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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.

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 β1, 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-β1 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.

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.

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.

Recent, research has displayed that the disorders of miR-18b are related to ischemic stroke. Here, we aimed to investigate the underlying neuroprotective mechanism of miR-18b in cerebral ischemia/reperfusion (I/R) injury. Oxygen-glucose deprivation/reperfusion (OGDR) model in vitro and middle cerebral artery occlusion (MCAO) model in vivo were established to simulate cerebral I/R injury. RT-PCR, western blotting, CCK-8, TUNEL, and TTC staining assays were applied in this study to explore the effect of miR-18b on cerebral I/R injury. Results displayed that miR-18b expression was reduced after cerebral I/R injury. Besides, miR-18b showed neuroprotective effects on cerebral I/R injury both in vitro and in vivo, These neuroprotective effects included promoting cell viability, decreasing cell apoptosis, reducing the production of inflammatory cytokines in SH-SY 5Y cells after OGDR and depressing MCAO-induced infarct size, neurological deficits and apoptotic cells in mice. Moreover, miR-18b negatively regulated ANXA3 expression, and its neuroprotection on cerebral I/R injury was overturned by ANXA3. Additionally, increasing miR-18b or decreasing ANXA3 promoted the activation of the PI3K/Akt signaling pathway in SH-SY 5Y cells after cerebral I/R injury. In conclusion, these data indicate that miR-18b protects against cerebral I/R injury by inhibiting ANXA3 and activating PI3K/Akt pathway, which provides a promising therapeutic target for ischemic stroke therapy.

Bone cancer-induced pain (BCP) is a challenging clinical problem because traditional therapies are often only partially effective. Annexin A3 (ANXA3) is highly expressed in microglia in the spinal cord, and its expression is upregulated during BCP. However, the roles of microglial ANXA3 in the development and maintenance of BCP and the underlying molecular mechanisms remain unclear. This study was performed on male mice using a metastatic lung BCP model. Adeno-associated virus shANXA3 (AAV-shANXA3) was injected intrathecally 14 days before and 7 days after bone cancer induction, and relevant pain behaviors were assessed by measuring the paw withdrawal mechanical threshold, paw withdrawal thermal latency, and spontaneous hind limb lifting. ANXA3 protein expression was downregulated in microglial N9 cells by lentiviral transfection (LV-shANXA3). ANXA3, hypoxia-inducible factor-1α (Hif-1α), vascular endothelial growth factor (VEGF) expression levels, and Hif-1α transactivation activity regulated by ANXA3 were measured. As a result, ANXA3 was expressed in microglia, and its expression significantly increased during BCP. ANXA3 knockdown reversed pain behaviors but did not prevent pain development. Moreover, ANXA3 knockdown significantly reduced Hif-1α and VEGF expression levels in vitro and in vivo. And overexpression of Hif-1α or VEGF blocked the effects of AAV-shANXA3 on BCP. ANXA3 knockdown in N9 cells significantly decreased the p-PKC protein expression in the cocultured neurons. Finally, ANXA3 overexpression significantly increased Hif-1α transactivation activity in 293T cells. Therefore, microglial ANXA3 downregulation alleviates BCP by inhibiting the Hif-1α/VEGF signaling pathway, which indicates that ANXA3 may be a potential target for the treatment of BCP.

Background Cystic fibrosis (CF) remains without a definitive cure. Novel therapeutics targeting the causative defect in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are in clinical use. Lumacaftor/ivacaftor is a CFTR modulator approved for patients homozygous for the CFTR variant p.Phe508del, but there are wide variations in treatment responses preventing prediction of patient responses. We aimed to determine changes in gene expression related to treatment initiation and response. Methods Whole-blood transcriptomics was performed using RNA-Seq in 20 patients with CF pre- and 6 months post-lumacaftor/ivacaftor (drug) initiation and 20 non-CF healthy controls. Correlation of gene expression with clinical variables was performed by stratification via clinical responses. Results We identified 491 genes that were differentially expressed in CF patients (pre-drug) compared with non-CF controls and 36 genes when comparing pre-drug to post-drug profiles. Both pre- and post-drug CF profiles were associated with marked overexpression of inflammation-related genes and apoptosis genes, and significant under-expression of T cell and NK cell-related genes compared to non-CF. CF patients post-drug demonstrated normalized protein synthesis expression, and decreased expression of cell-death genes compared to pre-drug profiles, irrespective of clinical response. However, CF clinical responders demonstrated changes in eIF2 signaling, oxidative phosphorylation, IL-17 signaling, and mitochondrial function compared to non-responders. Top overexpressed genes (MMP9 and SOCS3) that decreased post-drug were validated by qRT-PCR. Functional assays demonstrated that CF monocytes normalized calcium (increases MMP9 expression) concentrations post-drug. Conclusions Transcriptomics revealed differentially regulated pathways in CF patients at baseline compared to non-CF, and in clinical responders to lumacaftor/ivacaftor.

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.

IntroductionThe specific pathogenesis of ankylosing spondylitis (AS) remains unclear, and our study aimed to investigate the possible pathogenesis of AS.Materials and MethodsTwo 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.ResultsANXA3 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.ConclusionUpregulated of ANXA3, SORL1, and neutrophils may be a key factor in the progression of Ankylosing spondylitis.

Store‑operated 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 five‑fold 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.

Annexin A3 (ANXA3), an annexin family member, contains 36 kDa and 33 kDa isoforms. Similar to other annexin members, ANXA3 plays an important role in the development of human diseases. Recent studies have reported that abnormal ANXA3 expression is closely associated with the development, progression, metastasis, drug resistance and prognosis of several malignant tumours, such as breast cancer, lung cancer and hepatocellular carcinoma. ANXA3 exerts its role by regulating cell proliferation, migration and apoptosis via the phosphatidylinositol-3 kinase/Akt, nuclear factor-κB (NF-κB), c-JUN N-terminal kinase, extracellular signal-regulated kinase and hypoxia-inducible factor-1 signalling pathways. ANXA3 may act as a novel target for the early diagnosis and treatment of tumours. The present review summarises the recent progress in the role of ANXA3 and its regulatory pathways in tumours.

Annexin A3 (ANXA3), an annexin family member, contains 36 kDa and 33 kDa isoforms. Similar to other annexin members, ANXA3 plays an important role in the development of human diseases. Recent studies have reported that abnormal ANXA3 expression is closely associated with the development, progression, metastasis, drug resistance and prognosis of several malignant tumours, such as breast cancer, lung cancer and hepatocellular carcinoma. ANXA3 exerts its role by regulating cell proliferation, migration and apoptosis via the phosphatidylinositol-3 kinase/Akt, nuclear factor-κB (NF-κB), c-JUN N-terminal kinase, extracellular signal-regulated kinase and hypoxia-inducible factor-1 signalling pathways. ANXA3 may act as a novel target for the early diagnosis and treatment of tumours. The present review summarises the recent progress in the role of ANXA3 and its regulatory pathways in tumours.

Annexin A3 (ANXA3), also known as lipocortin III and placental anticoagulant protein III, has been reported to be dysregulated in tumor tissues and cancer cell lines, and harbors pronounced diagnostic and prognostic value for certain malignancies, such as breast, prostate, colorectal, lung and liver cancer. Aberrant expression of ANXA3 promotes tumor cell proliferation, invasion, metastasis, angiogenesis, and therapy resistance to multiple chemotherapeutic drugs including platinum-based agents, fluoropyrimidines, cyclophosphamide, doxorubicin, and docetaxel. Genetic alterations on the ANXA3 gene have also been reported to be associated with the propensity to form certain inherited, familial tumors. These diverse functions of ANXA3 in tumors collectively indicate that ANXA3 may serve as an attractive target for novel anticancer therapies and a powerful diagnostic and prognostic biomarker for early tumor detection and population risk screening. In this review, we dissect the role of ANXA3 in cancer in detail.

Ochratoxin A (OTA), one of the most abundant food-contaminating mycotoxins, is a possible carcinogen to humans. We previously demonstrated that long-term (40 weeks) OTA exposure induces the malignant transformation of human gastric epithelium cells (GES-1) in vitro. However, the specific mechanism underlying OTA-induced gastric carcinogenesis is complex. In the present study, we used 2-DE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI/TOF MS) combined with bioinformatics and immunoblotting to investigate the differentially expressed proteins between GES-1 and OTA-malignant transformed GES-1 cells (OTA-GES-1T cells) in vitro. We found that four differentially expressed proteins were identified after malignant transformation, including actin, cytoplasmic 1 (ACTB), F-actin-capping protein subunit alpha-1 (CAPZA1), Annexin A3 (ANXA3), thioredoxin peroxidase B from red blood cells (TPx-B) and Fibrinogen beta B (Fibrinogen β). Among the differentially expressed proteins, the effect of Annexin A3 was analyzed by MTT assay, western blot, cell cycle analysis, wound healing assay, Transwell assay, and colony formation assay in OTA-GES-1T cells. The results showed that inhibition of Annexin A3 by siRNA effectively prevented the proliferation, migration, and invasion abilities of OTA-GES-1T cells. Collectively, the results of this study will guide future research on OTA carcinogenicity.

The cellular and molecular mechanisms underlying onset and development of schizophrenia have not yet been completely elucidated, but the association of disturbed neuroplasticity and inflammation has gained particular relevance recently. These mechanisms are linked to annexins functions. ANXA3, particularly, is associated to inflammation and membrane metabolism cascades. The aim was to determine the ANXA3 levels in first-onset drug-naive psychotic patients. We investigated by western blot the protein expression of annexin A3 in platelets of first-onset, drug-naive psychotic patients (diagnoses according to DSM-IV: 28 schizophrenia, 27 bipolar disorder) as compared to 30 age- and gender-matched healthy controls. Annexin A3 level was lower in schizophrenia patients as compared to healthy controls (p

Annexin A3 (ANXA3) is highly expressed in different types of cancers, but the impact of ANXA3 in bone tumors is still not clear. In the present study, the expression of ANXA3 in osteosarcoma cells was first confirmed by cellular immunofluorescence. Reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) and western blot analysis were used to detect the expression of ANXA3 in osteoblasts in the osteosarcoma cell lines U2OS and HOS. Furthermore, small interfering (si)‑RNA were transfected into U2OS and HOS cells via a liposome‑mediated method. Then once ANXA3 had been successfully downregulated in U2OS and HOS cells, the cells were collected and total protein was extracted after 48 h of transfection. Western blot analysis was used to confirm successful ANXA3 transfection into osteosarcoma cells and the apoptotic rate of HOS and U2OS was detected by flow cytometry. The expression of ANXA3 in the osteosarcoma cell lines HOS and U2OS were first observed by confocal laser scanning microscopy, and was then detected by RT‑qPCR and western blotting. The mRNA and protein levels of ANXA3 in the osteosarcoma cell lines HOS and U2OS were significantly increased compared with osteoblasts, particularly in HOS cells. When siRNA was transfected into HOS and U2OS cells, the protein expression level of ANXA3 was measured via western blotting. The results indicated that the expression of ANXA3 was significantly decreased. In addition, to determine whether ANXA3 knockdown induced cell apoptosis, the present study analyzed the apoptotic rate by flow cytometry. The results revealed that ANXA3 knockdown markedly increased HOS and U2OS cell apoptosis. To the best of our knowledge, the present study is the first to confirm that ANXA3 is highly expressed in the osteosarcoma cell lines HOS and U2OS. In addition, downregulation of ANXA3 expression in HOS and U2OS cells could increase apoptotic ability.

Annexins are highly conserved and ubiquitous in various somatic cell types. They are involved in membrane transport and a range of calcium-regulated activities on the cell membrane surface, including vesicular transport, membrane fusion in exocytosis, signal transduction, and formation of calcium channels. They also regulate inflammatory response, cell differentiation, and interaction between cytoskeletal proteins. In this study, for the first time, an ANX3 gene from Artemia sinica ( As-anx3) was cloned. The As-anx3 full-length complementary DNA comprises 1,024 bp and has a 948 bp open reading frame encoding a 315-amino-acid polypeptide with four ANX domains. The profiles of both As-ANX3 mRNA and protein expression exhibited peaks at the 0 hr stage and had the same significant downregulation trend throughout the post-diapause embryo development stage. The ERK1/2, the phosphorylation levels of ERK1/2, and cell cycle-related protein (CDK4) expressions were analyzed by western blot analysis. The results showed that CDK4 presented a significantly ascending trend from 0 and 40 hr, although the phosphorylation levels of ERK1/2 did not increase significantly. The transcriptional and protein expressions of As-ANX3 were highly upregulated when the temperature was lowered from 25 to 15°C, but the expressions showed a gradual downward trend when the temperature was further lowered to 5°C. These results indicated that As-ANX3 plays a crucial role in restarting diapause and low-temperature stress in A. sinica.

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.

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.

Introduction: Childhood asthma is one of the most common pediatric diseases, and its incidence is increasing. Annexin A3 (ANXA3) is a member of the Annexin family, a well-known polygenic family of membrane binding proteins. Bioinformation analysis showed that ANXA3 was highly expressed in asthmatic patients, suggesting the effects of ANXA3 on asthma, whereas the mechanism is still unclear. Methods: A inflammatory response model of bronchial epithelial BEAS-2B cells induced by LPS was constructed. Immunoblot and quantitative PCR assays were performed to detect the expression levels of ANXA3 in control or LPS-induced BEAS-2B cells. MTT, flow cytometry (FCM), and Immunoblot assays were respectively conducted to detect the effects of ANXA3 on survival and apoptosis of LPS-induced BEAS-2B cells. qPCR and ELISA assays were performed to detect the expression of TNF-α, IL-6, and IL-8. Additionally, Immunoblot assays were performed to detect the effects of ANXA3 on HIF1α and NLRP3 inflammasome in BEAS-2B cells. Results: We found ANXA3 was overexpressed in LPS-induced BEAS-2B cells. ANXA3 ablation promoted the survival of LPS-induced BEAS-2B cells and suppressed the inflammatory response of LPS-induced BEAS-2B cells. Importantly, we noticed ANXA3 inhibited HIF1α-induced NLRP3 inflammasome activity, and increasing the expression of HIF-α rescued the effects of ANXA3 depletion on asthma. Conclusion: ANXA3 enhanced LPS-triggered inflammation of human bronchial epithelial cells by regulating hypoxia-inducible factor-1α (HIF1α)-mediated NLRP3 inflammasome activation, and thought ANXA3 as a promising molecular target for acute asthma treatment.

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., ANAX3 promoted cell growth and migration of melanoma via activation of Hif‐1α/VEGF signalling pathway.

Organ-specific cell-penetrating peptides (CPPs) are a class of molecules that can be highly effective at delivering therapeutic cargoes, and they are currently of great interest in cancer treatment strategies. Herein, we describe a new CPP (amino acid sequence serine-isoleucine-tyrosine-valine, or SIWV) that homes to glioblastoma multiforme (GBM) brain tumor tissues with remarkable specificity in vitro and in vivo. The SIWV sequence was identified from an isoform of annexin-A3 (AA3H), a membrane-interacting human protein. The mechanism of intracellular permeation is proposed to follow a caveolin-mediated endocytotic pathway, based on in vitro and in vivo receptor inhibition and genetic knockdown studies. Feasibility as a targeting agent for therapeutics is demonstrated in a GBM xenograft mouse model, where porous silicon nanoparticles (pSiNPs) containing the clinically relevant anticancer drug SN-38 are grafted with SIWV via a poly-(ethylene glycol) (PEG) linker. The formulation shows enhanced in vivo targeting ability relative to a formulation employing a scrambled control peptide, and significant (P < 0.05) therapeutic efficacy relative to free SN-38 in the GBM xenograft animal model.

Pancreatic cancer (PC) is a lethal cancer in the digestive system. microRNAs (miRNAs) have been demonstrated to participate in PC progression. In this context, we, thus, aimed to explore the mechanism of miR-382 in epithelial mesenchymal transition (EMT) and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. Gene expression data sets GSE16515, GSE71989, and GSE32676 were screened out, with the findings showing the significance of miR-382 and annexin A3 (Anxa3) in PC. A total of 115 PC patients were selected for determination of miR-382 and Anxa3 expression with lowly expressed miR-382 and highly expressed Anxa3 found via RT-quantitative PCR and Western blot analysis. Additionally, negative correlation was found between miR-382 and Anxa3 in PC. Dual-luciferase reporter gene assay and in situ hybridization results confirmed that miR-382 negatively regulated Anxa3. miR-382 targeted Anxa3 and suppressed PC progression by blocking the PI3K/Akt signaling pathway. After a series of gain- and loss-of function approaches, upregulation of miR-382 or silencing of Anxa3 inhibited the EMT and lymph node metastasis, as evidenced by increased level of E-cadherin and decreased level of N-cadherin, vimentin, vascular endothelial growth factor(VEGFR)-3, VEGF-C, and VEGF-D. Overexpression of miR-382 or downregulation of Anxa3 was shown to inhibit colony formation, migration, and invasion abilities of PC cells. Further, tumor xenograft in nude mice in vivo also confirmed the inhibitory role of miR-382 and silenced Anxa3 in lymph node metastasis in PC. Thus, this study provides promising therapeutic targets for PC treatment.NEW & NOTEWORTHY This study focused on the mechanism of miR-382 in epithelial mesenchymal transition and lymph node metastasis in PC in relation to Anxa3 and the PI3K/Akt signaling pathway. We found the inhibitory role of miR-382 in PC in vitro and in vivo.

Graphical abstract, 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.

The purpose of this study was to explore the role of ANXA3 in lung cancer cell resistance to oxaliplatin (OXA).After adding different concentrations of Ox, A549, and A549/Ox cell viability were examined using cell counting kit-8 (CCK-8) assay, and the mRNA and protein expressions of ANXA3 were analyzed by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot, respectively. After treating cells with 5 μg/mL and 15 μg/mL Ox for 24 hours and knocking down ANXA3, qRT-PCR, CCK8, flow cytometry, transwell, and BrdU assays were performed to examine ANXA3 expression level, cell viability, apoptosis, migration, and proliferative capacities, respectively. In addition, Western blot was performed to detect the protein expression of c-caspase 3.The higher the concentration of Ox added, the worse the cell viability. Meanwhile, ANXA3 expression in A549/Ox cells was found remarkably higher than that in normal A549 cells. After treated with different concentrations of Ox for 24 hours, the cell viability, migration capacity and cell proliferation of A549 cells were found remarkably decreased, while the opposite results were observed in cell apoptosis and C-caspase 3 protein expression, and the Ox treatment group was evidently lower than control group.Knockdown of ANXA3 may be able to inhibit the resistance of LCa cells to OXA.

The expression of Annexin A3 (ANXA3), a member of a family of calcium‐binding proteins, is restricted to neutrophils. Its abundance is increased in septic patients, yet the functional relevance of ANXA3 in this context remains unknown. Upon reviewing evidence from public transcriptome repositories and the literature, we hypothesize that during sepsis ANXA3 could mediate neutrophils microbicidal activity while also prolonging their survival, and thus also possibly contributing to organ damage., Summary According to publicly available transcriptome datasets, the abundance of Annexin A3 (ANXA3) is robustly increased during the course of sepsis; however, no studies have examined the biological significance or clinical relevance of ANXA3 in this pathology. Here we explored this interpretation gap and identified possible directions for future research. Based on reference transcriptome datasets, we found that ANXA3 expression is restricted to neutrophils, is upregulated in vitro after exposure to plasma obtained from septic patients, and is associated with adverse clinical outcomes. Secondly, an increase in ANXA3 transcript abundance was also observed in vivo, in the blood of septic patients in multiple independent studies. ANXA3 is known to mediate calcium‐dependent granules–phagosome fusion in support of microbicidal activity in neutrophils. More recent work has also shown that ANXA3 enhances proliferation and survival of tumour cells via a Caspase‐3‐dependent mechanism. And this same molecule is also known to play a critical role in regulation of apoptotic events in neutrophils. Thus, we posit that during sepsis ANXA3 might either play a beneficial role, by facilitating microbial clearance and resolution of the infection; or a detrimental role, by prolonging neutrophil survival, which is known to contribute to sepsis‐mediated organ damage.

Background Cutaneous Lupus Erythematosus (CLE) is an autoimmune disease. It can be limited to the skin or be one of manifestations of systemic LE (SLE). The typical histopathologic pattern in CLE/SLE is interface dermatitis, which can also be observed in dermatomyositis (DM). While LE may affect any organ system, DM most commonly affect muscles and skin. The aim of this study was to investigate the whole proteome of skin inflammatory foci in the cohort of CLE and DM patients in a comparative, hypothesis-free manner and identify disease-unique molecular mechanisms. Methods CLE (n=6), DM (n=5) patients and controls (n=6) were recruited at diagnosis or disease exacerbation. Skin biopsies were acquired, examined by a pathologist and selected inflammatory foci were laser micro-dissected. The total protein content was analyzed by mass-spectrometry, further analysis was performed by string-db.org platform. Certain proteomic findings were confirmed by immunohistochemistry (IHC). Results CLE infiltrates were more protein rich in comparison to DM lesions. There ratio of 5x upregulated proteins in LE/DM was 60, while ratio for DM/LE was 13. Our results confirmed high abundance of (IFN)-regulated proteins both in CLE and DM, including: IFIT1, IFIT2, IFIT3, MX1, MX2, OAS2, OAS3, STAT1,- STAT2, DDX58, DDX60 and EIF2AK2. Proteins expressed differentially in CLE covered complement proteins (C1b), including membrane attack complex (MAC) (C5, C6, C7, C8A and B) and complement regulators (CFHR1, CFHR2, CFHR5), as well as regulators of coagulation: thrombospondin 2 (THBS2), thrombin (F2), fibrinogen (F12) and annexin A3 (ANXA3). Importantly, we identified interleukin (IL) -16 as the only detectable and highly abundant cytokine in the CLE lesions and confirmed this finding by IHC. Conclusions Our data confirm evidence on IFN-regulated processes in CLE/SLE. Importantly, we identified IL-16 as a novel cytokine most strongly upregulated locally in the skin lesions. Moreover, we identified activation of MAC, complement regulating proteins as well as involvement of coagulation/fibrinolysis system. The study brings information on novel pathways involved in the inflammatory foci of the skin lesions in CLE patients. Our findings are of interest in further search of new therapeutic targets.

Background: As a member of annexin family proteins, annexin A3 (ANXA3) has 36-kDa and 33-kDa isoforms, which plays important roles in aggressiveness, tumorigenesis and drug resistance in tumors. 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. Current work established the potential role and regulation mechanism of 33-kDa ANXA3 in hepatocarcinoma. Methods: The expression patterns of ANXA3, CRKL, Rac1, c-Myc and pAkt were analyzed in hepatocarcinoma specimens by western blotting; The biological function of 33-kDa ANXA3 in hepatocarcinoma cell growth, metastasis, apoptosis, angiogenesis, chemoresistance and the underlying molecular mechanism were investigated using gain-of-function strategyin vitro or in vivo. Findings: 33-kDa ANXA3 protein was significantly upregulated in cancerous tissues compared with paracancerous non-tumor 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 proliferative, colony forming, migrative and invasive abilities of HepG2 cells through downregulating CRKL, Rap1b, Rac1, pMEK, pERK2 and c-Myc in ERK pathway; inhibited angiogenesis ability of HepG2 cells through inactivating PI3K/Akt-HIF pathway; induced apoptosis and enchanced 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 cancerous tissues compared with paracancerous non-tumor liver tissues of hepatocarcinoma patients. Interpretation: 33-kDa ANXA3 plays vital role in hepatocarcinoma malignancy, angiogenesis and chemoresistance. It is of potential use in prognosis and therapeutics for hepatocarcinoma. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81672737, 81272186, 31900517) and Natural Science Foundation of Liaoning (LZ2019003, 20181550168, LQ2017001). Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: Tissue use and study protocol were approved by the Medical Ethics Committee of Dalian Medical University. Informed consent was obtained from each patient. All experiment methods were performed in accordance with the relevant guidelines and regulations.