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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

LncNFYB promotes the proliferation of rheumatoid arthritis fibroblast-like synoviocytes via LncNFYB/ANXA2/ERK1/2 axis.

Long noncoding RNAs (lncRNAs) are specifically expressed in different diseases and regulate disease progression. To explore the functions of rheumatoid arthritis (RA)-specific lncRNA, we determined the lncRNA expression profile of fibroblast-like synoviocytes (FLS) obtained from patients with RA and osteoarthritis (OA) using a LncRNA microarray and identified up-regulated LncNFYB in RA as a potential therapeutic target. Using gain- and loss-of-function studies, LncNFYB was proven to promote FLS proliferation and cell cycle progress but not affect their invasion, migration, and apoptotic abilities. Further investigation discovered that LncRNA could combine with annexin A2 (ANXA2) and enhance the level of phospho-ANXA2 (Tyr24) in the plasma membrane area, which induced the activation of ERK1/2 to promote proliferation. These findings provide new insights into the biological functions of LncNFYB on modification of FLS, which may be exploited for the therapy of RA.

Bufalin targeting CAMKK2 inhibits the occurrence and development of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) accounts for about 15% of primary liver cancer, and the incidence rate has been rising 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 find its signature genes and therapeutic drug. Here, we studied that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the occurrence and metastasis of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway. METHODS: IC50 of bufalin in ICC cells was determined by CCK8 and invasive and migratory abilities were verified by wound healing, cell cloning, transwell and Western blot. IF and IHC verified the expression of CAMKK2 between ICC patients and normal subjects. BLI and pull-down demonstrated the binding ability of bufalin and CAMKK2. Bioinformatics predicted whether CAMKK2 was related to the Wnt/ß-catenin pathway. SKL2001, an activator of ß-catenin, verified whether bufalin acted through this pathway. In vitro and in vivo experiments verified whether overexpression of CAMKK2 affects the proliferative and migratory effects of ICC. Transmission electron microscopy verified mitochondrial integrity. Associated Ca2+ levels verified the biological effects of ANXA2 on ICC. RESULTS: It was found that bufalin inhibited the proliferation and migration of ICC, and CAMKK2 was highly expressed in ICC, and its high expression was positively correlated with poor prognosis.CAMKK2 is a direct target of bufalin, and is associated with the Wnt/ß-catenin signaling pathway, which was dose-dependently decreased after bufalin treatment. In vitro and in vivo experiments verified that CAMKK2 overexpression promoted ICC proliferation and migration, and bufalin reversed this effect. CAMKK2 was associated with Ca2+, and changes in Ca2+ content induced changes in the protein content of ANXA2, which was dose-dependently decreasing in cytoplasmic ANXA2 and dose-dependently increasing in mitochondrial ANXA2 after bufalin treatment. In CAMKK2 overexpressing cells, ANXA2 was knocked down, and we found that reversal of CAMKK2 overexpression-induced enhancement of ICC proliferation and migration after siANXA2. CONCLUSIONS: Our results suggest that bufalin targeting CAMKK2 promotes mitochondrial dysfunction and inhibits the proliferation and migration of intrahepatic cholangiocarcinoma through Wnt/ß-catenin signal pathway. Thus, bufalin, as a drug, may also be used for cancer therapy in ICC in the future.

Promoter Hypomethylation Upregulates ANXA2 Expression in Pancreatic Cancer and is Associated with Poor Prognosis.

Pancreatic cancer (PC) is one of the world's most aggressive and deadly cancers, owing to non-specific early clinical symptoms, late-stage diagnosis, and poor survival. Therefore, it is critical to identify specific biomarkers for its early diagnosis. Annexin A2 (ANXA2) is a calcium-dependent phospholipid-binding protein that has been reported to be upregulated in several cancer types, making it an emerging biomarker and potential cancer therapeutic target. However, the mechanism underlying the regulation of ANXA2 overexpression is still unclear. It is well established that genetic and epigenetic alterations may lead to widespread dysregulation of gene expression. Hence, in this study, we focused on exploring the regulatory mechanism of ANXA2 by investigating the transcriptional profile, methylation pattern, somatic mutation, and prognostic value of ANXA2 in PC using several bioinformatics databases. Our results revealed that the expression levels of ANXA2 were remarkably increased in PC tissues comparing to normal tissues. Furthermore, the high expression of ANXA2 was significantly related to the poor prognosis of PC patients. More importantly, we demonstrated for the first time that the ANXA2 promoter is hypomethylated in PC tissues compared to normal tissues which may result in ANXA2 overexpression in PC. However, more experimental research is required to corroborate our findings.

Annexin A2 Stabilizes Oncogenic JAG1 Intracellular Domain by Inhibiting Proteasomal Degradation in Glioblastoma Cells.

Glioblastoma (GBM) is the most lethal brain cancer, causing inevitable deaths of patients owing to frequent relapses of cancer stem cells (CSCs). The significance of the NOTCH signaling pathway in CSCs has been well recognized; however, there is no NOTCH-selective treatment applicable to patients with GBM. We recently reported that Jagged1 (JAG1), a NOTCH ligand, drives a NOTCH receptor-independent signaling pathway via JAG1 intracellular domain (JICD1) as a crucial signal that renders CSC properties. Therefore, mechanisms regulating the JICD1 signaling pathway should be elucidated to further develop a selective therapeutic regimen. Here, we identified annexin A2 (ANXA2) as an essential modulator to stabilize intrinsically disordered JICD1. The binding of ANXA2 to JICD1 prevents the proteasomal degradation of JICD1 by heat shock protein-70/90 and carboxy-terminus of Hsc70 interacting protein E3 ligase. Furthermore, JICD1-driven propagation and tumor aggressiveness were inhibited by ANXA2 knockdown. Taken together, our findings show that ANXA2 maintains the function of the NOTCH receptor-independent JICD1 signaling pathway by stabilizing JICD1, and the targeted suppression of JICD1-driven CSC properties can be achieved by blocking its interaction with ANXA2.

Association of pro-fibrinolytic receptor AnnexinA2 with tissue plasminogen activator/Inhibitor-1 in pre-eclampsia.

Background: The clinical manifestations of pre-eclampsia are related to placental anti-angiogenic factor alteration. These variations are mainly due to the alteration of plasminolytic components. The study aims to compare the expression of plasminolytic components in the placenta of women with and without pre-eclampsia. Material and Methods: The study included pregnant women with pre-eclampsia as PE group (n = 30) and without pre-eclampsia as a control group (n = 30). Placental bed biopsy tissues were collected. AnxA2, tPA, PAI-1 expression in the placental villous tissue was quantitatively evaluated using immunohistochemistry, western blot, and real time-PCR analysis. Results: The results of the study showed a significant decrease in the expression of ANXA2 and increased expression of tPA and PAI-1 in PE group compared to control group (p<0.005). AnxA2 expression showed positive correlation with tPA (r=+0.895, p=0.002) and negative correlation with PAI-1(r=-0.905, p=0.020) in control group whereas in the PE group AnxA2 expression was negatively correlated with tPA ((r=-0.801, p=0.016) and PAI-1 (R=-0.831, P=0.010). Conclusion: Decreased AnxA2 with increased expression of PAI-1 and tPA may be responsible for the altered fibrinolytic activity and play a significant role in pre-eclampsia pathogenesis.