Proteome-Wide Mendelian Randomization Analysis Identified Potential Drug Targets for Atrial Fibrillation.

Background Finding effective and safe therapeutic drugs for atrial fibrillation (AF) is an important concern for clinicians. Proteome-wide Mendelian randomization analysis provides new ideas for finding potential drug targets. Methods and Results Using a proteome-wide Mendelian randomization approach, we assessed the genetic predictive causality between thousands of proteins and AF risk and found that genetically predicted plasma levels of phosphomevalonate kinase, tumor necrosis factor ligand superfamily member 12, sulfhydryl oxidase 2, interleukin-6 receptor subunit alpha, and low-affinity immunoglobulin gamma Fc region receptor II-b might decrease AF risk, while genetically predicted plasma levels of beta-mannosidase, collagen alpha-1(XV) chain, ANXA4 (annexin A4), COF2 (cofilin-2), and RAB1A (Ras-related protein Rab-1A) might increase AF risk (P<3.4×10-5). By using different Mendelian randomization methods and instrumental variable selection thresholds, we performed sensitivity analyses in 30 scenarios to test the robustness of positive findings. Replication analyses were also performed in independent samples to further avoid false-positive findings. Drugs targeting tumor necrosis factor ligand superfamily member 12, interleukin-6 receptor subunit alpha, low-affinity immunoglobulin gamma Fc region receptor II-b, and annexin A4 are approved or in development. The results of the phenome-wide Mendelian randomization analysis showed that changing the plasma levels of phosphomevalonate kinase, cofilin-2, annexin A4, Ras-related protein Rab-1A, sulfhydryl oxidase 2, and collagen alpha-1(XV) chain did not increase the risk of other diseases while decreasing the risk of AF. Conclusions We found a significant causal association between genetically predicted levels of 10 plasma proteins and AF risk. Four of these proteins have drugs targeting them that are approved or in development, and our results suggest the potential for these drugs to treat AF or cause AF. Sulfhydryl oxidase 2, low-affinity immunoglobulin gamma Fc region receptor II-b, and beta-mannosidase have not been suggested by previous laboratory or epidemiological studies to be associated with AF and may reveal new pathophysiological pathways as well as therapeutic targets for AF.

LncRNA HOXD-AS1 promotes oral squamous cell carcinoma by sponging miR-203a-5p.

OBJECTIVE: In the present study, we aimed to explore lncRNA HOXD cluster antisense RNA 1 (HOXD-AS1) expression in oral squamous cell carcinoma (OSCC) tissues, its biological roles, and the underlying potential mechanisms in OSCC progression. MATERIALS AND METHODS: HOXD-AS1 expression in paired OSCC and non-tumor tissues from 60 OSCC patients was determined by RT-qPCR. The effects of HOXD-AS1 and miR-203a-5p overexpression on expression of Annexin A4, a validated target of miR-203a-5p, were analyzed by RT-qPCR and Western blot. The roles of HOXD-AS1, miR-203a-5p, and Annexin A4 in the invasion and migration of OSCC cells were analyzed by Transwell assays. RESULTS: HOXD-AS1 overexpression in OSCC predicted poor survival. HOXD-AS1 was predicted to interact with miR-203a-5p, but its expression was not significantly correlated with miR-203a-5p. HOXD-AS1 overexpression increased Annexin A4 expression, while miR-203a-5p overexpression decreased Annexin A4 expression in OSCC cells. Transwell assays showed that invasion and migration of OSCC cells were enhanced by HOXD-AS1 and Annexin A4 overexpression but were reduced by miR-203a-5p overexpression. In addition, miR-203a-5p overexpression suppressed the role of HOXD-AS1 in cell movement and Annexin A4 expression. CONCLUSIONS: HOXD-AS1 may upregulate Annexin A4 by sponging miR-203a-5p in OSCC to promote cancer cell invasion and migration.

Interplay of membrane crosslinking and curvature induction by annexins.

Efficient plasma membrane repair (PMR) is required to repair damage sustained in the cellular life cycle. The annexin family of proteins, involved in PMR, are activated by Ca2+ influx from extracellular media at the site of injury. Mechanistic studies of the annexins have been overwhelmingly performed using a single annexin, despite the recruitment of multiple annexins to membrane damage sites in living cells. Hence, we investigate the effect of the presence of the crosslinking annexins, annexin A1, A2 and A6 (ANXA1, ANXA2 and ANXA6) on the membrane curvature induction of annexin A4 (ANXA4) in model membrane systems. Our data support a mechanistic model of PMR where ANXA4 induced membrane curvature and ANXA6 crosslinking promotes wound closure. The model now can be expanded to include ANXA1 and ANXA2 as specialist free edge membrane crosslinkers that act in concert with ANXA4 induced curvature and ANXA6 crosslinking.

Evidence of an Annexin A4 mediated plasma membrane repair response to biomechanical strain associated with glaucoma pathogenesis.

Glaucoma is a common neurodegenerative blinding disease that is closely associated with chronic biomechanical strain at the optic nerve head (ONH). Yet, the cellular injury and mechanosensing mechanisms underlying the resulting damage have remained critically unclear. We previously identified Annexin A4 (ANXA4) from a proteomic analyses of human ONH astrocytes undergoing pathological biomechanical strain that mimics glaucomatous conditions. Annexins are a family of calcium-dependent phospholipid binding proteins with key functions in plasma membrane repair (PMR); an active mechanism to limit and mend cellular injury that involves membrane and cytoskeletal reorganizations. However, a role for direct membrane damage and PMR has not been well studied in the context of biomechanical strain, such as that associated with glaucoma. Here we report that this moderate strain surprisingly damages cell membranes to increase permeability in a calcium-dependent manner, and induces rapid aggregation of ANXA4 at injury sites. ANXA4 loss-of-function increases permeability, while exogenous ANXA4 reduces it. Furthermore, ANXA4 aggregation is associated with F-actin dynamics in vitro, and remarkably this interaction and aggregation signature is also observed in the glaucomatous ONH in patient samples. Together these studies link moderate biomechanical strain with direct membrane damage and actin dynamics, and identify an active PMR role for ANXA4 in new model of cell injury associated with glaucoma pathogenesis.

Circular RNA circ_0000592 elevates ANXA4 expression via sponging miR-1179 to facilitate tumor progression in gastric cancer.

Increasing evidence indicated that dysregulated circular RNAs were implicated in the progression of multiple malignancies. However, the function of circ_0000592 in gastric cancer (GC) progression and its associated mechanism remain poorly understood. Quantitative real-time PCR and Western blot assay were performed to detect RNA and protein expression. Cell proliferation, migration and invasion were analyzed by 5-Ethynyl-2'-deoxyuridine staining assay, Transwell migration assay and Transwell invasion assay, respectively. The glucose/lactate assay kit was used to assess the rates of glucose consumption and lactate production. The interaction between microRNA-1179 (miR-1179) and circ_0000592 or Annexin A4 (ANXA4) was confirmed by dual-luciferase reporter assay and RNA pull-down assay. Xenograft tumor model was established to investigate the effect of circ_0000592 on tumor growth in vivo. Circ_0000592 expression was elevated in GC tissues and cells. Circ_0000592 knockdown hampered cell proliferation, migration, invasion and glycolysis of GC cells. MiR-1179 was a direct target of circ_0000592, and circ_0000592 silencing-mediated effects in GC cells were partly reversed by the knockdown of miR-1179. MiR-1179 interacted with the 3' untranslated region (3'UTR) of ANXA4. Circ_0000592 silencing reduced ANXA4 expression partly by upregulating miR-1179 in GC cells. ANXA4 overexpression partly overturned circ_0000592 knockdown-induced effects in GC cells. Circ_0000592 depletion markedly suppressed xenograft tumor growth in vivo. Circ_0000592 contributed to GC progression through regulating miR-1179/ANXA4 axis, which provided novel potential biomarkers and therapeutic targets for GC treatment.

Simultaneous membrane binding of Annexin A4 and A5 suppresses 2D lattice formation while maintaining curvature induction.

HYPOTHESIS: Annexin A4 and A5 (ANXA4, ANXA5), both shown to be required for efficient plasma membrane repair (PMR) in living cells, bind as trimers to anionic membranes in the presence of calcium. Both annexins induce membrane curvature and self-assemble into crystal arrays on membranes, observations that have been associated with PMR. However, in-vitro studies of annexins have traditionally been performed using single annexins, despite the recruitment of multiple annexins to the damage site in cells. Hence, we study the potential cooperativity of ANXA4 and ANXA5 during membrane binding. EXPERIMENTS: Laser injury experiments were performed on MCF7 cells transfected to transiently express labelled ANXA4 and ANXA5 to study the localization of the proteins at the damage site. Using free-edged DOPC/DOPS (9:1) membranes we investigated the annexin-induced membrane rolling by fluorescence microscopy and the lateral arrangement of annexin trimers on the membrane surface by atomic force microscopy (AFM). FINDING: ANXA4 and ANXA5 colocalise at the damage site of MCF7 cells during repair. A (1:1) mixture of ANXA4 and ANXA5 induces membrane rolling with a time constant intermediate between the value for the pure annexins. While binding of the pure annexins creates crystal lattices, the (1:1) mixture generates a random arrangement of trimers. Thus, curvature induction remains as a functional property of annexin mixtures in PMR rather than crystal formation.

Membrane-cytoplasm translocation of annexin A4 is involved in the metastasis of colorectal carcinoma.

Annexin A4 (ANXA4) is a Ca2+- and phospholipid-binding protein that belongs to the annexin family, which is involved in the development of multiple tumour types via NF-κB signalling. In this study, we verified the high expression and membrane-cytoplasm translocation of ANXA4 in colorectal carcinoma (CRC). Calcium/calmodulin-dependent protein kinase II gamma (CAMK2γ) was found to be important for high ANXA4 expression in CRC, whereas carbonic anhydrase (CA1) promoted ANXA4 aggregation in the cell membrane. An increased Ca2+ concentration attenuated the small ubiquitin-like modifier (SUMO) modification of cytoplasmic ANXA4 and ANXA4 stabilization, and relatively high expression of ANXA4 promoted CRC tumorigenesis and epithelial-mesenchymal transition (EMT).

Annexin A4 trimers are recruited by high membrane curvatures in giant plasma membrane vesicles.

The plasma membrane (PM) of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the PM is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including PM repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant PM vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations, in which we extract molecular information from atomistic scale simulations as input to our macroscopic scale simulations, furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

p53 and ANXA4/NF­κB p50 complexes regulate cell proliferation, apoptosis and tumor progression in ovarian clear cell carcinoma.

Annexin IV (ANXA4) is highly expressed in ovarian clear cell carcinoma (OCCC); however, its underlying molecular mechanism in OCCC remains unknown. The present study aimed to identify the molecule that ANXA4 may act on and to determine its underlying molecular mechanism. Immunohistochemistry, co­immunoprecipitation and western blotting were performed to detect the expression and interaction of ANXA4, and its associated proteins. Furthermore, MTT assay, flow cytometry, western blotting and gene expression profile enrichment analysis were performed to identify the potential role and molecular mechanism of ANXA4 in OCCC. The results demonstrated that ANXA4 and nuclear factor­κ­light­chain­enhancer of activated B cells (NF­κB) p50 nuclear expression levels were significantly higher in OCCC tissues compared with other subtypes of ovarian cancer, such as serous and mucinous. In addition, a significantly positive correlation was observed between ANXA4 and NF­κB p50 expression in OCCC; however, the expression levels of mutant p53 and ANXA4 were negatively correlated in a linear manner. These results suggest that ANXA4 and NF­κB p50 may be potential independent risk factors for poor prognosis. ANXA4 and NF­κB p50 were demonstrated to interact and their expression was co­localized. The cBioPortal database was used to construct a protein­protein interaction network between ANXA4, NF­κB p50 and p53, and functional pathway analysis indicated that the genes were predominantly enriched in the cell cycle and during apoptosis. Transfection of the ANXA4 gene increased the expression of NF­κB p50, as well as its downstream targets, Cyclin D1 and B­cell lymphoma­2 (Bcl­2). Furthermore, transfection of the ANXA4 gene increased proliferation and decreased apoptosis of OCCC cells. Treatment with the NF­κB inhibitor, BAY 11­7082, decreased Cyclin D1 and Bcl­2 expression levels. Collectively, the results of the present study suggest that wild p53 activates ANXA4 transcription, promotes its expression and enhances NF­κB p50 and ANXA4 interaction. This in turn activates the NF­κB signaling pathway, promotes cell cycle progression and inhibits apoptosis, thus contributing to the malignant progression of OCCC. Thus, ANXA4 and NF­κB p50 may be used as prognostic biomarkers, and may be molecular therapeutic targets in OCCC.

Clinical Significance of Annexin A4 as a Biomarker in the Early Diagnosis of Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most prevalent cancer worldwide. Early detection of HCC is crucial to improve prognosis and survival. Nearly 30 % of HCC patients present with normal serum alpha fetoprotein (AFP), which highlights the need for new biomarkers for HCC. Annexin A4 (ANXA4) is one of the annexin family with high expressions found in gastric, liver, lung, colorectal and ovarian cancers. AIM: to evaluate the clinical significance of ANXA4 in the early diagnosis of HCC. METHODS: Thirty patients with hepatitis C virus (HCV) related HCC were enrolled in this study. They were stage A according to Barcelona Clinic Liver Cancer (BCLC) staging and they were grade A or B according to Child Pugh Classification. Twenty patients with HCV-related liver cirrhosis and 20 healthy persons seronegative for both HCV and HBV served as control group. ANXA4 and AFP were measured in serum of all cases. RESULTS: Serum ANXA4 level was significantly higher in HCC patients compared to patients with liver cirrhosis and healthy controls (188, IQR 42-428 and 23, IQR 24-33 and and 21, IQR 22-24 ng Ì· ml, respectively). By using the ROC curve, the area under the curve of ANXA4 was 0.972 and the best cut-off value was115 ng/ml, with sensitivity 95% and specificity 80%. CONCLUSION: The serum level of ANXA4 might be a good biomarker for the early detection of HCC.
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GSK3ß-Ikaros-ANXA4 signaling inhibits high-glucose-induced fibroblast migration.

Previous studies showed that the activation of Wnt signaling reduced high glucose (HG)-mediated fibroblast damage, but the molecular basis for this phenomenon remains elusive. This study aimed to analyze the level of phosphorylation of GSK3ß Ser9 (pGSK3ß Ser9) during HG damage. Moreover, the phosphomimic form of pGSK3ß Ser9 was expressed to analyze its effect on cell migration via the phosphorylation of Ikaros. The results revealed that HG treatment significantly reduced the pGSK3ß Ser9 level. The overexpression of GSK3ß Ser9D and GSK3ß Ser9A accelerated and inhibited fibroblast cell migration, respectively. P110α knockdown or treatment with SP600125, an inhibitor of JNK, also reduced the pGSK3ß Ser9 level under HG condition. Treatment with SP600125 inhibited the migration of fibroblasts, but not in GSK3ß Ser9D-expressing cells. Further, yeast two-hybrid screening and biochemical analysis identified that GSK3ß interacted and phosphorylated Ikaros at Ser391. Besides, GSK3ß Ser9D, but not GSK3ß Ser9A, activated Ikaros Ser391 phosphorylation. Expressing Ikaros or ß-catenin significantly promoted cell migration, suggesting that GSK3ß modulated cell migration partially via the activation of Ikaros besides ß-catenin signaling under HG condition. The expression of the phosphomimic form of Ikaros Ser391D resulted in a significant increase in the extent of cell migration compared with Ikaros under HG condition. Moreover, the Ikaros Ser391D DNA-binding affinity toward the ANXA4 promoter increased, and ANXA4 suppression promoted cell migration. In conclusion, the results of this study provided a new regulatory mechanism by which GSK3ß negatively regulated human skin fibroblast cell migration.

ANXA4 Activates JAK-STAT3 Signaling by Interacting with ANXA1 in Basal-Like Breast Cancer.

Annexin A4 (encoded by the ANXA4 gene) is a calcium ion (Ca2+)- and phospholipid-binding protein of the Annexin family. In this study, we checked the expression profile of ANXA4 in basal-like breast cancer (BLBC) and its association with survival outcomes using pan-cancer data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. Then, using MDA-MB-231 and MDA-MB-468 cells, we explored the functional role of ANXA4 in regulating a cancer-related signaling pathway and identified potential partners of ANXA4. The results showed that expression of total ANXA4 and the two dominant ANXA4 protein-coding transcripts (ENST00000409920.5 and ENST00000394295.4) was consistently upregulated in tumor tissues compared with normal breast tissues. BLBC patients with high ANXA4 expression had significantly worse overall survival, progression-free survival, and disease-free survival than those with low ANXA4 expression. ANXA4 could positively modulate cyclin D1 expression and G1/S progression in the two cell lines. An in vivo tumor model showed that ANXA4 inhibition significantly slowed the growth of tumors derived from the two BLBC cell lines. ANXA4 could increase JAK1 expression and STAT3 phosphorylation (Y705). ANXA4 colocalized with ANXA1 in some MDA-MB-231 cells. A co-immunoprecipitation assay confirmed direct binding between ANXA4 and ANXA1. Knockdown of ANXA1 reduced JAK1 expression and STAT3 phosphorylation and impaired ANXA4-induced upregulation of JAK1 and p-STAT3. In conclusion, this study revealed that aberrant ANXA4 upregulation is associated with poor survival in BLBC. ANXA4 could activate JAK-STAT3 signaling by elevating the expression of JAK1 and p-STAT3, which was mediated by direct interaction with ANXA1.

Annexin A4 N-terminal peptide inhibits adenylyl cyclase 5 and limits ß-adrenoceptor-mediated prolongation of cardiac action potential.

Adenylyl cyclases (AC) are essential for the normal and pathophysiological response of many cells. In cardiomyocytes, the predominant AC isoforms are AC5 and AC6. Specific AC5 inhibition was suggested as an option for the treatment of heart failure potentially advantageous over ß-blockers. We previously reported an interaction between the calcium-binding protein annexin A4 (ANXA4) and AC5 in human embryonic kidney 293 (HEK293) cells and an inhibition of cyclic adenosine monophosphate (cAMP) production in cardiomyocytes. Here, we investigated whether ANXA4 is able to differentiate between AC5 and AC6. In transfected HEK293 cells, ANXA4 specifically co-immunoprecipitated with AC5 and not with AC6, via its N-terminal domain. Both ANXA4 and a peptide comprising the ANXA4 N-terminal sequence (A4N1-22 ) decreased the cAMP production in AC5 and not in AC6 expressing cells. In line with ACs inhibition, in myocytes from ANXA4-deficient mice, ß-adrenoceptor (ßAR) stimulation led to a higher increase of the L-type calcium current (ICaL ) and to an excessive action potential duration (APD) prolongation as compared to wild-type cardiomyocytes. This enhanced response was reversed in the presence of A4N1-22 peptide likely via specific AC5 inhibition. We conclude that via the N-terminal domain ANXA4 inhibits AC5 not AC6, and that A4N1-22 as a specific AC5 inhibitor could serve as a novel therapeutic tool for the treatment of AC5-linked diseases.

Avian uterine fluid proteome: Exosomes and biological processes potentially involved in sperm survival.

Uterine fluid is an aqueous milieu to which sperm are exposed during their storage and ascent. In this study, a bottom-up proteomic strategy and bioinformatic analysis of hen uterine fluid was performed to improve the understanding of this fluid and its potential role in sperm survival mechanisms. The proteomic data were submitted to ProteomeXchange. Among the 913 proteins identified, 160 are known to be secreted and 640 are referenced in exosomes databases. We isolated exosomes from the avian uterine fluid, analyzed them using electron microscopy, and targeted several exosomes markers (ANXA1/2/4/5, VCP, HSP90A, HSPA8, PARK7, and MDH1) using immunoblotting. Electron microscopy and immunohistochemistry were also used to analyze uterovaginal junctions for the exosomal proteins ANXA4, VCP, and PARK7. Exosomes were observed both at the surface epithelium and inside sperm storage tubules. Our data were compared with two previously published studies on proteomic of hen uterine fluid, and with one study describing the proteomic content of rooster seminal plasma and sperm. In conclusion, we demonstrated for the first time that avian uterine fluid contains exosomes. These may play a key role in preserving sperm functions within the female genital tract. Their presence in the sperm storage tubules may represent an important mechanism regarding interaction between the female genital tract and sperm.

Annexin A4 inhibits sulfatide-induced activation of coagulation factor XII.

BACKGROUND: Factor XII (FXII) is a plasma serine protease that initiates the intrinsic pathway of blood coagulation upon contact with anionic substances, such as the sulfated glycolipid sulfatide. Annexins (ANXs) have been implicated in the regulation of the blood coagulation reaction by binding to anionic surfaces composed of phospholipids and sulfated glycoconjugates, but their physiological importance is only partially understood. OBJECTIVE: To test the hypothesis that ANXs are involved in suppressing the intrinsic pathway initiated by sulfatide, we examined the effect of eight recombinant ANX proteins on the intrinsic coagulation reaction and their sulfatide binding activities. METHODS: Recombinant ANXs were prepared in Escherichia coli expression systems and their anticoagulant effects on the intrinsic pathway initiated by sulfatide were examined using plasma clotting assay and chromogenic assay. ANXA4 active sites were identified by alanine scanning and fold deletion in the core domain. RESULTS AND CONCLUSIONS: We found that ANXA3, ANXA4, and ANXA5 strongly inhibited sulfatide-induced plasma coagulation. Wild-type and mutated ANXA4 were used to clarify the molecular mechanism involved in inhibition. ANXA4 inhibited sulfatide-induced auto-activation of FXII to FXIIa and the conversion of its natural substrate FXI to FXIa but showed no effect on the protease activity of FXIIa or FXIa. Alanine scanning showed that substitution of the Ca2+ -binding amino acid residue in the fourth fold of the core domain of ANXA4 reduced anticoagulant activity, and deletion of the entire fourth fold of the core domain resulted in complete loss of anticoagulant activity.

miR-203 Inhibits the Invasion and EMT of Gastric Cancer Cells by Directly Targeting Annexin A4.

Many studies have shown that downregulated miR-203 level is in a variety of cancers including gastric cancer (GC). However, the precise molecule mechanisms of miR-203 in GC have not been well clarified. In the current study, we investigated the biological functions and molecular mechanisms of miR-203 in GC cell lines. We found that miR-203 is downregulated in GC tissues and cell lines. Moreover, the low level of miR-203 was associated with increased expression of annexin A4 in GC tissues and cell lines. The invasion and EMT of GC cells were suppressed by overexpression of miR-203. However, downregulation of miR-203 promoted invasion and EMT of GC cells. Bioinformatics analysis predicted that annexin A4 was a potential target gene of miR-203. Next, luciferase reporter assay confirmed that miR-203 could directly target annexin A4. Consistent with the effect of miR-203, downregulation of annexin A4 by siRNA inhibited the invasion and EMT of GC cells. Introduction of annexin A4 in GC cells partially blocked the effects of miR-203 mimic. Introduction of miR-203 directly targeted annexin A4 to inhibit the invasion and EMT of GC cells. Overall, reactivation of the miR-203/annexin A4 axis may represent a new strategy for overcoming metastasis of GC.

ANXA4 promotes trophoblast invasion via the PI3K/Akt/eNOS pathway in preeclampsia.

The inadequate trophoblast invasion is associated with the development of preeclampsia (PE). Considering that annexin A4 (ANXA4) enhances tumor invasion, we aimed to explore the functional role of ANXA4 in trophoblast cells and to examine the underlying mechanism. ANXA4 expression in PE placentas was analyzed using immunohistochemistry and Western blotting. Cell proliferation, invasion, and apoptosis were determined using a MTT assay, Transwell assay, and flow cytometry, respectively. The expression levels of matrix metalloproteinase (MMP)-2, MMP-9, phosphoinositide 3-kinase (PI3K), Akt, phosphorylated (p)-Akt, and phosphorylated endothelial nitric oxide synthase (p-eNOS) were detected by Western blotting. Placentas were prepared for pathological examination using hematoxylin and eosin staining and apoptosis determination using the TUNEL method. Expression of ANXA4, PI3K, p-Akt and p-eNOS was downregulated in human PE placentas and PE placenta-derived extravillous cytotrophoblasts (EVCTs). Furthermore, ANXA4 overexpression promoted cell proliferation and invasion, inhibited cell apoptosis, and upregulated protein expression of PI3K, p-Akt, and p-eNOS in human trophoblast cells HTR-8/SVneo and JEG-3. By contrast, ANXA4 knockdown exerted the opposite effects. Furthermore, inhibition of the PI3K/Akt pathway by LY294002 abrogated the ANXA4 overexpression-mediated effects on trophoblast behavior. Furthermore, eNOS knockdown abrogated the ANXA4 overexpression-induced promotion of cell invasion and MMP2/9 expression. Additionally, in N-nitro-l-arginine methyl ester (l-NAME)-induced PE rats, ANXA4 overexpression alleviated PE progression, accompanied by an increase in expression of PI3K, p-Akt, and p-eNOS in rat placentas. Our findings demonstrate that ANXA4 expression is downregulated in PE. ANXA4 may promote trophoblast invasion via the PI3K/Akt/eNOS pathway.

Anxa4 mediated airway progenitor cell migration promotes distal epithelial cell fate specification.

Genetic studies have shown that FGF10/FGFR2 signaling is required for airway branching morphogenesis and FGF10 functions as a chemoattractant factor for distal epithelial cells during lung development. However, the detail downstream cellular and molecular mechanisms have not been fully characterized. Using live imaging of ex vivo cultured lungs, we found that tip airway epithelial progenitor cells migrate faster than cleft cells during airway bud formation and this migration process is controlled by FGFR2-mediated ERK1/2 signaling. Additionally, we found that airway progenitor cells that migrate faster tend to become distal airway progenitor cells. We identified that Anxa4 is a downstream target of ERK1/2 signaling. Anxa4-/- airway epithelial cells exhibit a "lag-behind" behavior and tend to stay at the stalk airways. Moreover, we found that Anxa4-overexpressing cells tend to migrate to the bud tips. Finally, we demonstrated that Anxa4 functions redundantly with Anxa1 and Anxa6 in regulating endoderm budding process. Our study demonstrates that ERK1/2/Anxa4 signaling plays a role in promoting the migration of airway epithelial progenitor cells to distal airway tips and ensuring their distal cell fate.

Lewis(y) antigen-mediated positive feedback loop induces and promotes chemotherapeutic resistance in ovarian cancer.

The present study aimed to investigate the association between Lewis(y) antigen and chemoresistance in ovarian cancer and to elucidate the underlying molecular mechanisms. Lewis(y) expression in chemoresistant ovarian cancer tissues and cells was detected by immunohistochemistry. α1,2­fucosyltransferase (FUT1) expression in different ovarian cancer chemotherapy-resistant cells was analyzed by reverse transcription-quantitative PCR (RT-qPCR). Genes differentially expressed in the chemoresistant and sensitive groups were screened using a gene chip followed by validation using RT-qPCR and western blot analysis. We found that Lewis(y) and FUT1 expression in ovarian cancer cells was significantly increased following the induction of drug resistance. The positive expression rate and intensity of Lewis(y) in ovarian cancer chemoresistant tissues were also significantly higher than those in the sensitive group. Compared with the non-resistant cell lines, the differentially expressed genes were mainly enriched in the terms related to the transmembrane receptor protein tyrosine kinase signaling pathway and positive regulation of cell proliferation. Interaction network analysis predicted genes participating in the regulation of apoptotic processes. The highly differential expression of Annexin A4 (ANXA4), BCL2 interacting killer (BIK), transmembrane 4 L six family member 4 (TM4SF4) and pleckstrin homology-like domain family A member 1 (PHLDA1) was validated using RT-qPCR in ovarian cancer cell lines. Finally, ANXA4 expression was increased at both the mRNA and protein level in the drug­resistant cells, and in addition, ANXA4 contained a Lewis(y) structure. The expression of Bcl-2 and other anti-apoptotic proteins increased with the increase of Lewis(y) expression. After blocking Lewis(y) using an antibody, the expression of the involved signaling pathway and apoptosis-related proteins decreased significantly. These findings provide strong evidence that Lewis(y) is a component of the structure of the ANXA4 membrane protein. Its overexpression can abnormally activate signaling pathways and regulate the expression of a number of factors, forming a positive feedback loop to induce the chemoresistance of ovarian cancer cells, and ultimately promoting the progression of ovarian cancer.

Toosendanin mediates cisplatin sensitization through targeting Annexin A4/ATP7A in non-small cell lung cancer cells.

Cisplatin (CDDP) is used in the treatment of non-small cell lung cancer (NSCLC), but due to the development of resistance, the benefit has been limited. Toosendanin (TSN) has shown therapeutic effects on NSCLC; however, the role of TSN on CDDP sensitization in NSCLC remains unknown. The antitumor effects of TSN and CDDP sensitization mediated by TSN were explored. TSN was added in various amounts to measure dose- and time-dependent cytotoxicity. Intracellular CDDP was detected by high-performance liquid chromatography. The protein levels of ATP7A, ATP7B, hCTR1, MRP-2, P-gp and Annexin A4 (Anxa4) were analyzed. The tests were conducted using normal NSCLC (A549 cell line) and CDDP-resistant cells (A549/DDP cell line). Anxa4 promotes CDDP resistance by regulating ATP7A, so Anxa4 was overexpressed and silenced and also transfected with pcMV6 or siRNA/ATP7A, respectively. Mechanistic investigations revealed that TSN decreased relative viability in NSCLC cells. Remarkably, TSN significantly enhanced CDDPsensitization in invalid doses. TSN downregulated Anxa4 expression, enhanced intracellular CDDP, and had no effect on MRP-2, P-gp, ATP7A, ATP7B or hCTR1. Subsequently, overexpression of Anxa4 led to a significant decrease in intracellular CDDP concentration. The adjustment of CDDP concentration regulated by TSN disappeared in Anxa4 or ATP7A-silenced cells. TSN also enhanced CDDP sensitization in single ATP7A-overexpressing cells, but had no effect on cells with simultaneous ATP7A overexpression and Anxa4 silencing. The present study suggests that TSN can mediate CDDP sensitization in NSCLC through downregulation of Anxa4.