariate analysis also identified low HECTD1 mRNA expression level as an independent risk factor for disease-free (hazard ratio: 1.54, 95% confidence interval: 1.11-2.13, P=0.009) and overall (hazard ratio: 1.50, 95% confidence interval: 1.01-2.24, P=0.046) survival among breast cancer patients. There was no association of HECTD1 protein expression with HECTD1 mRNA expression and prognosis. In conclusion, we identified low expression of HECTD1 mRNA as an independent poor prognostic factor in breast cancer and showed that HECTD1 mRNA expression was inversely correlated with genes involved in mitochondrial cellular respiratory function in breast cancer.

STRIPAK regulation of katanin microtubule severing in the Caenorhabditis elegans embryo.

Microtubule severing plays important role in cell structure and cell division. The microtubule severing protein katanin, composed of the MEI-1/MEI-2 subunits in Caenorhabditis elegans, is required for oocyte meiotic spindle formation; however, it must be inactivated for mitosis to proceed as continued katanin expression is lethal. Katanin activity is regulated by 2 ubiquitin-based protein degradation pathways. Another ubiquitin ligase, HECD-1, the homolog of human HECTD1/HECT domain E3 ubiquitin protein ligase 1, regulates katanin activity without affecting katanin levels. In other organisms, HECD-1 is a component of the striatin-interacting kinase phosphatase complex, which affects cell proliferation and a variety of signaling pathways. Here we conducted a systematic screen of how mutations in striatin-interacting kinase phosphatase components affect katanin function in C. elegans. Striatin-interacting kinase phosphatase core components (FARL-11, CASH-1, LET-92, and GCK-1) were katanin inhibitors in mitosis and activators in meiosis, much like HECD-1. By contrast, variable components (SLMP-1, OTUB-2) functioned as activators of katanin activity in mitosis, indicating they may function to alter striatin-interacting kinase phosphatase core function. The core component CCM-3 acted as an inhibitor at both divisions, while other components (MOB-4, C49H3.6) showed weak interactions with katanin mutants. Additional experiments indicate that katanin may be involved with the centralspindlin complex and a tubulin chaperone. HECD-1 shows ubiquitous expression in the cytoplasm throughout meiosis and early development. The differing functions of the different subunits could contribute to the diverse functions of the striatin-interacting kinase phosphatase complex in C. elegans and other organisms.

Vitamin C Inhibits Ubiquitination of Glutamate Transporter 1 (GLT-1) in Astrocytes by Downregulating HECTD1.

Excitatory neurotoxicity caused by the accumulation of glutamate in the synaptic cleft is an important cause of Parkinson's disease (PD). Astrocyte glutamate transporter 1 (GLT-1) is the main transporter responsible for transporting glutamate, and investigations toward the regulation of GLT-1 in astrocytes can reveal important insights. Vitamin C (VC) has important protective effects on the brain, but its effect on the regulation of GLT-1 expression is unclear. The purpose of this study was to explore any regulatory effect of VC on GLT-1 expression in astrocytes and to clarify the possible mechanism of such regulation. We found that GLT-1 expression was impaired in 1-methyl-4-phenylpyridinium iodide (MPP+)-treated astrocytes, and the transport capacity for glutamate was significantly reduced. Pretreatment with VC restored the GLT-1 expression in the MPP+-treated astrocytes. Intraperitoneal VC administration in a PD murine model confirmed that GLT-1 expression was restored in midbrain tissue. The VC-dependent rescue of GLT-1 expression in the MPP+-treated astrocytes was shown to be due to inhibition of GLT-1 ubiquitination. Transcriptome sequence analysis revealed a number of differentially expressed genes as a result of VC treatment on MPP+-treated astrocytes, including the downregulation of HECT Domain E3 ubiquitin protein ligase 1 (Hectd1). After knocking down Hectd1, the impaired GLT-1 expression caused by MPP+ was alleviated, while overexpression of Hectd1 significantly reduced the expression of GLT-1. After overexpression of Hectd1, VC could no longer increase GLT-1 expression of MPP+-treated astrocytes, indicating that HECTD1 is essential for VC regulation of GLT-1. Thus, VC reduces the ubiquitination of GLT-1 in astrocytes by inhibiting the expression of HECTD1. Our findings have identified a novel mechanism by which VC regulates the expression of GLT-1 in astrocytes.

Hsa_circ_0090002 regulates miR-186-5p/HECTD1 axis to mediate brain microvascular endothelial cell dysfunction.

BACKGROUND: Ischemic injury is a common nervous disease associated with the dysfunction of human brain microvascular endothelial cells (HBMECs). Circular RNAs (circRNAs) have key roles in ischemic injury. This research aims to investigate the role and mechanism of circ_0090002 in ischemic injury. METHODS: HBMECs were stimulated by oxygen-glucose deprivation (OGD). Circ_0090002, microRNA-186-5p (miR-186-5p), and homologous to the E6-AP Carboxyl Terminus domain E3 ubiquitin ligase 1 (HECTD1) levels were detected by quantitative reverse transcription polymerase chain reaction or Western blotting. Cell viability and migration were determined using Cell Counting Kit-8 (CCK-8) assay and wound healing assay. Flow cytometry and caspase-3 activity assay were used for apoptosis analysis. The oxidative injury and cell toxicity were assessed by reactive oxygen species (ROS) and lactic dehydrogenase (LDH) release assay kits, respectively. The interaction was investigated by dual-luciferase reporter, RNA immunoprecipitation (RIP) and RNA pull-down assays. In vivo assay was performed in rats. RESULTS: Circ_0090002 expression was reduced in OGD-stimulated HBMECs. Circ_0090002 overexpression attenuated OGD-induced reduction of cell viability and migration but elevation of apoptosis, oxidative stress and cell toxicity. Circ_0090002 sponged miR-186-5p and miR-186-5p overexpression reversed the protective role of circ_0090002 against the OGD-induced cell injury. MiR-186-5p targeted HECTD1miR-186-5p knockdown mitigated cell damages in by increasing HECTD1 level in OGD-treated HBMECs. Circ_0090002 could upregulate the HECTD1 expression via regulating miR-186-5p. Circ_0090002 inhibited infarct volume of brain in rats. CONCLUSION: These results demonstrated that circ_0090002 mitigated OGD-induced cell dysfunction in HBMECs by targeting the miR-186-5p/HECTD1 axis.

Overexpression of BIRC6 driven by EGF-JNK-HECTD1 signaling is a potential therapeutic target for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is an aggressive and highly lethal disease. The lack of targeted therapies and poor patient outcome have fostered efforts to discover new molecular targets to treat patients with TNBC. Here, we showed that baculoviral IAP repeat containing 6 (BIRC6) is overexpressed and positively correlated with epidermal growth factor (EGF) receptor (EGFR) in TNBC cells and tissues and that BIRC6 overexpression is associated with poor patient survival. Mechanistic studies revealed that BIRC6 stability is increased by EGF-JNK signaling, which prevents ubiquitination and degradation of BIRC6 mediated by the E3 ubiquitin ligase HECTD1. BIRC6 in turn decreases SMAC expression by inducing the ubiquitin-proteasome pathway, thereby antagonizing apoptosis and promoting the proliferation, colony formation, tumorsphere formation, and tumor growth capacity of TNBC cells. Therapeutically, the PEGylated cationic lipid nanoparticle (pCLN)-assisted delivery of BIRC6 small interfering RNA (siRNA) efficiently silences BIRC6 expression in TNBC cells, thus suppressing TNBC cell growth in vitro and in vivo, and its antitumor activity is significantly superior to that of the EGFR inhibitor gefitinib. Our findings identify an important regulatory mechanism of BIRC6 overexpression and provide a potential therapeutic option for treating TNBC.

Circular RNA circ_HECTD1 regulates cell injury after cerebral infarction by miR-27a-3p/FSTL1 axis.

Cerebral infarction is a common cerebrovascular disease caused by neural cell injury, with high mortality worldwide. Circular RNAs HECT domain E3 ubiquitin-protein ligase 1 (circ_HECTD1) has been reported to be related to the oxygen-glucose deprivation/reperfusion (OGD/R)-caused neuronal damage in cerebral ischemia. This study is designed to explore the role and mechanism of circ_HECTD1 in OGD/R-induced cell injury in cerebral ischemia. Circ_HECTD1, microRNA-27a-3p (miR-27a-3p), and Follistatin-like 1 (FSTL1) level were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The localization of circ_HECTD1 was analyzed by subcellular fractionation assay. Cell proliferative ability and apoptosis were assessed by 5-ethynyl-2'-deoxyuridine (EdU), 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), and flow cytometry assays. The protein levels of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), Cleaved poly-ADP-ribose polymerase (PARP), and FSTL1 were examined by western blot assay. The binding relationship between miR-27a-3p and circ_HECTD1 or FSTL1 was predicted by starbase 3.0 then verified by a dual-luciferase reporter assay. Circ_HECTD1 and FSTL1 were highly expressed, and miR-27a-3p was decreased in OGD/R-treated HT22 cells. Moreover, circ_HECTD1 knockdown could boost cell proliferative ability and repress apoptosis in OGD/R-triggered HT22 cells in vitro. Mechanical analysis discovered that circ_HECTD1 could regulate FSTL1 expression by sponging miR-27a-3p. Circ_HECTD1 deficiency could mitigate OGD/R-induced HT22 cell damage by modulating the miR-27a-3p/FSTL1 axis, providing a promising therapeutic target for cerebral infarction treatment.

The deubiquitinase TRABID stabilizes the K29/K48-specific E3 ubiquitin ligase HECTD1.

Ubiquitin is a versatile posttranslational modification, which is covalently attached to protein targets either as a single moiety or as a ubiquitin chain. In contrast to K48 and K63-linked chains, which have been extensively studied, the regulation and function of most atypical ubiquitin chains are only starting to emerge. The deubiquitinase TRABID/ZRANB1 is tuned for the recognition and cleavage of K29 and K33-linked chains. Yet, substrates of TRABID and the cellular functions of these atypical ubiquitin signals remain unclear. We determined the interactome of two TRABID constructs rendered catalytic dead either through a point mutation in the catalytic cysteine residue or through removal of the OTU catalytic domain. We identified 50 proteins trapped by both constructs and which therefore represent candidate substrates of TRABID. The E3 ubiquitin ligase HECTD1 was then validated as a substrate of TRABID and used UbiCREST and Ub-AQUA proteomics to show that HECTD1 preferentially assembles K29- and K48-linked ubiquitin chains. Further in vitro autoubiquitination assays using ubiquitin mutants established that while HECTD1 can assemble short homotypic K29 and K48-linked chains, it requires branching at K29/K48 in order to achieve its full ubiquitin ligase activity. We next used transient knockdown and genetic knockout of TRABID in mammalian cells in order to determine the functional relationship between TRABID and HECTD1. This revealed that upon TRABID depletion, HECTD1 is readily degraded. Thus, this study identifies HECTD1 as a mammalian E3 ligase that assembles branched K29/K48 chains and also establishes TRABID-HECTD1 as a DUB/E3 pair regulating K29 linkages.

Involvement of HECTD1 in LPS-induced astrocyte activation via σ-1R-JNK/p38-FOXJ2 axis.

BACKGROUND: Astrocytes participate in innate inflammatory responses within the mammalian central nervous system (CNS). HECT domain E3 ubiquitin protein ligase 1 (HECTD1) functions during microglial activation, suggesting a connection with neuroinflammation. However, the potential role of HECTD1 in astrocytes remains largely unknown. RESULTS: Here, we demonstrated that HECTD1 was upregulated in primary mouse astrocytes after 100 ng/ml lipopolysaccharide (LPS) treatment. Genetic knockdown of HECTD1 in vitro or astrocyte-specific knockdown of HECTD1 in vivo suppressed LPS-induced astrocyte activation, whereas overexpression of HECTD1 in vitro facilitated LPS-induced astrocyte activation. Mechanistically, we established that LPS activated σ-1R-JNK/p38 pathway, and σ-1R antagonist BD1047, JNK inhibitor SP600125, or p38 inhibitor SB203580 reversed LPS-induced expression of HECTD1, thus restored LPS-induced astrocyte activation. In addition, FOXJ2 functioned as a transcription factor of HECTD1, and pretreatment of primary mouse astrocytes with BD1047, SB203580, and SP600125 significantly inhibited LPS-mediated translocation of FOXJ2 into the nucleus. CONCLUSIONS: Overall, our present findings suggest that HECTD1 participates in LPS-induced astrocyte activation by activation of σ-1R-JNK/p38-FOXJ2 pathway and provide a potential therapeutic strategy for neuroinflammation induced by LPS or any other neuroinflammatory disorders.

HectD1 controls hematopoietic stem cell regeneration by coordinating ribosome assembly and protein synthesis.

Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.

Downregulation of circular RNA HECTD1 induces neuroprotection against ischemic stroke through the microRNA-133b/TRAF3 pathway.

AIMS: Circular RNAs (circRNAs) have been shown to play crucial roles in various biological processes and human diseases. However, their exact functions in ischemic stroke remain largely unknown. In this study, we explored the functional role of circRNA HECTD1 (circ-HECTD1) and its underlying mechanism in cerebral ischemia/reperfusion injury. METHODS: Mouse middle cerebral artery occlusion (MCAO) model and oxygen-glucose deprivation (OGD) model in HT22 cells were used to mimic the cerebral ischemia/reperfusion injury. Brain infarct volume, flow cytometry, caspase 3 activity, NF-κB activity, and TUNEL staining were performed to evaluate the function of circ-HECTD1. Luciferase report assay was used to explore the regulatory mechanism. FINDINGS: The results showed that the expression of circ-HECTD1 and tumor necrosis factor receptor-associated factor 3 (TRAF3) was remarkably up-regulated, while miR-133b was down-regulated in oxygen-glucose deprivation (OGD)-induced HT22 cells and mouse middle cerebral artery occlusion (MCAO) model. circ-HECTD1 knockdown relieved OGD-caused neuronal cell death in vitro. Simultaneously, circ-HECTD1 knockdown improved cerebral infarction volume and neuronal apoptosis in MCAO mice. circ-HECTD1 was able to negatively regulate the expression of miR-133b, and TRAF3 is one of the targets of miR-133b. Upregulation of miR-133b inhibited the expression of TRAF3 in OGD-stimulated cells, whereas circ-HECTD1 upregulation reversed this effect. Furthermore, upregulation of miR-133 was able to inhibit OGD-caused cell apoptosis and NF-κB activation, whereas upregulation of circ-HECTD1 attenuated these effects of miR-133b mimics. SIGNIFICANCE: Taken together, circ-HECTD1 knockdown inhibited the expression of TRAF3 by targeting miR-133b, thereby attenuating neuronal injury caused by cerebral ischemia.

circDLPAG4/HECTD1 mediates ischaemia/reperfusion injury in endothelial cells via ER stress.

Background: Vascular endothelial cell dysfunction, characterized by cell apoptosis and migration, plays a crucial role in ischaemia/reperfusion (I/R) injury, a common aspect of cardiovascular diseases. Recent studies have suggested that non-coding RNAs, such as circular RNAs (circRNA), play a role in cell dysfunction in I/R injury, although the detailed mechanism is unclear.Methods: Human umbilical vein endothelial cells (HUVECs) were used for in vitro I/R model. Protein expression was detected by western blotting (WB) and immunocytochemistry. The CRISPR/Cas9 system, WB, cell viability assays, Hoechst staining and a 3D migration model were used to explore functional changes. RNA expression was evaluated using quantitative real-time PCR and a FISH assay combined with lentivirus transfection regulating circRNAs and miRNAs. A mouse myocardial I/R model using C57 mice was established to confirm the in vitro findings.Results: In HUVECs, I/R induced a significant time-dependent decrease in HECTD1 associated with an approximately 45% decrease in cell viability and increases in cell apoptosis and migration, which were attenuated by HECTD1 overexpression. I/R-induced upregulation of endoplasmic reticulum stress was also attenuated HECTD1 overexpression. Moreover, miR-143 mimics inhibited HECTD1 expression, which was restored by circDLGAP4 overexpression, providing insight as to the molecular mechanism of I/R-induced HECTD1 in endothelial cell dysfunction.Conclusion: Our results suggest a critical role for circDLGAP4 and HECTD1 in endothelial cell dysfunction induced by I/R, providing novel insight into potential therapeutic targets for the treatment of myocardial ischaemia.

CircHECTD1 mediates pulmonary fibroblast activation via HECTD1.

BACKGROUND: Circular RNA (circRNA), a new class of noncoding RNA, has been shown to be important in silicosis due to its unique role as a transcription regulator or as a sponge of small RNA regulators. Here, the mechanisms underlying circHECTD1/HECTD1 in fibroblast activation and subsequent fibrosis induced by SiO2 were investigated. METHODS: Primary human pulmonary fibroblasts (HPF-a) were utilized, combined with quantitative real-time PCR (qRT-PCR) and fluorescence in situ hybridization (FISH) assays. LC3B-LV-RFP lentivirus was used to evaluate the role of autophagy. The CRISPR/Cas9 system was applied to specifically knock down HECTD1, combined with MTT, BrdU, and migration assays, to explore the functional changes induced by SiO2. RESULTS: After exposure to SiO2, the circHECTD1 level was decreased, which was associated with an increase in HECTD1 in HPF-a cells. SiO2-induced autophagy was reversed by either circHECTD1 overexpression or HECTD1 knockdown in HPF-a cells, with restored SiO2-induced fibroblast activation, proliferation, and migration via downstream autophagy. The lungs of mice exposed to SiO2 confirmed the upregulation of HECTD1 in pulmonary fibroblasts. CONCLUSIONS: Our data suggested a link between circHECTD1/HECTD1 and fibroblast activation with subsequent fibrosis induced by SiO2, providing novel insight into the potential of circHECTD1/HECTD1 to be a therapeutic target for silicosis.

Hectd1 is essential for embryogenesis in mice.

Many aspects of the functional role of the E3 ubiquitin ligase Hectd1 in embryogenesis and in cell biology still remain to be elucidated. In order to contribute to this task we now report the generation of a new transgenic mouse model for Hectd1 using the gene trap strategy. The HECT domain deletion mutant mouse was created by inserting a ß-geo cassette into the Hectd1 locus. Mice homozygous for Hectd1-mutant showed early embryonic lethality with abnormal placental development and defective of neural tube closure resulting in exencephaly. The thickness of the placenta of both Hectd1-mutant homozygous and heterozygous mice was distinctly thinner than that of wildtype mice, the difference being most pronounced in the labyrinth layer of the placenta. We also addressed the temporal and spatial expression profiles of Hectd1 in adult tissues by X-gal staining. Hectd1 expression was detected in specific cell populations of most but not all tissues of the adult organism. Furthermore, the expression of Hectd1 was regulated by insulin and by both heat and hypoxia. Thus, our studies reveal that Hectd1 is indispensable for normal embryogenesis and fetal survival. The generation of this new Hectd1 mutant mouse model provides ample opportunities to study the function of Hectd1 in mammalian cells in detail.

Reduced maternal vitamin A status increases the incidence of normal aortic arch variants.

While common in the general population, the developmental origins of "normal" anatomic variants of the aortic arch remain unknown. Aortic arch development begins with the establishment of the second heart field (SHF) that contributes to the pharyngeal arch arteries (PAAs). The PAAs remodel during subsequent development to form the mature aortic arch and arch vessels. Retinoic acid signaling involving the biologically active metabolite of vitamin A, plays a key role in multiple steps of this process. Recent work from our laboratory indicates that the E3 ubiquitin ligase Hectd1 is required for full activation of retinoic acid signaling during cardiac development. Furthermore, our study suggested that mild alterations in retinoic acid signaling combined with reduced gene dosage of Hectd1, results in a benign aortic arch variant where the transverse aortic arch is shortened between the brachiocephalic and left common carotid arteries. These abnormalities are preceded by hypoplasia of the fourth PAA. To further explore this interaction, we investigate whether reduced maternal dietary vitamin A intake can similarly influence aortic arch development. Our findings indicate that the incidence of hypoplastic fourth PAAs, as well as the incidence of shortened transverse arch are increased with reduced maternal vitamin A intake during pregnancy. These studies provide new insights as to the developmental origins of these benign aortic arch variants.