Erratum: Whole-transcriptome analysis of aluminum-exposed rat hippocampus and identification of ceRNA networks to investigate neurotoxicity of Al.

[This corrects the article DOI: 10.1016/j.omtn.2021.11.010.].

The mechanism of miR-665 targeting GNB3 in aluminum-induced neuronal apoptosis.

BACKGROUND: Aluminum exerts neurotoxic effects through various mechanisms, mainly manifested as impaired learning and memory function. METHODS: Forty SD rats were divided into 0, 10, 20, and 40 mM maltol aluminum [Al(mal)3] groups. Cell experiments are divided into 0, 100, 200, and 400 µM Al(mal)3 dose group and control, Al(mal)3, Al(mal)3+inhibitor NC, Al(mal)3+miR-665 inhibitor intervention group. Water maze was used to detect the learning and memory function of rats, HE staining was used to observe the morphology and number of neurons in the CA1 area of the rat hippocampus, Flow cytometry was used to detect the apoptosis of PC12 cells, PCR and Western blotting were used to detect the expression of Caspase3, miR-665 and GNB3/PI3K/AKT proteins. The target binding relationship between miR-665 and GNB3 was verified by double luciferase reporter gene experiment. RESULTS: In vivo experimental results showed that with the increase of Al(mal)3 concentration, the escape latency of rats was prolonged, the target quadrant dwell time was shortened, and the number of crossing platform was reduced. Moreover, the arrangement of neurons was loose and the number decreased; the expression of Caspase3 and miR-665 increased, while the expression of GNB3/PI3K/AKT proteins decreased. In vitro experiments, with the increase of Al(mal)3 concentration, apoptosis rate of PC12 cells increased, the expression of Caspase3, miR-665 and GNB3/PI3K/AKT proteins were consistent with rat results. After inhibiting miR-665 in the intervention group experiment, apoptosis rate of PC12 cells in the aluminum exposure group decreased, the expression of Caspase3 and miR-665 decreased, and the expression of GNB3/PI3K/AKT proteins increased. CONCLUSION: MiR-665 plays an important role in aluminum induced neuronal apoptosis by targeting GNB3 and regulating the PI3K/AKT pathway.

Zinc as a Mediator Through the ROCK1 Pathway of Cognitive Impairment in Aluminum-Exposed Workers: A Clinical and Animal Study.

Aluminum (Al) exposure was implicated in neurodegenerative diseases and cognitive impairment, yet the involvement of zinc (Zn) and its mechanism in Al-induced mild cognitive impairment (MCI) remains poorly understood. The objective is to explore the role of Zn in Al-induced cognitive impairment and its potential mechanisms. Montreal cognitive assessment (MoCA) test scores and serum Al, Zn from Al industry workers were collected. A mediation analysis was performed to evaluate the role of serum Zn among serum Al and MoCA test scores. Subsequently, an Al-exposure study was conducted on a rat model categorized into control, low-, medium-, and high-dose groups. After a Morris Water Maze test and detection of Al, Zn content in the hippocampus, integrated transcriptomic and proteomic analyses between the control group and the high-dose group were performed to identify the differentially expressed genes (DEPs), proteins (DEPs), and pathways. To corroborate these findings, quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting (WB) were selected to identify the gene and protein results. Zn overall mediates the relationship between serum Al and cognitive function (mediation effect 17.82%, effect value = - 0.0351). In the Al-exposed rat model, 734 DEGs, 18 miRNAs, 35 lncRNAs, 64 circRNAs, and 113 DEPs were identified between the high-dose group and the control group. Among them, ROCK1, DMD, and other four DEPs were identified as related to zinc finger proteins (ZNF). Co-enrichment analyses of the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) linked these changes to the RHOA/ROCK1 signaling axis. ZNF-related proteins Rock1, DMD, and DHX57 in the high-dose group were downregulated (p = 0.006, 0.003, 0.04), and the expression of Myl9, Rhoa, miR431, and miR182 was also downregulated (p = 0.003, 0.032, 0.032, and 0.046). These findings also show correlations between Al, Zn levels in the hippocampus, water maze performance, and expressions of Myl9, Rhoa, miR431, miR182, DMD, ROCK1, and DHX57, with both negative and positive associations. Based on the results, we determined that Zn was involved in Al-induced MCI in Al workers and Al-exposed rat models. Al exposure and interaction with Zn could trigger the downregulation of ZNF of ROCK1, DMD, and DHX57. miR431, miR182 regulate RHOA/ROCK1 was one of the Zn-involved pathways in Al-induced cognitive impairment.

The role of Nrf2/HO-1 signal pathway in regulating aluminum-induced apoptosis of PC12 cells.

BACKGROUND: Aluminum has definite neurotoxicity and can lead to apoptosis of nerve cells, but the specific mechanism remains to be further explored. The aim of this study was to investigate the role of Nrf2/HO-1 signaling pathway in neural cell apoptosis induced by aluminum exposure. METHODS: In this study, PC12 cells were used as the research object, aluminum maltol [Al(mal)3] was used as the exposure agent, and tert-butyl hydroquinone (TBHQ), an agonist of Nrf2, was used as the intervention agent to construct an in vitro cell model. Cell viability was detected by CCK-8 method, cell morphology was observed by light microscope, cell apoptosis was measured by flow cytometry, and expression of Bax and Bcl-2 proteins and Nrf2/HO-1 signaling pathway proteins were investigated by western blotting. RESULTS: With the increase of Al(mal)3 concentration, PC12 cell viability decreased, the early apoptosis rate and total apoptosis rate increased, the ratio of Bcl-2 and Bax protein expression decreased, and Nrf2/HO-1 pathway protein expression decreased. The use of TBHQ could activate the Nrf2/HO-1 pathway and reverse the apoptosis of PC12 cells induced by aluminum exposure. CONCLUSION: Nrf2/HO-1 signaling pathway plays a neuroprotective role in the apoptosis of PC12 cells caused by Al(mal)3, which provides a possible target for the intervention of aluminum induced neurotoxicity.

The association between blood lymphocyte NMDAR, group I mGluRs and cognitive function changes in occupationally aluminum-exposed workers and verification in rats.

BACKGROUND: Many studies have shown that occupational aluminum (Al) exposure could affect the cognitive functions of workers and cause mild cognitive impairment (MCI). Glutamate receptors (GluRs) play an important role in learning and memory functions. METHODS: 352 workers in a large Al production enterprise were investigated in this research. MMSE, CDT, DST, VFT, FOM were used to evaluate the cognitive functions of workers. Plasma Al levels as exposure indices were measured by Graphite Furnace Atomic Absorption Method (GFAAS). The expression of GluRs was measured by ELISA. Cognitive function comprehensive scores were obtained through factor analysis. Then a rat model of chronic AlCl3 exposure was established. The detection method of Al levels and protein expression were the same as mentioned-above. RESULTS: Compared with the Q1 group, the DST, VFT, and comprehensive cognitive function scores of the Q4 group were lower(P < 0.05). For every 1µg/L increase in plasma Al concentration, the risk of cognitive impairment increases 1.051 times (95 %CI:1.031,1.072). Both NMDAR1 and NMDAR2A protein expression level of Q1 group were higher than those of Q2, Q3, Q4 group (all P < 0.05). The mediating effect ratio of NMDAR1 between plasma Al levels and cognitive function comprehensive scores was a1*b1/c=11.30 %, and the mediating effect ratio of NMDAR2A was |a2*b2/c|=21.77 %. Compared with control group, the escape latency of rats in the high Al dose group was longer day by day (P < 0.05). With the increase of Al dose, the relative expression of NMDAR1, NMDAR2A, NMDAR2B, GluR1 and mGluR5 in cerebral cortex and lymphocytes of rats were decreased (P < 0.05). The result of correlation analysis on NMDAR1 protein expression between brain cortex and lymphocyte showed that the correlation coefficient is r = 0.646(P < 0.05). CONCLUSION: Taking together the results from both Al exposed workers and animal, there is a certain correlation between NMDAR1 protein contents of brain cortex and peripheral lymphocytes. We propose that lymphocyte NMDAR1 could be considered as a peripheral potential marker of cognitive impairment for further observation.

Whole-transcriptome analysis of aluminum-exposed rat hippocampus and identification of ceRNA networks to investigate neurotoxicity of Al.

Aluminum is a known neurotoxin that can induce Aß deposition and abnormal phosphorylation of tau protein, leading to Alzheimer disease (AD)-like damages such as neuronal damage and decreased learning and memory functions. In this study, we constructed a rat model of subchronic aluminum maltol exposure, and the whole-transcriptome sequencing was performed on the hippocampus of the control group and the middle-dose group. A total of 167 miRNAs, 37 lncRNAs, 256 mRNAs, and 64 circRNAs expression changed. The Kyoto Encyclopedia of Genes and Genomes showed that PI3K/AKT pathway was the most enriched pathway of DEGs, and IRS1 was the core molecule in the PPI network. circRNA/lncRNA-miRNA-mRNA networks of all DEGs, DEGs in the PI3K/AKT pathway, and IRS1 were constructed by Cytoscape. Molecular experiment results showed that aluminum inhibited the IRS1/PI3K/AKT pathway and increased the content of Aß and tau. In addition, we also constructed an AAV intervention rat model, proving that inhibition of miR-96-5p expression might resist aluminum-induced injury by upregulating expression of IRS1. In general, these results suggest that the ceRNA networks are involved in the neurotoxic process of aluminum, providing a new strategy for studying the toxicity mechanism of aluminum and finding biological targets for the prevention and treatment of AD.

The Role of PKC in Regulating NMDARs in Aluminum-Induced Learning and Memory Impairment in Rats.

Aluminum is a widespread environmental neurotoxicant that can induce Alzheimer's disease (AD)-like damage, such as neuronal injury and impairment of learning and memory. Several studies have shown that aluminum could reduce the synaptic plasticity, but its molecular mechanism remains unclear. In this study, rats were treated with aluminum maltol (Al(mal)3) to establish a toxic animal model and PMA was used to interfere with the expression of PKC. The Morris water maze and open field test were used to investigate the behavioral changes of the rats. Western blotting and RT-PCR were used to detect the expression levels of NMDAR subunits, PKC and CaMKII. The results showed that Al(mal)3 damaged learning and memory function and reduced anxiety in rats. During this process, the expression of PKC was downregulated and it inhibited the expression of NMDARs through the phosphorylation of CaMKII.

miR-29a/b1 Regulates BACE1 in Aluminum-Induced Aß Deposition in Vitro.

Aluminum is an environmental neurotoxin that comes extensively in contact with human beings. Animal and human studies demonstrated that aluminum exposure increases the deposition of beta amyloid proteins in the brain as it was observed in Alzheimer's disease. The purpose of this study was to investigate whether miR-29a/b1 affected the expression of beta-secrete enzymes (BACE1) in the process of amyloid ß-protein (Aß) deposition caused by aluminum exposure. The study was performed using two different cell lines. Our results showed that after rat primary cortical neurons were exposed to aluminum, BACE1 gene and protein levels increased to different degrees, and the expression level of Aß1-42 increased. In aluminum-exposed groups, the expression of miR-29a and miR-29b1 decreased, while the expression of amyloid protein Aß1-42 and BACE1 increased. In miRs transfection groups, the expression of amyloid protein Aß1-42 and BACE1 decreased. Aluminum may affect the expression of BACE1 by lowering miR-29a and miR-29b1. AEK293 cells were utilized in this research since they present elevated levels of miR-29a and miR-29b1. After HEK293 cells were exposed to aluminum alone, BACE1 mRNA and BACE1 protein expression levels increased with the increase of aluminum exposure dose (p < 0.05), and the level of Aß1-42 also increased (p < 0.05). Compared with the group exposed to aluminum alone at the same doses, the expression levels of BACE1 mRNA and BACE1 protein in the miRs transfected plus aluminum-exposed groups significantly decreased (p < 0.05), and the level of Aß1-42 also decreased (p < 0.05). This result is consistent with the investigation in rat primary neurons. The results of two types of cells showed that aluminum may cause abnormal down-regulation of the expressions of miR-29a and miR-29b1, thus negatively regulating the increase of BACE1 expression and finally leading to the increase of Aß.

Regulation of mGluR1 on the Expression of PKC and NMDAR in Aluminum-Exposed PC12 Cells.

Aluminum demonstrates clear neurotoxicity and can cause Alzheimer's disease (AD)-like symptoms, including cognitive impairment. One toxic effect of aluminum is a decrease in synaptic plasticity, but the specific mechanism remains unclear. In this study, PC12 cells were treated with Al(mal)3 to construct a toxic cell model. (S)-3,5-Dihydroxyphenylglycine (DHPG), α-methyl-4-carboxyphenylglycine (MCPG), and mGluR1-siRNA were used to interfere with the expression of metabotropic glutamate receptor subtype 1 (mGluR1). Polymerase chain reaction and western blotting were used to investigate the expression of mGluR1, protein kinase C (PKC), and N-methyl-D-aspartate receptor (NMDAR) subunits. ELISA was used to detect PKC enzyme activity. In PC12 cells, mRNA and protein expressions of PKC and NMDAR subunits were inhibited by Al(mal)3. Aluminum may further regulate the expression of NMDAR1 and NMDAR2B through mGluR1 to regulate PKC enzyme activity, thereby affecting learning and memory functions. Furthermore, the results implied that the mGluR1-PKC-NMDAR signaling pathway may predominately involve positive regulation. These findings provide new targets for studying the neurotoxic mechanism of aluminum.

Effect of aluminum combined with ApoEε4 on Tau phosphorylation and Aß deposition.

BACKGROUND: Aluminum is an environmental neurotoxin widely exposed to animals and humans. Studies have shown that Alzheimer's disease (AD) is characterized by abnormally phosphorylated tau and Aß deposition, aluminum exposure can lead to abnormal phosphorylated tau and Aß deposition. Numerous epidemiological data and studies have confirmed that ApoEε4 is a risk factor for AD. However, whether there is an interaction effect between aluminum and ApoEε4 has yet to be verified. METHODS: SH-SY5Y cells were exposed with AlCl3 and transfected with ApoEε4 respectively. The experimental groups included the blank control group, the low dose group (200 µM AlCl3), the medium dose group (400 µM AlCl3), the high dose group (800 µM AlCl3), empty plasmid group, ApoEε4 group and 400 µM AlCl3+ApoEε4 group. The cell viability was determined by CCK-8 kit after transfection for 48 h.The contents of total tau proteins, tau-181, tau-231, tau-262, tau-396 and Aß42, were determined by ELISA kit. The interaction between AlCl3 and ApoEε4 was analyzed by factorial design. RESULTS: With the increase of aluminum exposure, SH-SY5Y cell viability decreased, and the expression of the total tau, tau-181, tau-231, tau-262, tau-396 and Aß content increased. The viability of cells transfected with ApoEε4 is significantly lower than control group, and the expressions of total tau, tau-181, tau-231, tau-262, tau-396 and Aß in ApoEε4 transfected cells were significantly higher than control group. The viability of cells treated with AlCl3 plus ApoEε4 was lower than those treated with, either AlCl3, or ApoEε4. The expression of total tau, tau-181, tau-231, tau-262, tau-396 and Aß in the cells treated with AlCl3 plus ApoEε4 were significantly higher than those in other groups (p < 0.05). Moreover, analyzing data based on the factorial design, there was existed an interaction between AlCl3 and ApoEε4 (p < 0.05). CONCLUSION: Al and ApoEε4 gene can cause morphological changes of SH-SY5Y cells, reduce cell activity, and have obvious cytotoxic effects, and increase the phosphorylation levels of tau and the deposition of Aß increases. In the presence of both Al and ApoEε4 genes, the two factors interact with each other and show a synergistic effect.

Whole-Genome Sequencing Reveals Diverse Models of Structural Variations in Esophageal Squamous Cell Carcinoma.

Comprehensive identification of somatic structural variations (SVs) and understanding their mutational mechanisms in cancer might contribute to understanding biological differences and help to identify new therapeutic targets. Unfortunately, characterization of complex SVs across the whole genome and the mutational mechanisms underlying esophageal squamous cell carcinoma (ESCC) is largely unclear. To define a comprehensive catalog of somatic SVs, affected target genes, and their underlying mechanisms in ESCC, we re-analyzed whole-genome sequencing (WGS) data from 31 ESCCs using Meerkat algorithm to predict somatic SVs and Patchwork to determine copy-number changes. We found deletions and translocations with NHEJ and alt-EJ signature as the dominant SV types, and 16% of deletions were complex deletions. SVs frequently led to disruption of cancer-associated genes (e.g., CDKN2A and NOTCH1) with different mutational mechanisms. Moreover, chromothripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally mis-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromothripsis-derived double-minute chromosome formation (e.g., FGFR1 and LETM2) or BFB-affected chromosomes (e.g., CCND1, EGFR, ERBB2, MMPs, and MYC), with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification. Furthermore, analyses of copy-number alterations reveal high frequency of whole-genome duplication (WGD) and recurrent focal amplification of CDCA7 that might act as a potential oncogene in ESCC. Our findings reveal molecular defects such as chromothripsis and BFB in malignant transformation of ESCCs and demonstrate diverse models of SVs-derived target genes in ESCCs. These genome-wide SV profiles and their underlying mechanisms provide preventive, diagnostic, and therapeutic implications for ESCCs.

Genomic analyses reveal FAM84B and the NOTCH pathway are associated with the progression of esophageal squamous cell carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the sixth most lethal cancer worldwide and the fourth most lethal cancer in China. Genomic characterization of tumors, particularly those of different stages, is likely to reveal additional oncogenic mechanisms. Although copy number alterations and somatic point mutations associated with the development of ESCC have been identified by array-based technologies and genome-wide studies, the genomic characterization of ESCCs from different stages of the disease has not been explored. Here, we have performed either whole-genome sequencing or whole-exome sequencing on 51 stage I and 53 stage III ESCC patients to characterize the genomic alterations that occur during the various clinical stages of ESCC, and further validated these changes in 36 atypical hyperplasia samples. RESULTS: Recurrent somatic amplifications at 8q were found to be enriched in stage I tumors and the deletions of 4p-q and 5q were particularly identified in stage III tumors. In particular, the FAM84B gene was amplified and overexpressed in preclinical and ESCC tumors. Knockdown of FAM84B in ESCC cell lines significantly reduced in vitro cell growth, migration and invasion. Although the cancer-associated genes TP53, PIK3CA, CDKN2A and their pathways showed no significant difference between stage I and stage III tumors, we identified and validated a prevalence of mutations in NOTCH1 and in the NOTCH pathway that indicate that they are involved in the preclinical and early stages of ESCC. CONCLUSIONS: Our results suggest that FAM84B and the NOTCH pathway are involved in the progression of ESCC and may be potential diagnostic targets for ESCC susceptibility.

Artesunate altered cellular mechanical properties leading to deregulation of cell proliferation and migration in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is one of the most common types of cancer in China. Artesunate (ART) is used clinically as an anti-malarial agent and exhibits potent antiproliferative activity. In addition, ART has demonstrated remarkable antitumor activity, presenting a novel candidate for cancer chemotherapy. However, its effect on ESCC remains unknown. The present study analyzed the antitumor effects of ART in the KYSE-150 ESCC line by assessing cell proliferation, cell death, cell migration/invasion and the biomechanical properties of ART-treated KYSE-150 cells. ART treatment significantly suppressed the proliferation of KYSE-150 cells in a dose- and time-dependent manner, as assessed by MTT assay. Following treatment with 30 mg/l ART, the cell population in the G0/G1 phase and the level of cell apoptosis significantly increased from 54±1.5 to 68.1±0.3%, and from 4.53±0.58 to 12.45±0.62%, respectively. Furthermore, the cell migration and invasion of KYSE-150 cells treated with 30 mg/l ART was markedly inhibited. The cell membrane and biomechanical properties were investigated using atomic force microscopy, as targets of ART action. ESCC cells treated with 30 mg/l ART exhibited increased adhesive force, increased cytomembrane roughness and reduced elasticity compared with the control group (KYSE-150 cells without ART treatment). The biomechanical properties of KYSE-150 cells treated with 30 mg/l ART were similar to those of the SHEE normal human esophageal epithelial cell line. In conclusion, the present study demonstrated that ART may inhibit cell proliferation and migration in ESCC through changes in the biomechanical properties of the ESCC cells.

[Study of negative feedback between wild-type BRAF or RAFV600E and Mps1 in melanoma].

OBJECTIVE: To study the effect of Mps1 on BRAFWT/MEK/ERK pathway in the presence of wild type BRAF or BRAFV600E in melanoma. METHODS: Melanoma cells harboring BRAFWT genotype were transfected either with pBabe-puro-GST-BRAF-WT and/or pBabe-puro-GFP-Mps1-WT or pBabe-puro-GST-BRAFV600E and/or pBabe-puro-GFP-Mps1-WT, followed by Western blot to detect Mps1 and p-ERK expression. The melanoma cells harboring BRAFWT and BRAFV600E genotype were infected with pSUPER-Mps1 retrovirus to knockdown the endogenous Mps1 protein, followed by Western blot to detect Mps1 and p-ERK expression. Meanwhile, melanoma cells harboring BRAFV600E genotype were infected with pBabe-puro-GFP-Mps1 and Western blot was performed to detect Mps1 and p-ERK expression. RESULTS: In melanoma cells harboring BRAFWT genotype and transfected with pBabe-puro-GST-BRAF-WT and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels were notably reduced as compared to either negative control or empty vector. However, cells transfected with pBabe-puro-GST-BRAFV600E and pBabe-puro-GFP-Mps1-WT, phospho-ERK levels did not change significantly compared with either negative control or empty vector. Knockout of Mps1 in BRAF wild-type cell lines led to an increased ERK activity. However, there was no significant change of ERK activity in BRAFV600E cell lines in the absence of Mps1. The expression of p-ERK in BRAFV600E mutant cell lines infected with pBabe-puro-GFP-Mps1-WT did not show any significant difference from either negative control or empty vector. CONCLUSIONS: Based on these findings, it suggests that there exists an auto-regulatory negative feedback loop between the Mps1 kinase and BRAFWT/ERK signaling. Oncogenic BRAFV600E abrogates the regulatory negative feedback loop of Mps1 on the MAPK pathway.

Oncogenic B-Raf(V600E) abrogates the AKT/B-Raf/Mps1 interaction in melanoma cells.

Activating B-Raf mutations that deregulate the mitogen-activated protein kinase (MAPK) pathway commonly occur in cancer. Although B-Raf(V600E) induces increased Mps1 protein contributing to centrosome amplification and chromosome instability, the regulatory mechanisms of Mps1 in melanoma cells is not fully understood. Here, we report that Mps1/AKT and B-Raf(WT)/ERK signaling form an auto-regulatory negative feedback loop in melanoma cells; notably, oncogenic B-Raf(V600E) abrogates the negative feedback loop, contributing the aberrant Mps1 functions and tumorigenesis. Our findings raise the possibility that targeting the oncogenic B-Raf and Mps1, especially when used in combination could potentially provide great therapeutic opportunities for cancer treatment.