ALKBH5 targets ACSL4 mRNA stability to modulate ferroptosis in hyperbilirubinemia-induced brain damage.

Bilirubin-induced brain damage is a serious clinical consequence of hyperbilirubinemia, yet the underlying molecular mechanisms remain largely unknown. Ferroptosis, an iron-dependent cell death, is characterized by iron overload and lipid peroxidation. Here, we report a novel regulatory mechanism of demethylase AlkB homolog 5 (ALKBH5) in acyl-coenzyme A synthetase long-chain family member 4 (ACSL4)-mediated ferroptosis in hyperbilirubinemia. Hyperdifferential PC12 cells and newborn Sprague-Dawley rats were used to establish in vitro and in vivo hyperbilirubinemia models, respectively. Proteomics, coupled with bioinformatics analysis, first suggested the important role of ferroptosis in hyperbilirubinemia-induced brain damage. In vitro experiments showed that ferroptosis is activated in hyperbilirubinemia, and ferroptosis inhibitors (desferrioxamine and ferrostatin-1) treatment effectively alleviates hyperbilirubinemia-induced oxidative damage. Notably, we observed that the ferroptosis in hyperbilirubinemia was regulated by m6A modification through the downregulation of ALKBH5 expression. MeRIP-seq and RIP-seq showed that ALKBH5 may trigger hyperbilirubinemia ferroptosis by stabilizing ACSL4 mRNA via m6A modification. Further, hyperbilirubinemia-induced oxidative damage was alleviated through ACSL4 genetic knockdown or rosiglitazone-mediated chemical repression but was exacerbated by ACSL4 overexpression. Mechanistically, ALKBH5 promotes ACSL4 mRNA stability and ferroptosis by combining the 669 and 2015 m6A modified sites within 3' UTR of ACSL4 mRNA. Our findings unveil a novel molecular mechanism of ferroptosis and suggest that m6A-dependent ferroptosis could be an underlying clinical target for the therapy of hyperbilirubinemia.

ROS/mtROS promotes TNTs formation via the PI3K/AKT/mTOR pathway to protect against mitochondrial damages in glial cells induced by engineered nanomaterials.

BACKGROUND: As the demand and application of engineered nanomaterials have increased, their potential toxicity to the central nervous system has drawn increasing attention. Tunneling nanotubes (TNTs) are novel cell-cell communication that plays a crucial role in pathology and physiology. However, the relationship between TNTs and nanomaterials neurotoxicity remains unclear. Here, three types of commonly used engineered nanomaterials, namely cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2NPs), and multi-walled carbon nanotubes (MWCNTs), were selected to address this limitation. RESULTS: After the complete characterization of the nanomaterials, the induction of TNTs formation with all of the nanomaterials was observed using high-content screening system and confocal microscopy in both primary astrocytes and U251 cells. It was further revealed that TNT formation protected against nanomaterial-induced neurotoxicity due to cell apoptosis and disrupted ATP production. We then determined the mechanism underlying the protective role of TNTs. Since oxidative stress is a common mechanism in nanotoxicity, we first observed a significant increase in total and mitochondrial reactive oxygen species (namely ROS, mtROS), causing mitochondrial damage. Moreover, pretreatment of U251 cells with either the ROS scavenger N-acetylcysteine or the mtROS scavenger mitoquinone attenuated nanomaterial-induced neurotoxicity and TNTs generation, suggesting a central role of ROS in nanomaterials-induced TNTs formation. Furthermore, a vigorous downstream pathway of ROS, the PI3K/AKT/mTOR pathway, was found to be actively involved in nanomaterials-promoted TNTs development, which was abolished by LY294002, Perifosine and Rapamycin, inhibitors of PI3K, AKT, and mTOR, respectively. Finally, western blot analysis demonstrated that ROS and mtROS scavengers suppressed the PI3K/AKT/mTOR pathway, which abrogated TNTs formation. CONCLUSION: Despite their biophysical properties, various types of nanomaterials promote TNTs formation and mitochondrial transfer, preventing cell apoptosis and disrupting ATP production induced by nanomaterials. ROS/mtROS and the activation of the downstream PI3K/AKT/mTOR pathway are common mechanisms to regulate TNTs formation and mitochondrial transfer. Our study reveals that engineered nanomaterials share the same molecular mechanism of TNTs formation and intercellular mitochondrial transfer, and the proposed adverse outcome pathway contributes to a better understanding of the intercellular protection mechanism against nanomaterials-induced neurotoxicity.

Ferroptosis contributes to hemolytic hyperbilirubinemia­induced brain damage in vivo and in vitro.

Ferroptosis is driven by iron­dependent accumulation of lipid hydroperoxides, and hemolytic hyperbilirubinemia causes accumulation of unconjugated bilirubin and iron. The present study aimed to assess the role of ferroptosis in hemolytic hyperbilirubinemia­induced brain damage (HHIBD). Rats were randomly divided into the control, phenylhydrazine (PHZ) and deferoxamine (DFO) + PHZ groups, with 12 rats in each group. Ferroptosis­associated biochemical and protein indicators were measured in the brain tissue of rats. We also performed tandem mass tag­labeled proteomic analysis. The levels of iron and malondialdehyde were significantly higher and levels of glutathione (GSH) and superoxide dismutase activity significantly lower in the brain tissues of the PHZ group compared with those in the control group. HHIBD also resulted in significant increases in the expression of the ferroptosis­related proteins acyl­CoA synthetase long­chain family member 4, ferritin heavy chain 1 and transferrin receptor and divalent metal transporter 1, as well as a significant reduction in the expression of ferroptosis suppressor protein 1. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the differentially expressed proteins of rat brain tissues between the control and PHZ groups were significantly involved in ferroptosis, GSH metabolism and fatty acid biosynthesis pathways. Pretreatment with DFO induced antioxidant activity and alleviated lipid peroxidation­mediated HHIBD. In addition, PC12 cells treated with ferric ammonium citrate showed shrinking mitochondria, high mitochondrial membrane density, and increased lipid reactive oxygen species and intracellular ferrous iron, which were antagonized by pretreatment with ferrostatin­1 or DFO, which was reversed by pretreatment with ferrostatin­1 or DFO. The present study demonstrated that ferroptosis is involved in HHIBD and provided novel insights into candidate proteins that are potentially involved in ferroptosis in the brain during hemolytic hyperbilirubinemia.

Targeting Mitochondrial Dysfunction With LncRNAs in a Wistar Rat Model of Chronic Obstructive Pulmonary Disease.

BACKGROUND/AIM: Chronic obstructive pulmonary disease (COPD) has become a prominent healthcare issue in recent years. Cigarette smoking (CS) and fine particulate matter (PM2.5) are important causative factors for COPD. This study assessed the aberrant lncRNA profiles in the tissue of rats with COPD caused by CS or PM2.5 Materials and Methods: A COPD rat model was developed using CS (CSM) or PM2.5 (PMM), and lung tissue RNA was extracted. The Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) were used to investigate the correlations between the distinct lncRNAs and mRNA pathways. A coding-non-coding gene co-expression network (CNC) was constructed by establishing connections between differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs) associated with mitochondrial dysfunction and the inflammatory response. RESULTS: A quantitative real-time reverse transcription PCR (qRT-PCR) experiment was performed to verify the expression of the particular lncRNAs. Microarray analysis of lung tissue from the COPD model revealed that 123 and 444 lncRNAs were substantially raised and reduced in PMM vs. the control group (Ctrl), respectively, as were 621 and 1,178 mRNAs. Meanwhile, 81 and 340 lncRNAs were consistently raised and lowered in CSM vs. Ctrl, respectively, as were 408 and 931 mRNAs. GO enrichment and KEGG pathway analysis indicated that the COPD model was connected to inflammatory responses, mitochondrial dysfunction, and others. CONCLUSION: XR_340674, ENSRNOT00000089642, XR_597045, and XR_340651 were decreased, and XR_592469 was elevated. These lncRNAs were shown to be related to mitochondrial dysfunction in the lung tissue of animals exposed to CS or PM2.5.

Microglia-derived exosomal circZNRF1 alleviates paraquat-induced neuronal cell damage via miR-17-5p.

Paraquat (PQ) is an environmental poison that causes clinical symptoms similar to those of Parkinson's disease (PD) in vitro and in rodents. It can lead to the activation of microglia and apoptosis of dopaminergic neurons. However, the exact role and mechanism of microglial activation in PQ-induced neuronal degeneration remain unknown. Here, we isolated the microglia-derived exosomes exposed with 0 and 40 µM PQ, which were subsequently co-incubated with PQ-exposed neuronal cells to simulate intercellular communication. First, we found that exosomes released from microglia caused a change in neuronal cell vitality and reversed PQ-induced neuronal apoptosis. RNA sequencing data showed that these activated microglia-derived exosomes carried large amounts of circZNRF1. Moreover, a bioinformatics method was used to study the underlying mechanism of circZNRF1 in regulating PD, and miR-17-5p was predicted to be its target. Second, an increased Bcl2/Bax ratio could play an anti-apoptotic role. Bcl2 was predicted to be a downstream target of miR-17-5p. Our results showed that circZNRF1 plays an anti-apoptotic role by absorbing miR-17-5p and regulating the binding of Bcl2 after exosomes are internalized by dopaminergic neurons. In conclusion, we demonstrated a new intercellular communication mechanism between microglia and neurons, in which circZNRF1 plays a key role in protecting against PQ-induced neuronal apoptosis through miR-17-5p to regulate the biological process of PD. These findings may offer a novel approach to preventing and treating PD.

Evaluation of neurotoxicity and the role of oxidative stress of cobalt nanoparticles, titanium dioxide nanoparticles, and multiwall carbon nanotubes in Caenorhabditis elegans.

The widespread use of nanomaterials in daily life has led to increased concern about their potential neurotoxicity. Therefore, it is particularly important to establish a simple and reproducible assessment system. Representative nanomaterials, including cobalt nanoparticles (CoNPs), titanium dioxide nanoparticles (TiO2-NPs), and multiwall carbon nanotubes (MWCNTs), were compared in terms of their neurotoxicity and underlying mechanisms. In 0, 25, 50, and 75 µg/ml of these nanomaterials, the survival, locomotion behaviors, acetylcholinesterase (AchE) activity, reactive oxygen species production, and glutathione-S transferase 4 (Gst-4) activation in wildtype and transgenic Caenorhabditis elegans (C. elegans) were evaluated. All nanomaterials induced an imbalance in oxidative stress, decreased the ratio of survival, impaired locomotion behaviors, as well as reduced the activity of AchE in C. elegans. Interestingly, CoNPs and MWCNTs activated Gst-4, but not TiO2-NPs. The reactive oxygen species scavenger, N-acetyl-l-cysteine, alleviated oxidative stress and Gst-4 upregulation upon exposure to CoNPs and MWCNTs, and rescued the locomotion behaviors. MWCNTs caused the most severe damage, followed by CoNPs and TiO2-NPs. Furthermore, oxidative stress and subsequent activation of Gst-4 were involved in nanomaterials-induced neurotoxicity. Our study provides a comprehensive comparison of the neurotoxicity and mechanisms of typical nanomaterials, which could serve as a model for hazard assessment of environmental pollutants using C. elegans as an experimental model system.

Expression profiling of N6-methyladenosine-modified mRNA in PC12 cells in response to unconjugated bilirubin.

BACKGROUND: Abnormal methylation of N6-methyladenosine (m6A) is reportedly associated with central nervous system disorders. However, the role of m6A mRNA methylation in unconjugated bilirubin (UCB) neurotoxicity requires further research. METHODS: Rat pheochromocytoma PC12 cells treated with UCB were used as in vitro models. After the PC12 cells were treated with UCB (0, 12, 18, and 24 µM) for 24 h, the total RNA m6A levels were measured using an m6A RNA methylation quantification kit. The expression of m6A demethylases and methyltransferases was detected through western blotting. We determined the m6A mRNA methylation profile in PC12 cells exposed to UCB (0 and 18 µM) for 24 h using methylated RNA immunoprecipitation sequencing (MeRIP-seq). RESULTS: Compared with the control group, UCB (18 and 24 µM) treatment decreased the expression of the m6A demethylase ALKBH5 and increased the expression of the methyltransferases METTL3 and METTL14, which resulted in an increase in the total m6A levels in PC12 cells. Furthermore, 1533 m6A peaks were significantly elevated and 1331 peaks were reduced in the UCB (18 µM)-treated groups compared with those in the control group. Genes with differential m6A peaks were mainly enriched in protein processing in the endoplasmic reticulum, ubiquitin-mediated proteolysis, cell cycle, and endocytosis. Through combined analysis of the MeRIP-seq and RNA sequencing data, 129 genes with differentially methylated m6A peaks and differentially expressed mRNA levels were identified. CONCLUSION: Our study suggests that the modulation of m6A methylation modifications plays a significant role in UCB neurotoxicity.

The interplay between lncRNA NR_030777 and SF3B3 in neuronal damage caused by paraquat.

Paraquat (PQ) has been widely acknowledged as an environmental risk factor for Parkinson's disease (PD). However, the interaction between splicing factor and long non-coding RNA (lncRNA) in the process of PQ-induced PD has rarely been studied. Based on previous research, this study focused on splicing factor 3 subunit 3 (SF3B3) and lncRNA NR_030777. After changing the target gene expression level by lentiviral transfection technology, the related gene expression was detected by western blot and qRT-PCR. The expression of SF3B3 protein was reduced in Neuro-2a cells after PQ exposure, and the reactive oxygen species (ROS) scavenger N-acetylcysteine prevented this decline. Knockdown of SF3B3 reduced the PQ-triggered NR_030777 expression increase, and overexpression of NR_030777 reduced the transcriptional and translational level of Sf3b3. Then, knockdown of SF3B3 exacerbated the PQ-induced decrease in cell viability and aggravated the reduction of tyrosine hydroxylase (TH) protein expression. Overexpressing SF3B3 reversed the reduction of TH expression caused by PQ. Moreover, after intervention with the autophagy inhibitor Bafilomycin A1, LC3B-II protein expression was further increased in Neuro-2a cells with the knockdown of SF3B3, indicating that autophagy was enhanced. In conclusion, PQ modulated the interplay between NR_030777 and SF3B3 through ROS production, thereby impairing autophagic flux and causing neuronal damage.

A Novel M6A-Related Genes Signature Can Impact the Immune Status and Predict the Prognosis and Drug Sensitivity of Lung Adenocarcinoma.

Lung adenocarcinoma (LUAD) is a primary cause of cancer-related death around the world and has a poor outcome and high incidence. Treatment options are, however, restricted. One of the most critical factors in cancer and metastasis is the N6-methyladenine (m6A) alteration on RNA. This modification could alter gene expression and even function at numerous levels, such as the stability, translocation and translation of RNA splicing. This study aimed to construct an m6A-related genes signature to accurately predict the prognosis of LUAD patients. From TCGA datasets, the LUAD patient data and m6A-related genes were retrieved. LUAD patients' mutational features and differentially expressed genes (DEGs) were investigated. An univariate and LASSO model with m6A-related genes were constructed for the prediction of outcomes in LUAD. It was possible to develop a prognostic nomogram that could quantitatively predict LUAD patients' overall survival chances at 1, 3, and 5 years. Research into biological processes and cell pathways was carried out using GSEA. This study found six m6A-related DEGs in LUAD patients, and three of these DEGs(HNRNPC, IGFBP3 and IGF2BP1) were linked to the clinical outcomes of LUAD patients. We found that the overall survival rate for all LUAD patients with high-risk subgroup was considerably lower. According to ROC curves, the prognostic signature demonstrated a high degree of accuracy in predicting future outcomes. In addition, we created a novel nomogram achieved great accuracy with this one as well. The researchers also found that the novel signature might favorably modulate the immune response, and high-risk scores samples were more susceptible to numerous chemotherapeutic medicines. Overall, we developed a m6A-related gene prognostic signature that effectively predicted outcomes of LUAD patients and gave an immunological perspective for creating customized therapeutics.

Involvement of NEAT1/PINK1-mediated mitophagy in chronic obstructive pulmonary disease induced by cigarette smoke or PM2.5.

Background: This study sought to explore the underlying mechanism of long non-coding ribonucleic acid nuclear enriched abundant transcript 1 (NEAT1) and PTEN-induced kinase 1 (PINK1)-mediated mitophagy in chronic obstructive pulmonary disease (COPD) induced by cigarette smoke (CS) or fine particular matter (PM2.5). Methods: In total, 30 male Wistar Rats were divided into the following 3 groups: (I) the COPD group exposed to CS (CSM); (II) the COPD group exposed to PM2.5 (PMM); and (III) the control (Ctrl) group. Pulmonary function, the enzyme-linked immunoassay analysis results, the histopathology results, and the ultrastructures of the lung tissues were examined in the 3 groups, and NEAT1 expression levels and the mitophagy-related protein PINK1, Parkin, LC3B, and p62 levels were assessed by quantitative reverse transcription PCR (RT-qPCR) and Western blotting. The A549 cells were transfected with small interfering ribonucleic acid (siRNA) targeting NEAT1, and subsequently stimulated with CS extract (CSE) and PM2.5 suspension (PMS). Mitochondrial dysfunction and enhanced mitophagy were observed, and the expression of the NEAT1/PINK1 pathway was assessed by RT-qPCR and Western blotting. Results: Both the CSM and PMM groups had a lower tidal volume (VT), minute ventilation (MV), and a higher respiratory rate (f) than the Ctrl group. The interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha levels in the serum and bronchoalveolar lavage fluid of the CSM and PMM groups were significantly increased. The histological examination results revealed airway remodeling, the formation of pulmonary bullae, and emphysema in the CSM and PMM groups. Subsequently, the ultrastructures of the lung tissues in the CSM and PMM groups showed mitochondrial swelling and autophagosomes. Additionally, NEAT1 expression, the level of the mitophagy-related protein PINK1, Parkin, and the ratio of LC3-II/I increased synchronously. Further, NEAT1 siRNA blocked PINK1 expression, inhibited mitochondrial dysfunctions, and mitophagy activation in the A549 cells exposed to CSE or PMS. Conclusions: Our results suggest that CS and PM2.5 exposure induce mitochondrial dysfunction, and the NEAT1/PINK1 pathway plays a critical role in the occurrence and development of COPD by regulating mitophagy.

Paraquat-induced oxidative stress regulates N6-methyladenosine (m6A) modification of long noncoding RNAs in Neuro-2a cells.

Paraquat (PQ) is a ubiquitously applied herbicide. Long-term PQ exposure with low dose has been reported to induce abnormal expression of long non-coding RNAs (lncRNAs) in brain nerve cells, which could further lead to Parkinson's disease (PD). N6-methyladenosine (m6A) modification has recently been identified as having an important role in regulating the function of lncRNAs. However, how m6A modification regulates lncRNAs following PQ exposure remains largely unknown. Herein, this study reported m6A modification of lncRNAs in mouse neuroblastoma cells (Neuro-2a) following PQ induced reactive oxide species (ROS). M6A sequencing was performed to explore the m6A modificated pattern of lncRNAs in Neuro-2a cells which were treated with 200 µM PQ for 3 h. It was found that PQ hypermethylated total RNA and changed the expression of m6A methyltransferase and demethylase proteins, which leading to the alteration of m6A modification of lncRNAs. Furthermore, the functional analysis further revealed that N-acetyl-L-cysteine (NAC)，a ROS scavengers, partly reversed PQ-induced distinct m6A modificated pattern of lncRNAs. In addition, tow specific m6A modified lncRNAs were identified: cell division cycle 5-like (lncRNA CDC5L) and signal transducer and activator of transcription 3 (lncRNA STAT3), which could influence downstream autophagy related biological function. In summary, this work could potentially contribute to the new insight of lncRNAs m6A modification mechanism in the field of environmental toxicology.

N6-methyladenosine(m6A) demethylase FTO regulates cellular apoptosis following cobalt-induced oxidative stress.

Cobalt is an environmental toxicant that is known to damage human health. However, the molecular mechanisms underlying cobalt-induced neurotoxicity have not been elucidated in detail. In the present research, we used human neuroglioma H4 cells as an in vitro model. Cells were exposed to CoCl2 (0, 100, 200, 400 µM) for 24 h. We performed m6A sequencing techniques and constructed FTO-knockdown/FTO-overexpressing cells to investigate the role of FTO-mediated m6A modification in regulating apoptosis following CoCl2 induced oxidative stress. Our study has shown CoCl2 exposure led to the decrease of demethylase FTO as well as elevated oxidative stress. However, NAC treatment could partly reverse the reduction of FTO expression as well as the degree of ROS via eliminating oxidative stress. Meanwhile, MeRIP-seq and RNA-seq further revealed the potential function m6A modification in regulating apoptosis. More importantly, KEGG pathway and Gene ontology (GO) analyses further elucidated that the differentially m6A-modified genes were aggregated in apoptosis-related pathways. Mechanistic analysis indicated that knockdown of FTO facilitated CoCl2-induced apoptosis via caspase activation and G1/S cell cycle arrest. Nevertheless, overexpression of FTO partly attenuated the increased apoptosis following CoCl2 exposure. More notably, we observed that FTO regulated apoptosis in an m6A-dependent manner. Therefore, our findings reveal that CoCl2 induced ROS affected the m6A modification of apoptosis-related genes by decreasing the expression of FTO, thereby resulting in the activation of apoptosis. These findings provide important insights into CoCl2-induced apoptosis and m6A modification and propose a novel strategy for studying environmental toxicant-related neurodegeneration.

Epigenetics in neurodegenerative disorders induced by pesticides.

Neurodegenerative diseases are becoming major socio-economic burdens. However, most of them still have no effective treatment. Growing evidence indicates excess exposure to pesticides are involved in the development of various forms of neurodegenerative and neurological diseases through trigger epigenetic changes and inducing disruption of the epigenome. This review summaries studies on epigenetics alterations in nervous systems in relation to different kinds of pesticides, highlighting potential mechanism in the etiology, precision prevention and target therapy of various neurodegenerative diseases. In addition, the current gaps in research and future areas for study were also discussed.

Paraquat-induced oxidative stress regulates N6-methyladenosine (m6A) modification of circular RNAs.

Paraquat (PQ), a widely used herbicide and well-known oxidative stress inducer, has been linked to numerous neurodegenerative diseases, but the underlying mechanism(s) remains unknown. Circular RNAs (circRNAs) have recently been reported to be associated with oxidative stress in Parkinson's disease. Herein, we performed methylated RNA immunoprecipitation and RNA sequencing assays for mouse neuroblastoma (Neuro-2a) cells and successfully established a positive link between the alteration of circRNAs driven by m6A modification and PQ-induced oxidative stress. We observed oxidative stress and antioxidative stress present distinct m6A modification pattern of circRNAs as well as biological effect. Gene ontology and pathway analysis predicted that differentially m6A-methylated and expressed circRNAs are highly clustered in pathways associated with function and development of nervous system, including axon cargo transport, nervous system development, long-term potentiation, and neurotrophic signaling pathways. Moreover, we demonstrated that the alteration of m6A-methylated circRNAs upon PQ exposure could be partially reversed by N-acetylcysteine pretreatment. The mechanistic analysis further demonstrated that N-acetylcysteine pretreatment attenuated the decreased expression of target genes (UBC and PPP2CA) induced by PQ. These findings revealed distinct patterns of differentially m6A-modified circRNAs, indicating that m6A could participate in a specific regulatory network of circRNAs to modulate the expression of downstream genes in response to PQ-induced oxidative stress. In conclusion, our work established a link between m6A modification of circRNAs and PQ-induced oxidative stress, and further studies are required to explore the underlying molecular mechanisms associated with PQ-induced neurotoxicity.

Evaluations of Environmental Pollutant-Induced Mitochondrial Toxicity Using Caenorhabditis elegans as a Model System.

Environmental pollutants inevitably exert adverse effects on humans and other species. Quick identification and in-depth characterization of the pollutants are requisite objectives for clinicians and environmental health scientists. The nematode Caenorhabditis elegans has been utilized as a model organism for toxicity evaluation of environmental pollutants, due to its transparency, short lifespan, entire genome sequencing, and economical characteristics. However, few researchers have systematically addressed mitochondrial toxicity in response to toxicants, despite the critical role mitochondria play in energy production and respiration, as well as the generation of reactive oxygen species. Mitochondria are vulnerable to environmental pollutants, and their dysfunction contributes to cellular damage and toxicity in plethora of diseases. Here, we describe methods in step-by-step for mitochondrial toxicity evaluation in response to pollutants, including exposure of C. elegans to toxicants, mitochondrial ROS detection, mitochondrial morphology analysis, mitochondrial function analysis, such as ATP production and oxygen consumption, and gene expression studies, with the application of corresponding genetically modified strains.

Association Between Circular RNAs and Intracranial Aneurysm Rupture Under the Synergistic Effect of Individual Environmental Factors.

Introduction: To study the association between specific circular RNAs and rupture of intracranial aneurysm. To explore its clinical diagnostic significance and synergistic effects with individual environmental influencing factors. Methods: Three hundred and forty seven cases and controls were included in this study. Multivariate analysis was used to explore the main individual environmental factors. Intracranial aneurysm rupture related circular RNAs screened based on sequencing was verified in peripheral blood by PCR. ROC curve, logistic regression model and fork analysis were used to study the association, diagnostic values, and synergistic effects of circular RNA with intracranial aneurysms and individual environmental factors. Results: Smoking, hair dyeing, sitting time ≥6 h/day, single animal oil intake and hypertension are the main risk factors for intracranial aneurysm rupture; People with higher education, sleeping time ≥7 h/day, tea drinking, diabetes, higher levels of (hemoglobin, low density lipoprotein, serum calcium, and apolipoprotein-A1) have a low risk of intracranial aneurysm rupture. Hsa_circ_0008433 and hsa_circ_0001946 are closely related to intracranial aneurysm rupture and have certain clinical diagnostic significance (AUC = 0.726; 95% CI: 0.668~0.784). Hsa_circ_0008433 (OR = 0.497, 95% CI: 0.338~0.731), hsa_circ_0001946 (OR = 0.682, 95% CI: 0.509~0.914) were independent epigenetic factors affecting intracranial aneurysm rupture, and have a multiplicative interaction with age (OR = 3.052, 95% CI: 1.006~9.258). Conclusions: Low expressions of hsa_circ_0008433 and hsa_circ_0001946 are risk factors for intracranial aneurysms rupture and have good clinical diagnostic value. There was a multiplicative interaction between epigenetic score and age. The older and the higher the epigenetic score was, the more likely to have intracranial aneurysm rupture.

The negative role of histone acetylation in cobalt chloride-induced neurodegenerative damages in SHSY5Y cells.

Cobalt has been known for its neurotoxicity in numerous studies. However, the molecular mechanism underlying cobalt-induced neurotoxicity remains largely unknown. In this study, two neuroblastoma (SHSY5Y and N2a) cell lines and a phaeochromocytoma (PC12) line were used as in vitro models. Cells were treated for 24 h with 50, 100, 200, 300, 400 µM cobalt chloride (CoCl2) or cultured with 300 µM CoCl2 for 4, 8, 12 and 24 h to investigate the effects of histone acetylation on CoCl2-induced neurodegenerative damages. Our findings demonstrate that CoCl2 suppresses the acetylation of histone H3 and H4 in a time-dependent and dosage-dependent manner. Furthermore, CoCl2 selectively decreases the expression and activity of histone acetyltransferase (HAT) but has no effects on histone deacetylase (HDAC) in SHSY5Y cells. More importantly, we show that 100 ng/mL HDAC inhibitor trichostatin (TSA) pre-treatment partly attenuates 300 µM CoCl2-induced neurodegenerative damages in SHSY5Y cells. Mechanistic analyses show that CoCl2-induced neurodegenerative damages are associated with the dysfunction of APP, BACE1, PSEN1, NEP and HIF-1α genes, whose expression are partly mediated by histone modification. In summary, we demonstrate that histone acetylation is involved in CoCl2-induced neurodegenerative damages. Our study indicates an important connection between histone modification and the pathological process of neurodegenerative damages and provides a mechanism for cobalt-mediated epigenetic regulation.

Global N6-methyladenosine profiling of cobalt-exposed cortex and human neuroblastoma H4 cells presents epitranscriptomics alterations in neurodegenerative disease-associated genes.

Excessive exposure to cobalt (Co) is known to make adverse impact on the nervous system, but its detailed mechanisms of neurotoxicity have yet to be determined. In this study, C57BL/6 mice (0, 4, 8, 16 mg/kg CoCl2, 30 days) and human neuroblastoma H4 cells (0, 100, 400, 600 µM CoCl2) were used as in vivo and in vitro models. Our results revealed that CoCl2 intraperitoneal injection caused significant impairments in learning and memory, as well as pathological damage in the nervous system. We further certificated the alteration of m6A methylation induced by CoCl2 exposure. Our findings demonstrate for the first time, significant differences in the degree of m6A modification, the biological function of m6A-modified transcripts between cortex and H4 cell samples. Specifically, MeRIP-seq and RNA-seq elucidate that CoCl2 exposure results in differentially m6A-modified and expressed genes, which were enriched in pathways involving synaptic transmission, and central nervous system (CNS) development. Mechanistic analyses revealed that CoCl2 remarkably changed m6A modification level by affecting the expression of m6A methyltransferase and demethylase, and decreasing the activity of demethylase. We observed variation of m6A modification in neurodegenerative disease-associated genes upon CoCl2 exposure and identified regulatory strategy between m6A and potential targets mRNA. Our novel findings provide novel insight into the functional roles of m6A modification in neurodegenerative damage caused by environmental neurotoxicants and identify Co-mediated specific RNA regulatory strategy for broadening the epigenetic regulatory mechanism of RNA induced by heavy metals.

Reactive oxygen species regulate miR-17-5p expression via DNA methylation in paraquat-induced nerve cell damage.

There is emerging evidence suggesting that oxidative stress and DNA methylation can alter miRNA expression. However, little is known on the mechanism of miR-17-5p expression changes in paraquat (PQ)-induced nerve cell damage. In the present study, neuro-2a cells were pretreated with antioxidant N-acetylcysteine (NAC) or DNA methylation inhibitor decitabine (DAC), then exposed to different concentrations of PQ, while the expression levels of miR-17-5p were detected by qRT-PCR. Here, it is showed that PQ downregulated the expression of miR-17-5p dose-dependently in neuro-2a cells. The DNA methylation level was upregulated after PQ exposure, while downregulated with the pretreatment of NAC in the above content, detected by 5-mC immunofluorescence technique. The interaction effect of NAC and PQ in alternating DNA methylation level was further confirmed by flow cytometry. NAC and DAC individually had an interaction effect in PQ-induced nerve cell damage. After using NAC, PQ-induced ROS elevation and DNA methylation are reduced, thereby preventing the proapoptotic effect of miR-17-5p. Above all, PQ can induce DNA methylation variations through ROS production, leading to the downregulation of miR-17-5p expression in PQ-induced nerve cell damage.

Association of circular RNAs and environmental risk factors with coronary heart disease.

BACKGROUND: Coronary heart disease (CHD) is a complex disease caused by multi-factors and a major threat to human health. Circular RNAs (circRNAs) have critical roles in various biological processes and diseases. This study explores the independent role of circRNAs and their interaction with environmental factors in CHD. METHODS: A case-control study was conducted from March 2015 to September 2017 in Fuzhou, China. A total of 585 CHD patients and 585 gender- and age-matched healthy controls were enrolled. Questionnaire survey, health examination and molecular biology laboratory testing were conducted. Microarray technology and quantitative real-time polymerase chain reaction (PCR) were used to profile the expression levels of circRNAs. The area under the curve (AUC) of the receiver operating characteristic (ROC) was used to determine the diagnostic cut-offs. Multivariate logistic regression and multiplicative analysis were used to analyse the effects of environmental factors and hsa_circ_0008507, hsa_circ_0001946, hsa_circ_0000284 and hsa_circ_0125589 on CHD. RESULTS: The expression profile of circRNAs showed that 3423 circRNAs were differentially expressed at P < 0.05, but none pass multiple testing correction. qRT-PCR further confirmed the expression levels of hsa_circ_0008507, hsa_circ_0001946 and hsa_circ_0000284 in peripheral blood leukocytes in CHD cases were higher than those in non-CHD subjects (All p < 0.05). Hsa_circ_0008507 (OR = 1.29; 95% CI: 1.11-1.50), hsa_circ_0001946 (OR = 1.20; 95% CI: 1.01-1.42) and hsa_circ_0000284 (OR = 2.05; 95% CI: 1.32-3.19) were independent risk factors for CHD after controlling other common environmental risk factors. The AUC for hsa_circ_0008507, hsa_circ_0001946 and hsa_circ_0000284 was 0.75, 0.71 and 0.68, respectively. Compared with non-smoking individuals with low hsa_circ_0008507 expression, the smokers with high hsa_circ_0008507 expression showed the highest magnitude of OR in CHD risk. Additionally, a statistically significant multiplicative interaction was found between hsa_circ_0008507 and smoking for CHD. CONCLUSIONS: Hsa_circ_0008507, hsa_circ_0001946 and hsa_circ_0000284 were closely related to the occurrence and development of CHD. The combination of smoking and high hsa_circ_0008507 expression causes the occurrence and development of CHD.