ABSTRACT
Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments, ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.
Subject(s)
Annexin A3 , Hepacivirus , Hepatitis C , SS-B Antigen , Virus Internalization , Humans , Annexin A3/metabolism , Annexin A3/genetics , Autoantigens/metabolism , Autoantigens/genetics , HEK293 Cells , Hepacivirus/metabolism , Hepacivirus/physiology , Hepatitis C/metabolism , Hepatitis C/virology , Hepatitis C/genetics , Host-Pathogen Interactions , Lipid Droplets/metabolism , Lipid Droplets/virology , Ribonucleoproteins/metabolism , Ribonucleoproteins/genetics , Viral Core Proteins/metabolism , Viral Core Proteins/genetics , Viral Envelope Proteins/metabolism , Viral Envelope Proteins/geneticsABSTRACT
Acute lung injury (ALI) is a prevailing and deadly complication of sepsis coupled with increasing incidence and fatality rate. Annexin A3 (ANXA3) has been unraveled to be upregulated during sepsis. This study purposed to assess the role and the mechanism of ANXA3 in sepsis-induced ALI. After the construction of mouse model of sepsis, the pathological changes of mice lung tissues were estimated by H&E staining. ANXA3 expression in mice lung tissues and serum was examined. The degree of pulmonary edema and the levels of inflammatory factors in bronchoalveolar lavage fluid (BALF) were analyzed. In lipopolysaccharide (LPS)-induced mouse ALI model in vitro, CCK-8 assay measured cell viability and flow cytometry analysis detected cell apoptosis. Besides, ELISA assay detected the release of inflammatory cytokines. Western blot analyzed the expression of proteins associated with inflammation, apoptosis and extracellular-signal-regulated kinase (ERK)/ETS-like gene 1 (ELK1) signaling. Results revealed that ANXA3 was overexpressed in the lung tissues and serum of septic mice. Following the knockdown of ANXA3, sepsis-induced lung injury was alleviated, manifested as reduced lung edema, decreased inflammatory cell infiltration and inhibited cell apoptosis. Additionally, ANXA3 silence blocked ERK/ELK1 signaling both in sepsis mouse models and in vitro model of ALI induced by lipopolysaccharide (LPS). Moreover, the inhibitory effects of ANXA3 silencing on ERK/ELK1 signaling activation, the viability damage, inflammation and apoptosis in LPS-induced mouse ALI model in vitro were partially reversed by ERK activator. Collectively, depletion of ANXA3 exerted suppressive effects on the inflammation and apoptosis in sepsis-induced ALI through blocking ERK/ELK1 signaling.
Subject(s)
Acute Lung Injury , Sepsis , Animals , Mice , Acute Lung Injury/pathology , Annexin A3/metabolism , Apoptosis , Disease Models, Animal , Extracellular Signal-Regulated MAP Kinases/metabolism , Inflammation/pathology , Lipopolysaccharides/pharmacology , Lung/metabolism , Sepsis/metabolismABSTRACT
Tumor-associated macrophages (TAM) induce immunosuppression in laryngeal squamous cell carcinoma (LSCC). The interaction between LSCC cells and TAMs affects the progression of laryngeal cancer through exosomes, but the underlying molecular mechanism remains unclear. Proteomics analysis of TAMs isolated from human laryngeal tumor tissues obtained from patients with confirmed lymphatic metastasis revealed an upregulation of annexin A3 (ANXA3). In TAMs, ANXA3 promoted macrophages to polarize to an M2-like phenotype by activating the AKT-GSK3ß-ß-catenin pathway. In addition, ANXA3-rich exosomes derived from TAMs inhibited ferroptosis in laryngeal cancer cells through an ATF2-CHAC1 axis, and this process was associated with lymphatic metastasis. Mechanistically, ANXA3 in exosomes inhibited the ubiquitination of ATF2, whereas ATF2 acted as a transcription factor to regulate the expression of CHAC1, thus inhibiting ferroptosis in LSCC cells. These data indicate that abnormal ANXA3 expression can drive TAM reprogramming and promote an immunosuppressive microenvironment in LSCC. Meanwhile, ANXA3-rich exosomes inhibit ferroptosis of LSCC cells and promote lymphatic metastasis, thus promoting tumor progression.
Subject(s)
Annexin A3 , Exosomes , Ferroptosis , Laryngeal Neoplasms , Tumor-Associated Macrophages , Animals , Humans , Male , Mice , Annexin A3/metabolism , Cell Line, Tumor , Exosomes/metabolism , Laryngeal Neoplasms/pathology , Laryngeal Neoplasms/metabolism , Laryngeal Neoplasms/immunology , Lymphatic Metastasis/immunology , Lymphatic Metastasis/pathology , Squamous Cell Carcinoma of Head and Neck/metabolism , Squamous Cell Carcinoma of Head and Neck/pathology , Squamous Cell Carcinoma of Head and Neck/immunology , Tumor Microenvironment/immunology , Tumor-Associated Macrophages/metabolism , Tumor-Associated Macrophages/immunologyABSTRACT
BACKGROUND: Pyroptosis plays an important role in the pathological process of ischemic stroke (IS). However, the exact mechanism of pyroptosis remains unclear. This paper aims to reveal the key molecular markers associated with pyroptosis in IS. METHODS: We used random forest learning, gene set variation analysis, and Pearson correlation analysis to screen for biomarkers associated with pyroptosis in IS. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen and glucose deprivation/reoxygenation (OGD/R) models were constructed in vitro and in vivo. Cells were transfected with an Annexin A3 silencing (si-ANXA3) plasmid to observe the effects of ANXA3 on OGD/R + lipopolysaccharides (LPS)-induced pyroptosis. qRTâPCR and western blotting were used to detect the expression of potential biomarkers and pyroptotic pathways. RESULTS: Samples from a total of 170 IS patients and 109 healthy individuals were obtained from 5 gene expression omnibus databases. Thirty important genes were analyzed by random forest learning from the differentially expressed genes. Then, we investigated the relationship between the above genes and the pyroptosis score, obtaining three potential biomarkers (ANXA3, ANKRD22, ADM). ANXA3 and ADM were upregulated in the MCAO/R model, and the fold difference in ANXA3 expression was greater. Pyroptosis-related factors (NLRP3, NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, IL-1ß, and IL-18) were upregulated in the MCAO/R model. Silencing ANXA3 alleviated the expression of pyroptosis-related factors (NLRC4, AIM2, GSDMD-N, caspase-8, pro-caspase-1, cleaved caspase-1, and IL-18) induced by OGD/R + LPS or MCAO/R. CONCLUSION: This study identified ANXA3 as a possible pyroptosis-related gene marker in IS through bioinformatics and experiments. ANXA3 could inhibit pyroptosis through the NLRC4/AIM2 axis.
Subject(s)
Ischemic Stroke , Reperfusion Injury , Humans , Pyroptosis/genetics , Interleukin-18/metabolism , Interleukin-18/pharmacology , Caspase 1/metabolism , Caspase 1/pharmacology , Caspase 8/metabolism , Caspase 8/pharmacology , Ischemic Stroke/genetics , Lipopolysaccharides/pharmacology , Biomarkers , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Annexin A3/genetics , Annexin A3/metabolism , Annexin A3/pharmacologyABSTRACT
BACKGROUND: Cervical cancer, as one of the most common cancers in women, remains a major health threat worldwide. Annexin A3 (ANXA3), a component of the annexin family, is upregulated in numerous cancers, with no explicit role in cervical cancer. OBJECTIVE: This study aims to investigate the function of ANXA3 in cervical cancer. METHODS: Differential expression genes between the cervical cancer tissues of patients and the controls were analyzed in The Cancer Genome Atlas (TCGA) and Gene Expression Profiling Interactive Analysis (GEPIA) database. Using transfection approaches to either upregulate or downregulate ANXA3, its role in cell proliferation and chemosensitivity of human cervical cancer cell lines (HeLa and C33A) was evaluated. Furthermore, the binding activity between YAP1 and ANXA3 was also explored. RESULTS: Genomics analysis indicated that differential genes were mostly associated with cell cycle progression and DNA replication. ANXA3 was highly expressed in the cervical cancer tissues and closely linked to malignancy degree. Knockdown of ANXA3 in cervical cancer cells inhibited cell cycle progression. A similar result was observed in the reduction of cyclin D, CDK4, cyclin E, and CDK2 in cervical cancer cells with ANXA3 silencing. Cervical cancer cells obtained high sensitivity to cisplatin (DDP) when ANXA3 was downregulated. Conversely, these capabilities were the opposite in cervical cancer cells overexpressing ANXA3. Furthermore, the expression levels of ANXA3 and YAP1 were positively correlated. YAP1 upregulation was positively connected with malignant behaviors, which were reversed by ANXA3 downregulation. CONCLUSION: In light of our findings, targeting ANXA3 expressed in cervical cancer might contribute to more potential therapeutic strategies.
Subject(s)
Annexin A3 , Uterine Cervical Neoplasms , Female , Humans , Annexin A3/genetics , Annexin A3/metabolism , Cell Line, Tumor , Cell Proliferation/genetics , Drug Resistance, Neoplasm/genetics , Uterine Cervical Neoplasms/drug therapy , Uterine Cervical Neoplasms/geneticsABSTRACT
BACKGROUND: Osteosarcoma (OS) represents a common type of bone cancer. Long non-coding RNAs (LncRNAs) have shown their potential in therapeutic modalities for OS. This study's purpose was to reveal the action of lncRNA EBLN3P on OS growth and metastasis and its mechanism. METHODS: Expressions of EBLN3P/Hu antigen R (HuR)/Annexin A3 (ANXA3) were determined by RT-qPCR/Western blot. Proliferation/migration/invasion of OS cells were assessed via CCK-8/Transwell assays after interfering EBLN3P/ANXA3/HuR. The co-localization of EBLN3P/ANXA3/HuR cells was observed by FISH/immunofluorescence assays. Interplays among EBLN3P/ANXA3/HuR and the half-life period of ANXA3 were assessed by RNA immunoprecipitation/RNA pull-down/RNA stability experiment. The nude mouse xenograft model was established, followed by EBLN3P treatment to assess the function of EBLN3P on OS. RESULTS: EBLN3P/ANXA3 was highly expressed in OS cells. Silencing EBLN3P or ANXA3 limited the proliferation/migration/invasion of OS cells. Mechanically, EBLN3P/ANXA3 can bind to HuR, and EBLN3P enhanced ANXA3 mRNA stability by recruiting HuR, thus facilitating OS cell growth. Upregulated HuR or ANXA3 counteracted the suppressive action of silencing EBLN3P on OS cells. In vivo experiments revealed facilitated tumor growth and metastasis in vivo fomented by EBLN3P through manipulation of HuR/ANXA3. CONCLUSIONS: EBLN3P enhanced proliferative/migrative/invasive potentials of OS cells via increasing ANXA3 mRNA stability and protein level by recruiting HuR, which provided new potential therapeutic targets for OS clinical treatment. EBLN3P and ANXA3 might have potential roles in OS diagnosis, treatment, and prognosis. This study provided a theoretical reference for further clinical research in tumor surgery.
Subject(s)
Bone Neoplasms , Osteosarcoma , RNA, Long Noncoding , Animals , Mice , Humans , RNA, Long Noncoding/genetics , Cell Line, Tumor , Annexin A3 , Osteosarcoma/genetics , Cell Proliferation/genetics , Bone Neoplasms/genetics , Cell Movement/genetics , Gene Expression Regulation, NeoplasticABSTRACT
Annexin A3 (ANXA3), a member of Annexin family, is reported to mediate membrane transport and cancer development. However, the effect of ANXA3 on osteoclast formation and bone metabolism is still unclear. In this study, we found that knockdown of ANXA3 can significantly inhibit receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation through NF-κB signaling. ANXA3 downregulation abrogated the expression of osteoclast-specific genes, including Acp5, Mmp9 and Ctsk in osteoclast precursors. Moreover, lentiviral of shRNA against ANXA3 reversed the bone loss in osteoporosis using ovariectomized mice model. Mechanistically, we found that ANXA3 directly bound to RANK and TRAF6 to accelerate osteoclast differentiation by promoting their transcription and limiting degradation. In conclusion, we propose a fundamentally novel RANK-ANXA3-TRAF6 complex to effectively modulate the formation and differentiation of osteoclast to manipulate bone metabolism. The ANXA3-targeted therapeutic strategy may provide new insight for bone degrading-related diseases prevention and treatment.
Subject(s)
Bone Resorption , Osteoclasts , Mice , Animals , Osteoclasts/metabolism , TNF Receptor-Associated Factor 6/metabolism , Annexin A3/metabolism , Annexin A3/pharmacology , Bone and Bones/metabolism , Signal Transduction , NF-kappa B/metabolism , RANK Ligand/metabolism , Cell Differentiation , Bone Resorption/metabolism , OsteogenesisABSTRACT
Diabetes mellitus is a metabolic disorder that increases fracture risk and interferes with bone formation and impairs fracture healing. Genomic studies on diabetes and fracture healing are lacking. We used a weighted co-expression network analysis (WGCNA) method to identify susceptibility modules and hub genes associated with T2DM and fracture healing. First, we downloaded the GSE95849, GSE93213, GSE93215, and GSE142786 data from the Gene Expression Omnibus (GEO) website, analyzed differential expression genes and constructed a WGCNA network. Second, we screened out 30 hub genes, which were found to be enriched in neutrophil activation, translational initiation, RAGE receptor binding, propanoate metabolism, and other pathways through Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and gene set enrichment analysis (GSEA) analyses. Third, we searched for genes related to bone metabolism and fracture healing in the published genome-wide single nucleotide polymorphism (SNP) data, built a protein-protein interaction (PPI) network with hub genes, and found that they were associated with metabolic process, blood vessel development, and extracellular matrix organization. ANXA3 was identified as the biomarker based on gene expression and correlation analysis. And the AUC value of it was 0.947. Fourth, we explored that ANXA3 was associated with neutrophils in fracture healing process by single-cell RNA sequencing analysis. Finally, we collected clinical patient samples and verified the expression of ANXA3 by qRT-PCR in patents with T2DM and fracture non-union. In conclusion, this is the first genomics study on the effect of T2DM on fracture healing. Our study identified some characteristic modules and hub genes in the etiology of T2DM-associated fracture non-union, which may help to further investigate the molecular mechanisms. Up-regulated ANXA3 potentially contributed to fracture non-union in T2DM by mediating neutrophils. It can be a prognostic biomarker and potential therapeutic target.
Subject(s)
Diabetes Mellitus, Type 2 , Gene Regulatory Networks , Annexin A3 , Biomarkers , Gene Expression Profiling/methods , Gene Ontology , HumansABSTRACT
Exosomes play a critical role in intracellular communication. The biogenesis and function of exosomes are regulated by multiple biochemical factors. In the present study, we find that mechanical force promotes the biogenesis of exosomes derived from periodontal ligament stem cells (PDLSCs) and alters the exosomal proteome profile to induce osteoclastic differentiation. Mechanistically, mechanical force increases the level of exosomal proteins, especially annexin A3 (ANXA3), which facilitates exosome internalization to activate extracellular signal-regulated kinase (ERK), thus inducing osteoclast differentiation. Moreover, the infusion of exosomes derived from PDLSCs into mice promotes mechanical force-induced tooth movement and increases osteoclasts in the periodontal ligament. Collectively, this study demonstrates that mechanical force treatment promotes the biogenesis of exosomes from PDLSCs and increases exosomal protein ANXA3 to facilitate exosome internalization, which activates ERK phosphorylation, thus inducing osteoclast differentiation. Our findings shed light on new mechanisms for how mechanical force regulates the biology of exosomes and bone metabolism.
Subject(s)
Annexin A3 , Periodontal Ligament , Animals , Annexin A3/metabolism , Cell Differentiation/physiology , Extracellular Signal-Regulated MAP Kinases/metabolism , Mice , Osteoclasts , Osteogenesis/physiology , Stem Cells/metabolismABSTRACT
Radioresistance currently poses a significant challenge to successful disease control of nasopharyngeal carcinoma (NPC). We previously uncovered that annexin A3 (ANXA3), a calcium-dependent phospholipid binding protein, is underexpressed in radioresistant NPC cells and mouse xenografts. This study aims to further unravel the mechanistic basis underlying ANXA3-mediated radioresistance in NPC. We show that either innate ANXA3 downregulation or short hairpin RNA(shRNA)-based knockdown of ANXA3 confers resistance to ionizing radiation (IR) in NPC both in vitro and in mouse xenograft models in vivo, whereas radiosensitization was observed when ANXA3 was ectopically expressed. Mechanistically, ANXA3 knockdown dramatically enhances IR-induced epidermal growth factor receptor (EGFR) phosphorylation and nuclear translocation, leading to increased post-IR phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) concomitant with markedly accelerated DNA DSB repair. In addition, pretreatment with cetuximab efficiently abrogated the radioresistant phenotype of ANXA3-low cells as well as the ANXA3 knockdown-induced post-IR EGFR nuclear accumulation, suggesting that EGFR is an essential mediator for ANXA3 depletion-mediated radioprotection in NPC. Collectively, this work reveals for the first time a critical role of ANXA3 in radiation survival and DNA repair mechanism of NPC and provides mechanistic evidence to support ANXA3 as a potential therapeutic target to improve radiocurability for NPC.
Subject(s)
Annexin A3 , Nasopharyngeal Neoplasms , Animals , Annexin A3/genetics , Annexin A3/metabolism , Cell Line, Tumor , DNA , Down-Regulation/genetics , ErbB Receptors/genetics , ErbB Receptors/metabolism , Gene Expression Regulation, Neoplastic , Humans , Mice , Nasopharyngeal Carcinoma/genetics , Nasopharyngeal Carcinoma/pathology , Nasopharyngeal Carcinoma/radiotherapy , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/radiotherapy , RNA, Small Interfering , Radiation Tolerance/genetics , Radiation, Ionizing , Xenograft Model Antitumor AssaysABSTRACT
Introduction: The specific pathogenesis of ankylosing spondylitis (AS) remains unclear, and our study aimed to investigate the possible pathogenesis of AS. Materials and Methods: Two datasets were downloaded from the GEO database to perform differentially expressed gene analysis, GO enrichment analysis, KEGG pathway analysis, DO enrichment analysis, GSEA analysis of differentially expressed genes, and construction of diagnostic genes using SVM and WGCNA along with Hypoxia-related genes. Also, drug sensitivity analysis was performed on diagnostic genes. To identify the differentially expressed immune genes in the AS and control groups, we analyzed the composition of immune cells between them. Then, we examined differentially expressed genes in three AS interspinous ligament specimens and three Degenerative lumbar spine specimens using high-throughput sequencing while the immune cells were examined using the neutrophil count data from routine blood tests of 1770 HLA-B27-positive samples and 7939 HLA-B27-negative samples. To assess the relationship between ANXA3 and SORL1 and disease activity, we took the neutrophil counts of the first 50 patients with above-average BASDAI scores and the last 50 patients with below-average BASDAI scores for statistical analysis. We used immunohistochemistry to verify the expression of ANXA3 and SORL1 in AS and in controls. Results: ANXA3 and SORL1 were identified as new diagnostic genes for AS. These two genes showed a significant differential expression between AS and controls, along with showing a significant positive correlation with the neutrophil count. The results of high-throughput sequencing verified that these two gene deletions were indeed differentially expressed in AS versus controls. Data from a total of 9707 routine blood tests showed that the neutrophil count was significantly higher in AS patients than in controls (p < 0.001). Patients with AS with a high BASDAI score had a much higher neutrophil count than those with a low score, and the difference was statistically significant (p < 0.001). The results of immunohistochemistry showed that the expression of ANXA3 and SORL1 in AS was significantly higher than that in the control group. Conclusion: Upregulated of ANXA3, SORL1, and neutrophils may be a key factor in the progression of Ankylosing spondylitis.
Subject(s)
Spondylitis, Ankylosing , Annexin A3/metabolism , HLA-B27 Antigen/genetics , Humans , LDL-Receptor Related Proteins , Leukocyte Count , Membrane Transport Proteins , Neutrophils/metabolism , Spondylitis, Ankylosing/diagnosisABSTRACT
Cerebral amyloid angiopathy (CAA), characterized by cerebral vascular amyloid accumulation, neuroinflammation, microbleeds, and white matter (WM) degeneration, is a common comorbidity in Alzheimer disease and a prominent contributor to vascular cognitive impairment and dementia. WM loss was recently reported in the corpus callosum (CC) in the rTg-DI rat model of CAA. The current study shows that the CC exhibits a much lower CAA burden compared with the adjacent cortex. Sequential Window Acquisition of All Theoretical Mass Spectra tandem mass spectrometry was used to show specific proteomic changes in the CC with emerging WM loss and compare them with the proteome of adjacent cortical tissue in rTg-DI rats. In the CC, annexin A3, heat shock protein ß1, and cystatin C were elevated at 4 months (M) before WM loss and at 12M with evident WM loss. Although annexin A3 and cystatin C were also enhanced in the cortex at 12M, annexin A5 and the leukodystrophy-associated astrocyte proteins megalencephalic leukoencephalopathy with subcortical cysts 1 and GlialCAM were distinctly elevated in the CC. Pathway analysis indicated neurodegeneration of axons, reflected by reduced expression of myelin and neurofilament proteins, was common to the CC and cortex; activation of Tgf-ß1 and F2/thrombin was restricted to the CC. This study provides new insights into the proteomic changes that accompany WM loss in the CC of rTg-DI rats.
Subject(s)
Cerebral Amyloid Angiopathy , White Matter , Animals , Annexin A3/metabolism , Brain/metabolism , Cerebral Amyloid Angiopathy/metabolism , Cystatin C/metabolism , Proteomics , Rats , White Matter/metabolismABSTRACT
The parenchymal microglia possess different morphological characteristics in cerebral physiological and pathological conditions; thus, visualizing these cells is useful as a means of further investigating parenchymal microglial function. Annexin A3 (ANXA3) is expressed in microglia, but it is unknown whether it can be used as a marker protein for microglia and its physiological function. Here, we compared the distribution and morphology of parenchymal microglia labeled by ANXA3, cluster of differentiation 11b (CD11b), and ionized calcium-binding adaptor molecule 1 (Iba1) and measured the expression of ANXA3 in nonparenchymal macrophages (meningeal and perivascular macrophages). We also investigated the spatiotemporal expression of ANXA3, CD11b, and Iba1 in vivo and in vitro and the cellular function of ANXA3 in microglia. We demonstrated that ANXA3-positive cells were abundant and evenly distributed throughout the whole brain tissue and spinal cord of adult rats. The morphology and distribution of ANXA3-labeled microglia were quite similar to those labeled by the microglial-specific markers CD11b and Iba1 in the central nervous system (CNS). ANXA3 was expressed in the cytoplasm of microglia, and its expression was significantly increased in activated microglia. ANXA3 was almost undetectable in the nonparenchymal macrophages. Meanwhile, the protein and mRNA expression levels of ANXA3 in different regions of the CNS were different from those of CD11b and Iba1. Moreover, knockdown of ANXA3 inhibited the proliferation and migration of microglia, while overexpression of ANXA3 enhanced these activities. This study confirms that ANXA3 may be a novel marker for parenchymal microglia in the CNS of adult rats and enriches our understanding of ANXA3 from expression patterns to physiological function.
Subject(s)
Annexin A3/analysis , Central Nervous System/cytology , Microglia/chemistry , Nerve Tissue Proteins/analysis , Animals , Annexin A3/biosynthesis , Annexin A3/genetics , Biomarkers , CD11b Antigen/biosynthesis , CD11b Antigen/genetics , Calcium-Binding Proteins/biosynthesis , Calcium-Binding Proteins/genetics , Cell Cycle , Cell Movement , Cells, Cultured , Gene Knockdown Techniques , Genetic Vectors , Infarction, Middle Cerebral Artery/pathology , Lentivirus , Macrophages/chemistry , Microfilament Proteins/biosynthesis , Microfilament Proteins/genetics , Organ Specificity , RNA, Messenger/biosynthesis , Rats , Rats, Sprague-Dawley , TransfectionABSTRACT
Esophageal squamous cell carcinoma (ESCC) is one of the lethal malignancies commonly found in the eastern world, with overall five-year survival rates less than 25%. The present study aimed to investigate the biological function of annexin A3 (ANXA3) in ESCC cell proliferation. The mRNA and protein levels of ANXA3 in ESCC tissues and cell lines were determined by real-time PCR and Western blot, respectively. Lentiviral transduction was applied to overexpress or reduce ANXA3 expression in ESCC cell lines. The effect of ANXA3 on ESCC cell proliferation was evaluated by cell-counting kit-8 assay in vitro and tumor-bearing animal model in vivo. We found that ANXA3 was substantially upregulated in ESCC tissues compared to adjacent normal tissues as well as ESCC cell lines compared to normal esophageal endothelial cells. Suppression of ANXA3 significantly inhibited ESCC cell proliferation in vitro and tumor growth in vivo. We further revealed that NF-κB was involved in ANXA3-mediated ESCC cell proliferation. Our results suggest that ANXA3 acts as an oncogene in ESCC, and targeting ANXA3 or NF-κB may serve as potential therapeutic strategies for patients with ESCC.
Subject(s)
Annexin A3/physiology , Esophageal Neoplasms/metabolism , Esophageal Squamous Cell Carcinoma/metabolism , NF-kappa B/metabolism , Signal Transduction , Animals , Cell Line, Tumor , Humans , Mice , Mice, Inbred BALB C , Mice, NudeABSTRACT
Introduction: As a member of annexin family proteins, annexin A3 (ANXA3) has 36-kDa and 33-kDa isoforms. ANXA3 plays crucial roles in the tumorigenesis, aggressiveness and drug-resistance of cancers. However, previous studies mainly focused on the role of total ANXA3 in cancers without distinguishing the distinction between the two isoforms, the role of 33-kDa ANXA3 in cancer remains unclear. Objectives: Current work aimed to investigate the function and regulation mechanism of 33-kDa ANXA3 in hepatocarcinoma. Methods: The expressions of ANXA3, CRKL, Rac1, c-Myc and pAkt were analyzed in hepatocarcinoma specimens by Western blotting. The biological function of 33-kDa ANXA3 in the growth, metastasis, apoptosis, angiogenesis, chemoresistance of hepatocarcinoma cells with the underlying molecular mechanism were investigated using gain-of-function strategy in vitro or in vivo. Results: 33-kDa ANXA3 was remarkably upregulated in tumor tissues compared with corresponding normal liver tissues of hepatocarcinoma patients. Its stable knockdown decreased the in vivo tumor growing velocity and malignancy of hepatocarcinoma HepG2 cells transplanted in nude mice. The in vitro experimental results indicated 33-kDa ANXA3 knockdown suppressed the proliferation, colony forming, migration and invasion abilities of HepG2 cells through downregulating CRKL, Rap1b, Rac1, pMEK, pERK2 and c-Myc in ERK pathway; inhibited angiogenesisability of HepG2 cells through inactivating PI3K/Akt-HIF pathway; induced apoptosis and enhanced chemoresistance of HepG2 cells through increasing Bax/decreasing Bcl-2 expressions and inactivating caspase 9/caspase 3 in intrinsic apoptosis pathway. Accordingly, CRKL, Rac1, c-Myc and pAkt were also upregulated in hepatocarcinoma patients ' tumor tissues compared with corresponding normal liver tissues. Conclusions: The overexpression of 33-kDa ANXA3 is involved in the clinical progression of hepatocarcinoma and in the malignancy, angiogenesis and apoptosis of hepatocarcinoma cells. It is of potential use in hepatocarcinoma diagnosis and treatment.
Subject(s)
Annexin A3/metabolism , Apoptosis , Carcinoma, Hepatocellular/metabolism , Liver Neoplasms/metabolism , Animals , Carcinogenesis/metabolism , Carcinoma, Hepatocellular/pathology , Cell Movement , Cell Proliferation , Drug Resistance, Neoplasm , Female , Hep G2 Cells , Humans , Liver Neoplasms/pathology , MAP Kinase Signaling System , Male , Mice , Mice, Nude , Middle Aged , Phosphatidylinositol 3-Kinases/metabolism , Protein Isoforms/metabolism , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL) and can be classified into two types based on the amino acid at position 233 in Tax protein, which probably plays crucial roles in leukemogenesis. We previously revealed that L233-Tax-expressing cells secreted chemoattractants for endothelial cells and formed significantly larger tumors accompanying neovascularization than P233-Tax-expressing cells in athymic mice. In the present study, comparative proteomic analysis of the culture medium of Tax-expressing cells revealed that annexin A3 and probably extracellular matrix protein 1 served as chemoattractants. Conversely, L233-Tax-expressing cells were impaired in the secretion of collagen alpha-1 (I) chain precursor, which participates in tissue tension homeostasis, leading to tumor mass development. The analysis also demonstrated that both L233-Tax- and P233-Tax-expressing cells had deficits in the secretion of potentially antiangiogenic molecules, including pigment epithelium-derived factor and collagen alpha-1 (VIII) chain, and they produced complement component 3, which might participate in tumor cell proliferation, metastasis, and immune evasion. These findings provided novel insights into prognostication of EBL and the function of Tax in leukemogenesis induced by BLV.
Subject(s)
Annexin A3/metabolism , Collagen Type I/metabolism , Enzootic Bovine Leukosis/virology , Gene Products, tax/metabolism , Leukemia Virus, Bovine/genetics , Amino Acid Substitution , Animals , Cattle , Cell Line , Endothelial Progenitor Cells/metabolism , Endothelial Progenitor Cells/virology , Fibroblasts/metabolism , Fibroblasts/virology , Gene Products, tax/genetics , Mutation , Proteomics , RatsABSTRACT
Idiopathic pulmonary fibrosis (IPF) is a lethal, agnogenic interstitial lung disease with limited therapeutic options. To investigate vital genes involved in the development of IPF, we integrated and compared four expression profiles (GSE110147, GSE53845, GSE24206, and GSE10667), including 87 IPF samples and 40 normal samples. By reanalyzing these datasets, we managed to identify 62 upregulated genes and 20 downregulated genes in IPF samples compared with normal samples. Differentially expressed genes (DEGs) were analyzed by gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to illustrate relevant pathways of IPF, biological processes, molecular function, and cell components. The DEGs were then subjected to protein-protein interaction (PPI) for network analysis, serving to find 11 key candidate genes (ANXA3, STX11, THBS2, MMP1, MMP9, MMP7, MMP10, SPP1, COL1A1, ITGB8, IGF1). The result of RT-qPCR and immunohistochemical staining verified our finding as well. In summary, we identified 11 key candidate genes related to the process of IPF, which may contribute to novel treatments of IPF.
Subject(s)
Idiopathic Pulmonary Fibrosis/genetics , Annexin A3/genetics , Collagen Type I/genetics , Collagen Type I, alpha 1 Chain , Databases, Genetic , Gene Expression Profiling , Gene Ontology , Humans , Insulin-Like Growth Factor I/genetics , Integrin beta Chains/genetics , Matrix Metalloproteinases/genetics , Osteopontin/genetics , Protein Interaction Maps , Qa-SNARE Proteins/genetics , Thrombospondins/geneticsABSTRACT
Storeoperated calcium entry (SOCE) is important for refilling endoplasmic reticulum (ER) Ca2+ stores and Ca2+ signaling. Extracellular Ca2+ is conducted by highly Ca2+selective Orai channels that are activated by stromal interaction molecule proteins (STIMs), which are sensitive ER Ca2+ sensors. We found an approximately fivefold increase in annexin A3a (anxa3a) expression levels in stim2b knockout zebrafish. The present study investigated whether annexin A3 protein is involved in SOCE. We used Ca2+ imaging and electrophysiological recordings to determine the effect of annexin A2, A3, and A6 overexpression on SOCE and Orai-dependent Ca2+ currents (ICRAC) in cultured cells. Annexin A3 increased SOCE amplitude and potentiated Ca2+ ICRAC currents. These results suggest that annexin A3 is a positive modulator of SOCE.
Subject(s)
Annexin A3/metabolism , Calcium Channels , Calcium , Animals , Calcium/metabolism , Calcium Channels/genetics , Calcium Channels/metabolism , Calcium Signaling , ORAI1 Protein , Stromal Interaction Molecule 1/genetics , Zebrafish/metabolismABSTRACT
BACKGROUND: Melanoma, a relatively common malignancy, has become one of the tumors with the fastest rising incidence in recent years. The purpose of this study was to investigate the effect of Microglial Annexin A3 (ANXA3) on melanoma. METHODS: Serum samples were obtained from 20 patients with melanoma or 20 healthy controls. Kaplan-Meier survival analysis was performed. Transcriptome were used to analyze the correlation between ANXA3 expression and overall survival in patients with melanoma. Human melanoma cell lines WM-115 cells were transfected with ANXA3, si-ANXA3, ANXA3 + si-hypoxia inducible factor-1α (HIF-1α), si-ANXA3 + HIF-1α, and negative plasmids. Cell proliferation assay, cell invasion assay, and wound healing assay were performed on WM-115 cells. Lactate dehydrogenase (LDH) and caspase-3/9 activities were detected by commercial kits. Western blot and RT-PCR were used to detect the protein and mRNA expression of relation factors. RESULTS: ANXA3 expression was up-regulated in patients with melanoma in comparison with healthy controls. Over-expression of ANXA3 promoted cell growth and migration, and reduced cytotoxicity of WM-115 cells. Overall survival (OS) and disease-free survival (DFS) of patients with high ANXA3 expression were both lower than those of patients with low ANXA3 expression. Down-regulation of ANXA3 reduced cell growth and migration, and promoted cytotoxicity of WM-115 cells. ANXA3 induced vascular endothelial growth factor (VEGF) signaling pathway by activation of HIF-1α. CONCLUSION: In conclusion, our results indicated that ANXA3 promoted cell growth and migration of melanoma via activation of HIF-1α/VEGF signaling pathway.
Subject(s)
Annexin A3/metabolism , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Melanoma/metabolism , Melanoma/pathology , Skin Neoplasms/metabolism , Skin Neoplasms/pathology , Vascular Endothelial Growth Factor A/metabolism , Annexin A3/blood , Annexin A3/genetics , Case-Control Studies , Cell Line, Tumor , Cell Movement , Humans , Melanoma/mortality , Skin Neoplasms/mortality , Melanoma, Cutaneous MalignantABSTRACT
Accumulating evidence has indicated that inflammation is required for the initiation and progression of hepatocellular carcinoma (HCC). The annexin family protein, which has a highly similar structure, has been demonstrated to participate in pro- or anti-inflammatory regulation in the developing of tumours. However, the potential effects of ANXA3 in the immune microenvironment of HCC remain unknown. In present study, we found that increased ANXA3 expression is associated with a higher infiltrated neutrophil-lymphocyte ratio (iNLR) in HCC. Moreover, HCC patients with a high iNLR and high ANXA3 expression confer the highest risk of death. ANXA3 can be detected in both cell lysates and culture supernatants. However, the secretory ANXA3 did not directly regulate the iNLR. Further study demonstrated that ANXA3 upregulated the iNLR by inducing chemokine CXCL8 and CCL25 release from HCC cells. We further confirmed that ANXA3 promotes tumourigenesis and detected the same associations between ANXA3 and the iNLR or chemokines in vivo. Our findings indicate that ANXA3 regulates the chemokine to remodel the iNLR and promotes tumourigenicity in HCC. These results further expanded our understanding of ANXA3 in the microenvironment of HCC and might provide novel targets for the investigation of molecular treatments for HCC patients.