NUSAP1 promotes gastric cancer radioresistance by inhibiting ubiquitination of ANXA2 and is suppressed by miR-129-5p.

BACKGROUND: Radiotherapy is an important strategy for the treatment of advanced gastric cancer (GC), while the radioresistance limits its effectiveness. Nucleolar and spindle associated protein 1 (NUSAP1) was implicated in cancer progression and chemoresistance. However, the underlying mechanisms of NUSAP1 influencing GC radioresistance remain largely unknown. METHODS: Meta-analysis was conducted to systematically evaluate the prognostic value of NUSAP1 in human cancers. Gene set enrichment analysis (GSEA) was conducted using The Cancer Genome Atlas (TCGA) and gene expression omnibus (GEO) datasets. MRNA and protein expressions were detected by qRT-PCR and western blot, respectively. The radiosensitivity of GC cells was observed by colony formation, flow cytometry, comet, immunofluorescence, and animal assays. Immunoprecipitation assay and mass spectrometry were utilized to identify protein associations. MiRNAs binding with NUSAP1 were determined by starbase prediction, luciferase reporter, and RNA immunoprecipitation (RIP) assays. RESULTS: NUSAP1 high expression predicted worse overall survival (OS) and disease-free survival (DFS) with no statistical heterogeneity through the meta-analysis. Downregulation of NUSAP1 significantly increased GC radiosensitivity by inhibiting colony formation, DNA damage repair, and promoting apoptosis following irradiation. Additionally, NUSAP1 silencing combined with radiation resulted in a synergistic anti-tumor effect in xenograft mouse model. Mechanistically, NUSAP1 interacted with ANXA2, protecting it against protein degradation via impeding its ubiquitination process. NUSAP1 was confirmed as a target of miR-129-5p and negatively regulated by it. CONCLUSION: Our results suggested that NUSAP1 enhanced the radioresistance of GC cells. NUSAP1 could be a promising target to increase GC radiosensitivity.

AGR2 facilitates teratoma progression by regulating glycolysis via the AnXA2/EGFR axis.

Anterior gradient-2 (AGR2) is highly expressed in several tumors and plays an important role in tumor development. However, the biological function of AGR2 in teratomas has not yet been thoroughly studied. In this study, AGR2 was found to be upregulated in teratoma tissues and in human testicular teratoma cell lines by Western blotting and qRT-PCR assays. A DNA Methylation-Specific PCR assay demonstrated that AGR2 upregulation resulted from hypomethylated AGR2 in teratoma cells. NCC-IT and NT2-D1 cells were transfected with pcDNA-AGR2 or sh-AGR2 to obtain AGR2-overexpressed or -silenced cells, and cell proliferation, invasion and glycolysis were determined using CCK-8, 5-ethynyl-2'-deoxyuridine (EdU), Transwell assays, and commercial kits. The results revealed that overexpression of AGR2 promoted teratoma cell proliferation and invasion and elevated glycolysis levels evidencing by the increase in lactate secretion, glucose consumption, ATP levels and the expression of glycolysis-related proteins, while knockdown of AGR2 showed the opposite results. The interactions between AGR2 and annexin A2 (AnXA2), as well as between AnXA2 and epidermal growth factor receptor (EGFR) were verified by co-immunoprecipitation assay. Mechanistic studies revealed that AGR2 interacts with AnXA2 and increases the level of AnXA2 to recruit more AnXA2 to EGFR, there by promoting EGFR expression. A series of rescue experiments showed that knockdown of AnXA2 or EGFR weakened the promotional effects of AGR2 overexpression on the proliferation, invasion, and glycolysis of teratoma cells. Finally, tumorigenicity assays were performed using NT2-D1 cells stably transfected with either LV-NC-shRNA or LV-shAGR2. The results showed that AGR2 knockdown significantly inhibited teratoma tumor growth in vivo. In conclusion, our data suggested that AGR2 facilitates glycolysis in teratomas through promoting EGFR expression by interacting with AnXA2, thereby promoting teratoma cells proliferation and invasion.

Generation of B7-H3 isoform regulated by ANXA2/NSUN2/YBX1 axis in human glioma.

In recent years, in the development of emerging immunotherapy, B7-H3 is also termed as CD276 and has become a novel chimeric antigen receptor (CAR)-T target against glioma and other tumours, and aroused extensive attention. However, B7-H3 has three isoforms (2, 3 and 4Ig) with the controversial expression and elusive function in tumour especially glioma. The current study mainly focuses on the regulatory factors and related mechanisms of generation of different B7-H3 isoforms. First, we have determined that 2Ig is dominant in glioma with high malignancy, and 4Ig is widely expressed, whereas 3Ig shows negative expression in all glioma. Next, we have further found that RNA binding protein annexin A2 (ANXA2) is essential for B7-H3 isoform maintenance, but fail to determine the choice of 4Ig or 2Ig. RNA methyltransferase NOP2/Sun RNA methyltransferase 2 (NSUN2) and 5-methylcytosine reader Y-box binding protein 1 (YBX1) facilitate the production of 2Ig. Our findings have uncovered a series of factors (ANXA2/NSUN2/YBX1) that can determine the alternative generation of different isoforms of B7-H3 in glioma. Our result aims to help peers gain a clearer understanding of the expression and regulatory mechanisms of B7H3 in tumour patients, and to provide better strategies for designing B7H3 as a target in immunotherapy.

Sporadic Parathyroid Adenoma: A Pilot Study of Novel Biomarkers in Females.

Background and Objectives: Parathyroid adenoma is a distinct cause of primary hyperparathyroidism, with the vast majority being sporadic ones. Proteomic analysis of parathyroid adenomas has proposed a large number of related proteins. The aim of this study is to evaluate the immunohistochemical staining of ANXA2, MED12, MAPK1 and VDR in parathyroid adenoma tissue. Materials and Methods: Fifty-one parathyroid adenomas were analyzed for ANXA2, MED12, MAPK1 and VDR expressions. Tissue was extracted from formalin-fixed paraffin-embedded parathyroid adenoma specimens; an immunohistochemical study was applied, and the percentage of allocation and intensity were evaluated. Results: ANXA2 stained positively in 60.8% of all cell types, while MED12 had positive staining in 66%. MAPK1 expression was found to be negative in total, although a specific pattern for oxyphil cells was observed, as they stained positive in 17.7%. Finally, VDR staining was positive at 22.8%, based on nuclear staining. Conclusions: These immunohistochemical results could be utilized as biomarkers for the diagnosis of sporadic parathyroid adenoma. It is of great importance that a distinct immunophenotype of nodule-forming cells in a positive adenoma could suggest a specific pattern of adenoma development, as in hereditary patterns.

Molecular cloning and characterization of duck Annexin A2 and its effect on DTMUV replication.

Annexin A2 (ANXA2) is a multifaceted protein implicated in various stages of viral infections, particularly in envelope virus replication through mechanisms such as endocytosis and exocytosis. This study delves into the characterization and functional dynamics of duck ANXA2 (duANXA2). We successfully cloned the full-length coding sequence of duANXA2 and conducted a detailed structural analysis. The open reading frame (ORF) of duANXA2 is 1020 bp, encoding 339 amino acids and featuring 4 conserved domains. Phylogenetic tree analysis indicates that duANXA2 is most closely related to Gallus gallus, with significantly lesser homology to fish species. We evaluated the tissue-specific expression of duANXA2 in healthy ducks, noting its ubiquitous presence but varying expression levels across different organs, with notably high expression in the esophagus and immune organs. Upon infecting duck embryo fibroblast (DEF) cells with the duck Tembusu virus (DTMUV), a flavivirus causing ducks substantial mortality and a dramatic decline in egg production, we observed a pronounced upregulation of duANXA2. Functional assays demonstrated that overexpression of duANXA2 in DEF cells augments DTMUV replication, while its interference markedly reduces DTMUV replication. These findings underscore the role of duANXA2 as a facilitator of DTMUV replication, presenting it as a potential target for therapeutic intervention in managing DTMUV infections.

Machine learning algorithms integrate bulk and single-cell RNA data to unveil oxidative stress following intracerebral hemorrhage.

BACKGROUND: Increased oxidative stress (OS) activity following intracerebral hemorrhage (ICH) had significantly impacting patient prognosis. Identifying optimal genes associated with OS could enhance the understanding of OS after ICH. METHODS: We employed single-cell RNA sequencing (scRNA-seq) to investigate the heterogeneity of OS across various cellular tiers following ICH, aiming to acquire biological insights into ICH. We utilized AUCell, Ucell, singscore, ssgsea, and AddModuleScore algorithms, along with correlation analysis, to identify hub genes influencing high OS post-ICH. Furthermore, we employed four machine learning algorithms, eXtreme Gradient Boosting, Boruta, Random Forest, and Least Absolute Shrinkage and Selection Operator, to identify the optimal feature genes. To validate the accuracy of our analysis, we conducted validation in ICH animal experiments. RESULTS: After analyzing the scRNA-seq dataset using various algorithms, we found that OS activity exhibited heterogeneity across different cellular layers following ICH, with particularly heightened activity observed in monocytes. Further integration of bulk data and machine learning algorithms revealed that ANXA2 and COTL1 were closely associated with high OS after ICH. Our animal experiments demonstrated an increase in OS expression post-ICH. Additionally, the protein expression of ANXA2 and COTL1 was significantly elevated and co-localized with microglia. Pearson correlation coefficient analysis revealed a significant correlation between ANXA2 and OS, indicating strong consistency (r = 0.84, p < 0.05). Similar results were observed for COTL1 and OS (r = 0.69, p < 0.05). CONCLUSIONS: Following ICH, ANXA2 and COTL1 might penetrate the brain via monocytes, localize within microglia, and enhance OS activity. This might help us better understand OS after ICH.

A Novel tsRNA, m7G-3' tiRNA LysTTT, Promotes Bladder Cancer Malignancy Via Regulating ANXA2 Phosphorylation.

Emerging evidence indicates that transfer RNA (tRNA)-derived small RNAs (tsRNAs), originated from tRNA with high abundance RNA modifications, play an important role in many complex physiological and pathological processes. However, the biological functions and regulatory mechanisms of modified tsRNAs in cancer remain poorly understood. Here, it is screened for and confirmed the presence of a novel m7G-modified tsRNA, m7G-3'-tiRNA LysTTT (mtiRL), in a variety of chemical carcinogenesis models by combining small RNA sequencing with an m7G small RNA-modified chip. Moreover, it is found that mtiRL, catalyzed by the tRNA m7G-modifying enzyme mettl1, promotes bladder cancer (BC) malignancy in vitro and in vivo. Mechanistically, mtiRL is found to specifically bind the oncoprotein Annexin A2 (ANXA2) to promote its Tyr24 phosphorylation by enhancing the interactions between ANXA2 and Yes proto-oncogene 1 (Yes1), leading to ANXA2 activation and increased p-ANXA2-Y24 nuclear localization in BC cells. Together, these findings define a critical role for mtiRL and suggest that targeting this novel m7G-modified tsRNA can be an efficient way for to treat BC.

Ahnak in the prefrontal cortex mediates behavioral correlates of stress resilience and rapid antidepressant action in mice.

The prefrontal cortex (PFC) is a key neural node mediating behavioral responses to stress and the actions of ketamine, a fast-acting antidepressant. The molecular mechanisms underlying these processes, however, are not fully understood. Our recent study revealed a pivotal role of hippocampal Ahnak as a regulator of cellular and behavioral adaptations to chronic stress. However, despite its significant expression in the PFC, the contribution of cortical Ahnak to behavioral responses to stress and antidepressants remains unknown. Here, using a mouse model for chronic social stress, we find that Ahnak expression in the PFC is significantly increased in stress-resilient mice and positively correlated with social interaction after stress exposure. Conditional deletion of Ahnak in the PFC or forebrain glutamatergic neurons facilitates stress susceptibility, suggesting that Ahnak is required for behavioral resilience. Further supporting this notion, Ahnak expression in the PFC is increased after the administration of ketamine or its metabolite (2R, 6R)-hydroxynorketamine (HNK). Moreover, Ahnak deletion in forebrain glutamatergic neurons blocks the restorative behavioral effects of ketamine or HNK in stress-susceptible mice. This forebrain excitatory neuron-specific Ahnak deletion reduces the frequency of mini excitatory postsynaptic currents in layer II/III pyramidal neurons, suggesting that Ahnak may induce its behavioral effects via modulation of glutamatergic transmission in the PFC. Altogether, these data suggest that Ahnak in glutamatergic PFC neurons may be critical for behavioral resilience and antidepressant actions of ketamine or HNK in chronic social stress-exposed mice.

Oxidative stress-induced hypermethylation and low expression of ANXA2R: Novel insights into the dysfunction of melanocytes in vitiligo.

BACKGROUND: Vitiligo is a skin disorder with melanocyte destruction caused by complex interplay between multiple genetic and environmental factors. Recent studies have suggested DNA methylation is involved in the melanocyte damage, but the underlying mechanism remains unknown. OBJECTIVE: To explore the abnormal DNA methylation patterns in vitiligo lesional and nonlesional skin, and the mechanism of DNA methylation involved in vitiligo pathogenesis. METHODS: Initially, the genome-wide aberrant DNA methylation profiles in lesional and nonlesional skin of vitiligo were detect via Illumina methylation EPIC 850k Beadchip. Subsequently, a comprehensive analysis was conduct to investigate the genomic characteristics of differentially methylated regions (DMRs). Furthermore, the effects of key aberrant methylated genes on cell apoptosis and function of both melanocytes and keratinocytes were further identified and validated by western bloting, ELISA, and immunofluorescence. RESULTS: Compared with nonlesional skins, we discovered 79 significantly differentially methylated CpG sites in vitiligo lesions. These DMRs were mainly located in the gene body and the TS1500 region. Annexin A2 receptor (ANXA2R), a crucial gene in cell apoptosis, was hypermethylated in vitiligo lesions. Furthermore, we showed that ANXA2R displayed hypermethylation and low expression levels in both keratinocytes and melanocytes of vitiligo patients, and the hypermethylated-triggered downregulation of ANXA2R under oxidative stress induced melanocyte apoptosis, and inhibited the secretion of stem cell factor (SCF) from keratinocytes thus impaired the survival of melanocytes. CONCLUSIONS: Our study illustrates the DNA methylation modification in vitiligo, and further demonstrates the molecular mechanism of hypermethylated ANXA2R in the dysfunction of melanocytes under oxidative stress.

The novel DNA methylation marker FIBIN suppresses non-small cell lung cancer metastasis by negatively regulating ANXA2.

OBJECTIVES: The clinical T1 stage solid lung cancer with metastasis is a serious threat to human life and health. In this study, we performed RNA sequencing on T1 advanced-stage lung cancer and adjacent tissues to identify a novel biomarker and explore its roles in lung cancer. METHODS: Quantitative reversed-transcription PCR, reverse transcription PCR and Western blot, MSP and Methtarget were utilized to evaluate FIBIN expression levels at both the transcriptional and protein levels as well as its methylation status. Differential target protein was evaluated for relative and absolute quantitation by isobaric tags. Co-IP was performed to detect the interactions between target protein. Precise location and expression levels of target proteins were revealed by immunofluorescence staining and component protein extraction using specific kits, respectively. RESULTS: We reported that FIBIN was frequently silenced due to promoter hypermethylation in lung cancer. Additionally, both in vitro and in vivo experiments confirmed the significant anti-proliferation and anti-metastasis capabilities of FIBIN. Mechanistically, FIBIN decreased the nuclear accumulation of ß-catenin by reducing the binding activity of GSK3ß with ANXA2 while promoting interaction between GSK3ß and ß-catenin. CONCLUSION: Our findings firstly identify FIBIN is a tumor suppressor, frequently silenced due to promoter hypermethylation. FIBIN may serve as a predictive biomarker for progression or metastasis among early-stage lung cancer patients.

Hspb1 protects against severe acute pancreatitis by attenuating apoptosis and ferroptosis via interacting with Anxa2 to restore the antioxidative activity of Prdx1.

Acute pancreatitis (AP) is a common abdominal disease that typically resolves on its own, but the mortality rate dramatically increases when it progresses to severe acute pancreatitis (SAP). In this study, we investigated the molecular mechanism underlying the development of SAP from AP. We utilized two SAP models induced by pancreatic duct ligation and caerulein administration. Transcriptomic and proteomic analyses were subsequently performed to determine the mRNA and protein expression profiles of pancreatic samples from SAP and AP model and normal mice. To explore the role of Hspb1 in SAP, we used Hspb1 knockout (KO) mice, a genetically engineered chronic pancreatitis strain (T7D23A), Anxa2 KO mice, and acinar cell-specific Prdx1 knockout mice. Additionally, various in vivo and in vitro assays were performed to elucidate the molecular events and direct targets of Hspb1 in acinar cells. We found that Hspb1 expression was upregulated in AP samples but significantly reduced in acinar cells from SAP samples. KO or inhibition of Hspb1 worsened AP, while AAV8-Hspb1 administration mitigated the severity of SAP and reduced remote organ damage in mice. Furthermore, AAV8-Hspb1 treatment prevented the development of chronic pancreatitis. We found that KO or inhibition of Hspb1 promoted acinar cell death through apoptosis and ferroptosis but not necroptosis or autophagy by increasing reactive oxygen species (ROS) and lipid ROS levels. Mechanistically, Hspb1 directly interacted with Anxa2 to decrease its aggregation and phosphorylation, interact with the crucial antioxidant enzyme Prdx1, and maintain its antioxidative activity by decreasing Thr-90 phosphorylation. Notably, the overexpression of Hspb1 did not have a protective effect on acinar-specific Prdx1 knockout mice. In summary, our findings shed light on the role of Hspb1 in acinar cells. We showed that targeting Hspb1/Anxa2/Prdx1 could serve as a potential therapeutic strategy for SAP.

Activation of astrocytes by ANXA2-derived extracellular vesicles from lung cancer cells affects aggressiveness, immunotherapy response and microenvironment of lung cancer.

Background: ANXA2 has been extensively documented in relation to cancer. Nevertheless, the involvement of ANXA2 in lung carcinoma remains uncertain. Methods: Data from The Cancer Genome Atlas (TCGA) database was downloaded using open-access methods. The examination of publicly available data was conducted utilizing the R software. The mRNA level of specific molecules was detected using Real-time Quantitative PCR (qPCR). The protein level of specific molecules was detected using the Western blot assay. The cell proliferation ability of cancer cells was assessed using the CCK8 assay. The invasion and migration capability of cancer cells was assessed using the Transwell assay. Validation of exosomes extraction was conducted using electron microscopy and particle size analysis. Results: In this study, based on series experiments, we found that ANXA2 can promote the activation of neuroastrocytes cells CP-H122 through the exosome pathway. Also, we found that ANXA2 can be transmitted from A549 cells to CP-H122 through the exosomes pathway and further promote the activation of CP-H122. Activated CP-H122 cells further enhance the proliferation, invasion and metastasis of A549 cells. Meanwhile, we performed transcriptome sequencing to explore the downstream genes of ANXA2 to screen potential targets for follow-up studies. Analysis based on public data showed that ANXA2 was related to the worse survival performance and clinical features of lung cancer. Gene set enrichment analysis based on the Hallmark gene set indicated that the patient with high ANXA2 expression might have a higher activity of the apical surface, reactive oxygen species pathway, angiogenesis, TGF-ß signaling, MYC target, but lower activity of pancreas-ß cells. More important, our results showed that ANXA2 can affects immunotherapy response and reshape immune microenvironment of lung cancer. Conclusions: This study demonstrates that ANXA2 activates CP-H122 cells, affects A549 cell behavior, and impacts lung cancer prognosis and immunotherapy response.

FAXC interacts with ANXA2 and SRC in mitochondria and promotes tumorigenesis in cholangiocarcinoma.

Cholangiocarcinoma (CCA) is one of the most difficult malignancies to treat as the therapeutic options are limited. Although several driver genes have been identified, most remain unknown. In this study, we identified a failed axon connection homolog (FAXC), whose function is unknown in mammals, by analyzing serially passaged CCA xenograft models. Knockdown of FAXC reduced subcutaneous tumorigenicity in mice. FAXC was bound to annexin A2 (ANXA2) and c-SRC, which are tumor-promoting genes. The FAXC/ANXA2/c-SRC complex forms in the mitochondria. FAXC enhances SRC-dependent ANXA2 phosphorylation at tyrosine-24, and the C-terminal amino acid residues (351-375) of FAXC are required for ANXA2 phosphorylation. Transcriptome data from a xenografted CCA cell line revealed that FAXC correlated with epithelial-mesenchymal transition, hypoxia, and KRAS signaling genes. Collectively, these findings advance our understanding of CCA tumorigenesis and provide candidate therapeutic targets.

UBA3 promotes the occurrence and metastasis of intrahepatic cholangiocarcinoma through MAPK signaling pathway.

Intrahepatic cholangiocarcinoma (ICC) accounts for approximately 15% of primary liver cancers, and the incidence rate has been increasing in recent years. Surgical resection is the best treatment for ICC, but the 5-year survival rate is less than 30%. ICC signature genes are crucial for the early diagnosis of ICC, so it is especially important to identify signature genes. The aim of this study is to screen the signature genes of ICC and find the potential target for the treatment of ICC. We find that UBA3 is highly expressed in ICC, and knockdown of UBA3 inhibits ICC proliferation, invasion and migration. Mechanistic experiments show that UBA3 promotes ICC proliferation, invasion and migration by affecting ANXA2 through the MAPK signaling pathway. UBA3 is a target of bufalin, and bufalin targeting UBA3 inhibits ICC development and progression through the MAPK signaling pathway. In conclusion, our study shows that bufalin inhibits ICC by targeting UBA3, which has emerged as a new biomarker and potential therapeutic target for ICC.

The mechanistic role of natural antimicrobials in preventing Staphylococcus aureus invasion of MAC-T cells using an in vitro mastitis model.

BACKGROUND: Starting primarily as an inflammation of the mammary gland, mastitis is frequently driven by infectious agents such as Staphylococcus aureus. Mastitis has a large economic impact globally, which includes diagnostic, treatment, and the production costs not to mention the potential milk contamination with antimicrobial residues. Currently, mastitis prevention and cure depends on intramammary infusion of antimicrobials, yet, their overuse risks engendering resistant pathogens, posing further threats to livestock. METHODS: In our study we aimed to investigate, in vitro, using bovine mammary epithelial cells (MAC-T), the efficacy of the AuraShield an antimicrobial mixture (As) in preventing S. aureus attachment, internalisation, and inflammation. The antimicrobial mixture (As) included: 5% maltodextrin, 1% sodium chloride, 42% citric acid, 18% sodium citrate, 10% silica, 12% malic acid, 9% citrus extract and 3% olive extract (w/w). RESULTS AND DISCUSSION: Herein we show that As can significantly reduce both adherence and invasion of MAC-T cells by S. aureus, with no impact on cell viability at all concentrations tested (0.1, 0.2, 0.5, 1%) compared with untreated controls. The anti-apoptotic effect of As was achieved by significantly reducing cellular caspase 1, 3 and 8 activities in the infected MAC-T cells. All As concentrations were proven to be subinhibitory, suggesting that Ac can reduce S. aureus virulence without bacterial killing and that the effect could be dual including a host modulation effect. In this context, we show that As can reduce the expression of S. aureus clumping factor (ClfB) and block its interaction with the host Annexin A2 (AnxA2), resulting in decreased bacterial adherence in infection of MAC-T cells. Moreover, the ability of As to block AnxA2 had a significant decreasing effect on the levels of pro inflammatory cytokine released upon S. aureus interaction with MAC-T cells. CONCLUSION: The results presented in this study indicate that mixtures of natural antimicrobials could potentially be considered an efficient alternative to antibiotics in treating S. aureus induced mastitis.

Proteomic analysis identifies HSP90AA1, PTK2B, and ANXA2 in the human entorhinal cortex in Alzheimer's disease: Potential role in synaptic homeostasis and Aß pathology through microglial and astroglial cells.

Alzheimer's disease (AD), the most prevalent neurodegenerative disorder worldwide, is clinically characterized by cognitive deficits. Neuropathologically, AD brains accumulate deposits of amyloid-ß (Aß) and tau proteins. Furthermore, these misfolded proteins can propagate from cell to cell in a prion-like manner and induce native proteins to become pathological. The entorhinal cortex (EC) is among the earliest areas affected by tau accumulation along with volume reduction and neurodegeneration. Neuron-glia interactions have recently come into focus; however, the role of microglia and astroglia in the pathogenesis of AD remains unclear. Proteomic approaches allow the determination of changes in the proteome to better understand the pathology underlying AD. Bioinformatic analysis of proteomic data was performed to compare ECs from AD and non-AD human brain tissue. To validate the proteomic results, western blot, immunofluorescence, and confocal studies were carried out. The findings revealed that the most disturbed signaling pathway was synaptogenesis. Because of their involvement in synapse function, relationship with Aß and tau proteins and interactions in the pathway analysis, three proteins were selected for in-depth study: HSP90AA1, PTK2B, and ANXA2. All these proteins showed colocalization with neurons and/or astroglia and microglia and with pathological Aß and tau proteins. In particular, ANXA2, which is overexpressed in AD, colocalized with amoeboid microglial cells and Aß plaques surrounded by astrocytes. Taken together, the evidence suggests that unbalanced expression of HSP90AA1, PTK2B, and ANXA2 may play a significant role in synaptic homeostasis and Aß pathology through microglial and astroglial cells in the human EC in AD.

The upregulation of Annexin A2 by TLR4 pathway facilitates lipid accumulation and liver injury via blocking AMPK/mTOR-mediated autophagy flux during the development of non-alcoholic fatty liver disease.

BACKGROUND AND AIMS: Non-alcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. In this study, we aimed to investigate the role and regulatory mechanism of Annexin A2 (ANXA2) in the pathogenesis of NAFLD. METHODS: Histological analyses and ELISA were used to illuminate the expression of ANXA2 in NAFLD and healthy subjects. The role of ANXA2 was evaluated using high-fat diet (HFD)-fed mice via vein injection of adeno-associated viruses (AAV) knocking down ANXA2 or non-targeting control (NC) shRNAs. Moreover, HepG2 and LO2 cells were employed as in vitro hepatocyte models to investigate the expression and function of ANXA2. RESULTS: ANXA2 was confirmed to be one of three hub genes in liver injury, and its expression was positively correlated with NAFLD activity score (NAS) and macrophage infiltration in NAFLD. Moreover, ANXA2 was significantly upregulated in NAFLD patients and HFD-fed mice. LPS/TLR4 pathway strongly upregulated ANXA2 expression, which is mediated by direct ANXA2 promoter binding by TLR4 downstream NF-κB p65 and c-Jun transcription factors. Increased ANXA2 expression was correlated with decreased autophagy flux and autophagy was activated by the depletion of ANXA2 in the models of NAFLD. Furthermore, ANXA2 interference led to the activation of AMPK/mTOR signaling axis, which may play a causal role in autophagy flux and the amelioration of steatosis. CONCLUSIONS: ANXA2 is a pathological predictor and promising therapeutic target for NAFLD. ANXA2 plays a crucial role in linking inflammation to hepatic metabolic disorder and injury, mainly through the blockage of AMPK/mTOR-mediated lipophagy.

The lncRNA GAS5 upregulates ANXA2 to mediate the macrophage inflammatory response during atherosclerosis development.

Inflammatory macrophages play a crucial role in atherosclerosis development. The long non-coding RNA growth arrest-specific 5 (GAS5) regulates THP-1 macrophage inflammation by sponging microRNAs. The purpose of this study was to investigate the regulatory mechanism of GAS5 in atherosclerosis development. GSE40231, GSE21545, and GSE28829 datasets from the Gene Expression Omnibus database were integrated after adjusting for batch effect. Differential analysis was performed on the integrated dataset and validated using the Genotype-Tissue Expression and GSE57691 datasets. Potential biological functions of GAS5 and annexin A2 (ANXA2) were identified using gene set enrichment analysis (GSEA). ssGSEA, CIBERSORTx, and ImmuCellAI algorithms were used to identify immune infiltration in plaque samples. GAS5 and ANXA2 expression levels in RAW264.7 cells treated with oxidized low-density lipoprotein (ox-LDL) were measured by qRT-PCR and Western blot. Small interfering and short hairpin RNA were used to silence GAS5 expression. Plasmids of ANXA2 were used to establish ANXA2 overexpression. Apoptosis and inflammatory markers in macrophages were detected by Western blot. Aortic samples from APOE-/- mice were collected to validate the expression of GAS5 and ANXA2. GAS5 expression was significantly increased during atherosclerosis. GAS5 expression was positively correlated with macrophage activation and ANXA2 expression in plaques. Furthermore, ANXA2 upregulation was also related to the activation of macrophage. GSEA indicated similar biological functions for GAS5 and ANXA2 in plaques. Moreover, in vitro experiments showed that both GAS5 and ANXA2 contributed to macrophage apoptosis and inflammation. Rescue assays revealed that the inflammatory effects of GAS5 on macrophages were ANXA2-dependent. In vivo experiments confirmed the highly expression of Gas5 and Anxa2 in the plaque group. We identified the atherogenic roles of GAS5 and ANXA2 in the inflammatory response of macrophages. The inflammatory response in ox-LDL-treated macrophages was found to be mediated by GAS5-ANXA2 regulation, opening new avenues for atherosclerosis therapy.

Global crotonylome identifies EP300-regulated ANXA2 crotonylation in cumulus cells as a regulator of oocyte maturation.

Lysine crotonylation (Kcr), a newly discovered post-translational modification, played a crucial role in physiology and disease progression. However, the roles of crotonylation in oocyte meiotic resumption remain elusive. As abnormal cumulus cell development will cause oocyte maturation arrest and female infertility, we report that cumulus cells surrounding human meiotic arrested oocytes showed significantly lower crotonylation, which was associated with decreased EP300 expression and blocked cumulus cell expansion. In cultured human cumulus cells, exogenous crotonylation or EP300 activator promoted cell proliferation and reduced cell apoptosis, whereas EP300 knockdown induced the opposite effect. Transcriptome profiling analysis in human cumulus cells indicated that functions of crotonylation were associated with activation of epidermal growth factor receptor (EGFR) pathway. Importantly, we characterized the Kcr proteomics landscape in cumulus cells by LC-MS/MS analysis, and identified that annexin A2 (ANXA2) was crotonylated in cumulus cells in an EP300-dependent manner. Crotonylation of ANXA2 enhanced the ANXA2-EGFR binding, and then activated the EGFR pathway to affect cumulus cell proliferation and apoptosis. Using mouse oocytes IVM model and EP300 knockout mice, we further confirmed that crotonylation alteration in cumulus cells affected the oocyte maturation. Together, our results indicated that EP300-mediated crotonylation is important for cumulus cells functions and oocyte maturation.

FGF19 promotes nasopharyngeal carcinoma progression by inducing angiogenesis via inhibiting TRIM21-mediated ANXA2 ubiquitination.

PURPOSE: Nasopharyngeal carcinoma (NPC) has characteristics of high invasion and early metastasis. Most NPC patients present with locoregionally advanced illness when first diagnosed. Therefore, it is urgent to discover NPC biomarkers. Fibroblast growth Factor 19 (FGF19) plays a role in various physiological or pathological processes, including cancer. In this research, we discovered the importance of FGF19 in NPC, and clarified its role in tumour angiogenesis. METHODS: Western blotting, immunohistochemistry and ELISA were used to investigate FGF19 expression in NPC. Then we took CCK8, colony formation, Transwell and wound healing assays to identify the influence of FGF19 on NPC malignant behaviours. The proliferative and metastatic capacity of FGF19 were evaluated in nude mice and zebrafish. The role of FGF19 in angiogenesis was investigated by tube formation and Matrigel plug angiogenesis assays. We then evaluated the variation in Annexin A2(ANXA2) levels with the treatment of FGF19. Lastly, co-immunoprecipitation and ubiquitination assays were performed to identify the mechanisms involved. RESULTS: FGF19 levels were elevated in tissues and serum of NPC patients and were associated with poor clinical stages. High expression of FGF19 promoted NPC malignant behaviours. In particular, FGF19 expression was correlated with microvessel density in tissues and NPC-derived FGF19 could accelerate angiogenesis in vitro and in vivo. Mechanistically, FGF19 influenced ANXA2 expression to promote angiogenesis. Moreover, tripartite motif-containing 21(TRIM21) interacted with ANXA2 and was responsible for ANXA2 ubiquitination. CONCLUSION: FGF19 promoted NPC angiogenesis by inhibiting TRIM21-mediated ANXA2 ubiquitination. It may serve as a noninvasive biomarker for NPC and provides new insights for therapy.