Wnt3a Proteins Research Articles

WNT Signaling Cascade Proteins and LRP6 in the Formation of Various Types of Coronary Lesions in Patients With Coronary Artery Disease.

Assessment of WNT1, WNT3a, and LRP6 concentrations in patients with ischemic heart disease (IHD) and obstructive and non-obstructive coronary artery (CA) disease. This cross-sectional observational study included 50 IHD patients (verified by coronary angiography, CAG), of which 25 (50%) were men, mean age 64.9±8.1 years; 20 patients had non-obstructive CA disease (stenosis <50%), and 30 patients had hemodynamically significant stenosis. Concentrations of WNT1, WNT3a and LRP6 were measured in all patients. The concentrations of WNT1 and WNT3a proteins were significantly higher in patients with IHD and obstructive CA disease (p < 0.001), while the concentration of LRP6 was higher in the group with non-obstructive CA disease (p = 0.016). Data analysis of the group with obstructive CA disease showed a moderate correlation between WNT1 and LRP6 (ρ=0.374; p=0.042). Correlation analysis of all groups of patients with CA disease revealed a moderate association between the concentrations of WNT1 and uric acid (ρ=0.416; p=0.007). Regression analysis showed that risk factors for the development of IHD, such as increased body mass index, age, smoking, dyslipidemia, and hypertension, did not significantly influence the type of CA disease in IHD patients. According to ROC analysis, the obstructive form of IHD was predicted by a WNT3a concentration higher than 0.155 ng/ml and a LRP6 concentration lower than 12.94 ng/ml. IHD patients with non-obstructive CA disease had the greatest increase in LRP6, while patients with obstructive CA disease had significantly higher concentrations of the canonical WNT cascade proteins, WNT1 and WNT3a. According to the ROC analysis, a WNT3a concentration >0.155 ng/ml can serve as a predictor for the presence of hemodynamically significant CA stenosis in IHD patients (sensitivity 96.7%; specificity 70%), whereas a LRP6 concentration >12.94 ng/ml can predict the development of non-obstructive CA disease (sensitivity 76.7%; specificity 65%).

Abstract LB187: Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications

Abstract Lung cancer is the leading cause of cancer mortality worldwide, representing an unmet medical need. Receptor tyrosine kinase-like orphan receptors (RORs) are oncofetal antigens that act as receptors/co-receptors of WNT5A and other related noncanonical WNT proteins, in transducing β-catenin independent WNT signaling. Dysregulated ROR/WNT have been linked to cancer progression and therapy resistance. Recently, ROR receptors have drawn increasing attention as anti-cancer targets of antibody-drug conjugates (ADCs) due to their selective expression on the tumor cell surface. The ROR family contains ROR1 and ROR2, which share 58% sequence homology. However, their functional interactions between ROR/WNT5A and whether RORs have distinct or redundant roles in tumor phenotypes remain unclear. In this study, we sought to address these biological questions in NSCLC, and investigate the potential application of anti-ROR therapies. To this end, we used CRISPR/Cas9 to generate clones of ROR1 or ROR2 single knockouts (KOs) in the NSCLC LCLC-103H cell line and implemented an array of assays to characterize the effect of single ROR KOs. ROR KOs were confirmed by both western blot and flow cytometry. We found reduced ROR2 mean fluorescence intensity (MFI) on ROR1 KOs and increased ROR1 MFI on ROR2 KOs in flow cytometrical analysis. Phenotypically, ROR1 KOs did not affect tumor doubling time, but ROR2 KOs promoted cell proliferation in 2D cultures. Unlike 2D cultures, tumor 3D spheroid proliferation was significantly inhibited in both ROR1 and ROR2 KOs, implying that each ROR is indispensable in maintaining tumor growth featured by cell-cell/extracellular matrix interactions. Mechanistically, ROR1 KOs tended to decrease ROR2 and WNT5A/B protein expression, while ROR2 KOs caused compensatory upregulation of ROR1 and WNT5A/B. Both ROR1 and ROR2 KOs decreased SNAIL expression, which is a key driver of epithelial-to-mesenchymal plasticity (EMP). Furthermore, alterations in ROR1/2 signaling pathways were observed. We have identified pathways downstream of ROR1/2 including WNT, EMP, and survival, which may shed light on the biological roles of RORs in cancer and potential therapeutic intervention. Finally, the therapeutic application of ROR1/2 was evaluated by monomethyl auristatin F (MMAF) containing anti-ROR ADCs. ROR1 KOs demonstrated the on-target cytotoxicity of anti-ROR1 ADC, while ROR2 KOs showed increased sensitivity to anti-ROR1 ADC due to the increased ROR1 expression in ROR2 KOs. In vivo analysis is currently underway. Collectively, our results demonstrate that RORs have both distinct and compensatory functions in regulating tumor proliferation. We believe a greater understanding in ROR1/2 biology may lead to better targeted therapeutic applications. Citation Format: Wen-An Pan, Xin Guo, Christina Galang, Mira Ko, Jeff Kucharski, Nicole Miller, Haim Moskowitz, Jeff Watkins, Brian Lannutti, Katti Jessen, Hao Liu. Investigating the roles of ROR1/2 in non-small cell lung carcinoma NSCLC and potential therapeutic applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB187.

Significance of the Wnt signaling pathway in coronary artery atherosclerosis.

The progression of coronary atherosclerosis is an active and regulated process. The Wnt signaling pathway is thought to play an active role in the pathogenesis of several cardiovascular diseases; however, a better understanding of this system in atherosclerosis is yet to be unraveled. In this study, real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting were used to quantify the expression of Wnt3a, Wnt5a, and Wnt5b in the human coronary plaque, and immunohistochemistry was used to identify sites of local expression. To determine the pathologic significance of increased Wnt, human vascular smooth muscle cells (vSMCs) were treated with Wnt3a, Wnt5a, and Wnt5b recombinant proteins and assessed for changes in cell differentiation and function. RT-PCR and Western blotting showed a significant increase in the expression of Wnt3a, Wnt5a, Wnt5b, and their receptors in diseased coronary arteries compared with that in non-diseased coronary arteries. Immunohistochemistry revealed an abundant expression of Wnt3a and Wnt5b in diseased coronary arteries, which contrasted with little or no signals in normal coronary arteries. Immunostaining of Wnt3a and Wnt5b was found largely in inflammatory cells and myointimal cells. The treatment of vSMCs with Wnt3a, Wnt5a, and Wnt5b resulted in increased vSMC differentiation, migration, calcification, oxidative stress, and impaired cholesterol handling. This study demonstrates the upregulation of three important members of canonical and non-canonical Wnt signaling pathways and their receptors in coronary atherosclerosis and shows an important role for these molecules in plaque development through increased cellular remodeling and impaired cholesterol handling.

R-spondin-1 induces Axin degradation via the LRP6-CK1ε axis

R-spondins (RSPOs) are secreted signaling molecules that potentiate the Wnt/β-catenin pathway by cooperating with Wnt ligands. RSPO1 is crucial in tissue development and tissue homeostasis. However, the molecular mechanism by which RSPOs activate Wnt/β-catenin signaling remains elusive. In this study, we found that RSPOs could mediate the degradation of Axin through the ubiquitin–proteasome pathway. The results of Co-IP showed that the recombinant RSPO1 protein promoted the interaction between Axin1 and CK1ε. Either knockout of the CK1ε gene or treatment with the CK1δ/CK1ε inhibitor SR3029 caused an increase in Axin1 protein levels and attenuated RSPO1-induced degradation of the Axin1 protein. Moreover, we observed an increase in the number of associations of LRP6 with CK1ε and Axin1 following RSPO1 stimulation. Overexpression of LRP6 further potentiated Axin1 degradation mediated by RSPO1 or CK1ε. In addition, recombinant RSPO1 and Wnt3A proteins synergistically downregulated the protein expression of Axin1 and enhanced the transcriptional activity of the SuperTOPFlash reporter. Taken together, these results uncover the novel mechanism by which RSPOs activate Wnt/β-catenin signaling through LRP6/CK1ε-mediated degradation of Axin.Graphical

Is There a Correlation Between Serum SFRP5 and Wnt5a Proteins and Insulin Resistance in Iraqi Infertile Females Undergoing ICSI?

Is There a Correlation Between Serum SFRP5 and Wnt5a Proteins and Insulin Resistance in Iraqi Infertile Females Undergoing ICSI?

Activation of the Wnt/β-catenin/CYP1B1 pathway alleviates oxidative stress and protects the blood-brain barrier under cerebral ischemia/reperfusion conditions.

Abstract JOURNAL/nrgr/04.03/01300535-202407000-00033/figure1/v/2023-11-20T171125Z/r/image-tiff Accumulating evidence suggests that oxidative stress and the Wnt/β-catenin pathway participate in stroke-induced disruption of the blood-brain barrier. However, the potential links between them following ischemic stroke remain largely unknown. The present study found that cerebral ischemia leads to oxidative stress and repression of the Wnt/β-catenin pathway. Meanwhile, Wnt/β-catenin pathway activation by the pharmacological inhibitor, TWS119, relieved oxidative stress, increased the levels of cytochrome P450 1B1 (CYP1B1) and tight junction-associated proteins (zonula occludens-1 [ZO-1], occludin and claudin-5), as well as brain microvascular density in cerebral ischemia rats. Moreover, rat brain microvascular endothelial cells that underwent oxygen glucose deprivation/reoxygenation displayed intense oxidative stress, suppression of the Wnt/β-catenin pathway, aggravated cell apoptosis, downregulated CYP1B1 and tight junction protein levels, and inhibited cell proliferation and migration. Overexpression of β-catenin or knockdown of β-catenin and CYP1B1 genes in rat brain microvascular endothelial cells at least partly ameliorated or exacerbated these effects, respectively. In addition, small interfering RNA-mediated β-catenin silencing decreased CYP1B1 expression, whereas CYP1B1 knockdown did not change the levels of glycogen synthase kinase 3β, Wnt-3a, and β-catenin proteins in rat brain microvascular endothelial cells after oxygen glucose deprivation/reoxygenation. Thus, the data suggest that CYP1B1 can be regulated by Wnt/β-catenin signaling, and activation of the Wnt/β-catenin/CYP1B1 pathway contributes to alleviation of oxidative stress, increased tight junction levels, and protection of the blood-brain barrier against ischemia/hypoxia-induced injury.

Uterine WNTS modulates fibronectin binding activity required for blastocyst attachment through the WNT/CA2+ signaling pathway in mice

Adhesion of the implanting blastocyst involves the interaction between integrin proteins expressed by trophoblast cells and components present in the basement membrane of the endometrial luminal epithelium. Although several factors regulating integrins and their adhesion to fibronectin are already known, we showed that Wnt signaling is involved in the regulation of blastocyst adhesion through the trafficking of integrins expressed by trophoblast cells. Localization of Itgα5β1 by immunofluorescence and FN-binding assays were conducted on peri-implantation blastocysts treated with either Wnt5a or Wnt7a proteins. Both Wnt5a and Wnt7a induced a translocation of Itgα5β1 at the surface of the blastocyst and an increase in FN-binding activity. We further demonstrated that uterine fluid is capable of inducing integrin translocation and this activity can be specifically inhibited by the Wnt inhibitor sFRP2. To identify the Wnt signaling pathway involved in this activity, blastocysts were incubated with inhibitors of either p38MAPK, PI3K pathway or CamKII prior to the addition of Wnts. Whereas inhibition of p38MAPK and PI3K had not effect, inhibition of CamKII reduced FN-binding activity induced by Wnts. Finally, we demonstrated that inhibition of Wnts by sFRP2 reduced the binding efficiency of the blastocyst to uterine epithelial cells. Our findings provide new insight into the mechanism that regulates integrin trafficking and FN-binding activity and identifies Wnts as a key player in blastocyst attachment to the uterine epithelium.

Small Nucleolar RNA Host Gene 9 Promotes the Development of Breast Cancer by Regulating the miR-326/Wnt5a/β-Catenin Axis

Breast cancer (BC) is a highly prevalent aggressive malignancy in women worldwide, and the search for key targets in its pathogenesis is a focus of research. Long non-coding RNAs (lncRNAs) play an important role in many cancers, including breast cancer. This study aimed to investigate the role of lncRNA SNHG9 in BC. The expression of SNHG9 in BC cells was found to be higher than that of human mammary epithelium. SNHG9 was found to inhibit the proliferation, migration, and invasion of cells and promote apoptosis. It was also found that SNHG9 regulates the miR-326/Wnt5a/β-catenin axis to promote the development of BC. Dual luciferase reporter and RNA pull-down assays confirmed the interaction between SNHG9, Wnt5a, and miR-326. Western blot analysis indicated that the expression of Wnt5a, β-catenin, c-myc, and cyclin D1 decreased significantly after the silencing of SNHG9 and the overexpression of miR-326. On the contrary, Wnt5a, β-catenin, c-myc, and cyclin D1 proteins were significantly up-regulated after inhibiting miR-326 expression. These findings suggest that SNHG9 is a promising target for BC therapy.

Reduced mastication during growth inhibits cognitive function by affecting trigeminal ganglia and modulating Wnt signaling pathway and ARHGAP33 molecular transmission

Binding of brain-derived neurotrophic factor (BDNF) to its receptor tyrosine kinase B (TrkB) is essential for the development of the hippocampus, which regulates memory and learning. Decreased masticatory stimulation during growth reportedly increases BDNF expression while decreasing TrkB expression in the hippocampus. Increased BDNF expression is associated with Wnt family member 3A (Wnt3a) expression and decreased expression of Rho GTPase Activating Protein 33 (ARHGAP33), which regulates intracellular transport of TrkB. TrkB expression may be decreased at the cell surface and affects the hippocampus via BDNF/TrkB signaling. Mastication affects cerebral blood flow and the neural cascade that occurs through the trigeminal nerve and hippocampus. In the current study, we hypothesized that decreased masticatory stimulation reduces memory/learning in mice due to altered Wnt3a and ARHGAP33 expression, which are related to memory/learning functions in the hippocampus. To test this hypothesis, we fed mice a powdered diet until 14 weeks of age and analyzed the BDNF and TrkB mRNA expression in the right hippocampus using real-time polymerase chain reaction and Wnt3a and ARHGAP33 levels in the left hippocampus using western blotting. Furthermore, we used staining to assess BDNF and TrkB expression in the hippocampus and the number of nerve cells, the average size of each single cell and the area of intercellular spaces of the trigeminal ganglion (TG). We found that decreased masticatory stimulation affected the expression of BDNF, Wnt3a, ARHGAP33, and TrkB proteins in the hippocampus, as well as memory/learning. The experimental group showed significantly decreased numbers of neurons and increased the area of intercellular spaces in the TG. Our findings suggest that reduced masticatory stimulation during growth induces a decline in memory/learning by modulating molecular transmission mechanisms in the hippocampus and TG.

Synthesis and potential osteogenic applications of Wnt3a‐loaded ZIF‐8 nanoparticles

The Wnt signaling pathway plays a critical role in bone homeostasis, and the related protein therapy strategies have been reported to have great potential in osseointegration; however, they face formidable challenges such as complex external environments and unavoidable protein denaturation. In this work, we report a novel approach combining the synthesis of metal–organic frameworks (MOFs) and protein encapsulation in a one-pot process based on zeolitic imidazolate framework-8 (ZIF-8) and Wnt3a protein, with improved biomechanical behavior and enhanced protein biological response. This combination was designed to enhance the Wnt3a protein function through the improved chemical stability provided by the ZIF-8 crystals. Additionally, the zinc ions contained in the ZIF-8 crystals induced bone homeostasis, further favoring the osteogenesis. The results showed that the Wnt3a protein-loaded ZIF-8 crystals served as efficient drug delivery vehicles to promote osteogenesis, preventing protein denaturation. In particular, Wnt3a-loaded ZIF-8 nanoparticles (Wnt3a@ZIF-8 NPs) had higher efficacy on bone marrow mesenchymal stem cells (BMSCs) than ZIF-8 NPs or Wnt3a proteins, contributing to the osteogenesis through ZIF-8 crystals and intracellular Wnt3a proteins released from Wnt3a@ZIF-8 NPs. Furthermore, polymerase chain reaction (PCR) analysis showed that the osteogenic pathways were upregulated. Overall, the present one-pot process can open up new avenues to develop signaling protein-delivery systems for applications in protein therapy strategies.

Cytoplasmic localization of IRF5 induces Wnt5a/E-cadherin degradation and promotes gastric cancer cells metastasis.

IRF5, a nucleoplasm shuttling protein, is a pivotal transcription factor regulating immune system activity. It's well known that immunosuppression is involved in the development of gastric cancer. However, no data exist for the expression and function of IRF5 in gastric cancer. This study demonstrated that IRF5 was cytoplasm-enriched in gastric cancer cells. IRF5 promoted gastric cancer cell migration, which involved the inhibition of Wnt5a and E-cadherin proteins expression. IRF5 (LA) localized in nucleus had no significant effect on Wnt5a and E-cadherin expressions, while mutation of IRF5 (ΔNLS), which prevents IRF5 nuclear translocation, had more impact on these inhibitory effects. In addition, degradation rates of both Wnt5a and E-cadherin were enhanced by resiquimod, an IRF5 agonist. Further in vivo experiments indicated that IRF5 knockout of gastric cancer cells repressed their pulmonary metastasis in nude mice. Finally, the expression and clinical significance of IRF5 were analyzed using gastric cancer tissue microarrays, which suggested that the expression of IRF5 varied procedurally in different progressive stages of gastric cancer. Our data revealed that IRF5 cytoplasmic localization were associated with Wnt5a and E-cadherin degradation and gastric cancer cell metastasis. Inhibiting IRF5 expression and/or its cytoplasmic localization may provide a novel target for gastric cancer therapy.

Combination of untargeted and targeted proteomics for secretome analysis of L-WRN cells.

Organoid culture is a promising biomedical technology that requires specialized growth factors. Recently, a recombinant L-WRN cell line has been extensively used to generate conditioned medium (L-CM) for organoid culture. Nevertheless, methods for evaluating the stability of the L-WRN cells have been limited. In this study, a novel proteomics-based approach was developed to analyze the secretome of the cells. Serum-free L-CM was lyophilized, precipitated by trichloroacetic acid, and desalted prior to analysis by liquid chromatography-tandem mass spectrometry. Data-dependent acquisition (DDA) was conducted for the untargeted secretome profiling of the cells, and parallel reaction monitoring (PRM) was applied for the targeted quantification of the Wnt3A, R-spondin3, and noggin proteins (WRNs). This study also compared the performance of two types of PRM methods, namely MS1-independent PRM and MS1-dependent PRM, that can be executed on an Orbitrap instrument. The results showed that the growth of mouse intestinal organoids was closely related to the use of L-CM. The composition of L-CM could be markedly affected by the medium collection scheme. A total of 1725, 2302, and 2681 proteins were identified from the L-CM collected on day 5, day 9, and day 13, respectively. The MS1-independent PRM outperformed the MS1-dependent PRM and effectively quantified the WRNs with high repeatability and specificity. In conclusion, by integrating untargeted and targeted proteomics, this study develops a mass spectrometry-based method for the secretome analysis and quality control of the L-WRN cells. The methodology and findings of the present work will benefit future studies on organoids and secretomes.

Cocaine and Its Abstinence Condition Modulate Striatal and Hippocampal Wnt Signaling in a Male Rat Model of Drug Self-Administration.

Recent years have provided more and more evidence confirming the important role of Wnt/β-catenin signaling in the pathophysiology of mental illnesses, including cocaine use disorder. High relapse rates, which is a hallmark of drug addiction, prompt the study of changes in Wnt signaling elements (Wnt5a, Wnt7b, and Ctnnb1) in the motivational aspects of cocaine use and early drug-free period (3 days after the last exposure to cocaine). For this purpose, an animal model of intravenous cocaine self-administration and two types of drug-free period (extinction training and abstinence in the home cage) were used. The studies showed that chronic cocaine self-administration mainly disturbs the expression of Wnt5a and Ctnnb1 (the gene encoding β-catenin) in the examined brain structures (striatum and hippocampus), and the examined types of early abstinence are characterized by a different pattern of changes in the expression of these genes. At the same time, in cocaine self-administrated animals, there were no changes in the level of Wnt5a and β-catenin proteins at the tested time points. Moreover, exposure to cocaine induces a significant reduction in the striatal and hippocampal expression of miR-374 and miR-544, which can regulate Wnt5a levels post-transcriptionally. In summary, previous observations from experimenter-administered cocaine have not been fully validated in the cocaine self-administration model. Yoked cocaine administration appears to disrupt Wnt signaling more than cocaine self-administration. The condition of the cocaine-free period, the routes of drug administration, and the motivational aspect of drug administration play an important role in the type of drug-induced molecular changes observed. Furthermore, in-depth research involving additional brain regions is needed to determine the exact role of Wnt signaling in short-term and long-lasting plasticity as well as in the motivational aspects of cocaine use, and thus to assess its potential as a target for new drug therapy for cocaine use disorder.

Spatiotemporal distribution analyses of Wnt5a ligand and its receptors Ror2, Frizzled2, and Frizzled5 during tongue muscle development in prenatal mice

BackgroundThe mammalian tongue is a highly specialized muscular organ. The Wnt5a ligand regulates muscle development by mediating the activation of several noncanonical Wnt signaling pathways in a receptor context-dependent fashion. However, there is poor information on the expression and behavior of Wnt5a proteins during muscle development of the embryonic tongue. MethodsThe spatiotemporal distribution profiles of the Wnt5a ligand and its receptors, receptor tyrosine kinase-like orphan receptor 2 (Ror2), Frizzled2 (Fzd2), and Frizzled5 (Fzd5), in the developing tongue muscles of prenatal mice from embryonic day 12.5–18.5 were analyzed using immunofluorescence (IF) double staining of a target protein and desmin, a marker protein of myogenic cells. Immunolabeling images were subjected to digital detection analysis using the WinROOF 2018 version 4.19.0 image processing software when needed. ResultsIF signals of the Wnt5a ligand protein and its receptors Ror2 and Fzd2 were detected in developing myoblasts and myotubes of the embryonic tongue, but they were undetectable in mature myofibers equipped with sarcomere structures. Fzd2 expression was specific for desmin-positive developing muscle cells, whereas those of Ror2 and the Wnt5a ligand were widespread and nonselective for desmin-positive cells and that of Fzd5 was predominant in desmin-negative cells of the epithelium and subepithelial mesenchyme. ConclusionDeveloping muscle cells but not mature myofibers of the mouse embryonic tongue express the Wnt5a ligand and its receptors Ror2 and Fzd2, which may mediate Wnt5a signaling in the development processes of tongue muscle fibers.

Amelogenin as a regenerative endodontic molecule for immature teeth with apical periodontitis. An experimental study.

Vitality of the dentin-pulp complex depends on cell activity and signalling processes. Amelogenin protein regulates cell signalling pathways during tooth development by activating the Wnt/β-catenin intercellular signalling pathway. This study aimed to regenerate a vascularized pulp using recombinant amelogenin protein, in necrotic root canals by cell homing. Access opening was performed for a total of 120 root canals and were left open to become contaminated with oral microbes for 14 days then cleaned. Canals were divided into 2 groups; in the First group, the canals were filled with amelogenin, while in the 2nd group the canals were left empty. Samples were evaluated histologically and with immunodetection of Sox2, Oct4, Vascular endothelial growth factor (VEGF), Wnt1a, Wnt 3a, Wnt 10b, and Glial Fibrillary Acidic Proteins (GFAP). IC50 was used to determine the cytotoxicity of amelogenin. Regenerated dense cellular tissue was seen in the apical part of amelogenin-treated root canals, and regenerated delicate vascularized tissue was observed in the radicular and pulp chamber. Cells found in the regenerated soft tissue expressed Wnt family members that regulate stem cell pluripotency. Also, Sox2 and Oct4, Pluripotency markers, could be identified in the newly formed apical papilla and dental follicle. Furthermore, VEGF in the regenerated pulps indicated neovascularization. While the GFAP immune reactivity demonstrated that the neuro-sensory organ was being replicated in the regenerated dental pulps. Finally, IC50 test showed that recombinant amelogenin protein has a safe dose at high-level concentrations. Recombinant amelogenin protein induces pulp regeneration most likely from the Sox2 identified stem cells within the apical papilla and can enhance apex formation in non-vital immature teeth.

LncRNA FGD5-AS1 potentiates autophagy-associated doxorubicin resistance by regulating the miR-154-5p/WNT5A axis in osteosarcoma.

Osteosarcoma is prevalent in children and adolescent. The oncogenic function of long-chain noncoding RNA (lncRNA) FGD5 antisense RNA 1 (FGD5-AS1) has been reported. However, the function of FGD5-AS1 in doxorubicin-resistance in osteosarcoma remains to be illucidated. Quantitative real-time PCR (qRT-PCR) and western blot analysis (WB) were used to measure the expression of FGD5-AS1, miR-154-5p, WNT5A and autophagy proteins. MTT assay was used to assess cell viability and transwell assay was performed to evaluate migration. A nude mouse xenograft model was developed to verify the function of FGD5-AS1 in vivo. FGD5-AS1was upregulated in doxorubicin-resistant (DXR) osteosarcoma cells. Knockdown of FGD5-AS1 suppressed osteosarcoma cell proliferation, migration, and autophagy. FGD5-AS1 upregulated WNT5A expression via sponging miR-154-5p. Furthermore, FGD5-AS1 enhanced osteosarcoma cell chemotherapy resistance through upregulation of WNT5A by inhibiting miR-154-5p. Suppression of FGD5-AS1 significantly suppressed tumor growth in nude mice. FGD5-AS1 may promote chemoresistance through WNT5A-induced autophagy by sponging miR-154-5p in osteosarcoma cells.

Expression of hepatocyte nuclear factor 4 alpha, wingless-related integration site, and β-catenin in clinical gastric cancer

BACKGROUNDGastric cancer (GC) is the second most common cause of cancer-related deaths worldwide. Hepatocyte nuclear factor 4 alpha (HNF4α) that belongs to the nuclear hormone receptor superfamily, is overexpressed in GC tissues, and might be involved in the development of GC by regulating its downstream wingless-related integration site (WNT)/β-catenin signaling.AIMTo clarify the expression of HNF4α/WNT5a/β-catenin signaling proteins in clinical GC tissues. METHODSWe immunohistochemically stained pathological blocks of GC and matched para-cancerous tissues. The intensity of HNF4α, WNT5a and β-catenin staining in the tumor cells was determined according to cell rates and staining intensity. The correlations between GC and HNF4α, WNT5a, and β-catenin expression using chi-square and paired chi-square tests. Relationships between double-positive HNF4α and WNT5a expression and types of gastric tumor tissues were assessed using regression analysis. Correlations between HNF4α and WNT5a expression at the RNA level in GC tissues found in the TCGA database were analyzed using Pearson correlation coefficients. RESULTSWe found more abundant HNF4α and WNT5a proteins in GC, especially in mucinous adenocarcinoma and mixed GC than in adjacent tissues (P < 0.001). Low and high levels of cytoplasmic β-catenin respectively expressed in GC and adjacent tissues (P < 0.001) were not significantly associated with pathological parameters. CONCLUSIONThe expressions of HNF4α and WNT5a could serve as early diagnostic biomarkers for GC.

Neuroprotective Effect of Baicalein Against Oxaliplatin-Induced Peripheral Neuropathy: Impact on Oxidative Stress, Neuro-inflammation and WNT/β-Catenin Signaling.

Oxaliplatin, an effective anti-cancer agent used in the treatment of colorectal cancer, isassociated with severe dose-limiting side effects like peripheral neuropathy, which currently remains a major unmet clinical need. This study was designed to investigate the possible neuroprotective potential of a bioflavonoid, baicalein in an experimental model of oxaliplatin-induced peripheral neuropathy. Rats were administered with a dose of 4mg/kg oxaliplatin i.p. twice per week for four weeks, and were evaluated for behavioral and functional nerve parameters, followed by biochemical, immunohistochemical and western blot analysis. This study shows that baicalein reversed oxaliplatin-induced behavioral deficits and significantly prevented oxaliplatin-induced sensory nerve conduction deficits in rats. Molecular analysis revealed baicalein significantly strengthened the antioxidant defense system by enhancing the expression of MnSOD, HO-1, and GSH levels. Baicalein treatment neutralized the oxaliplatin-induced neuroinflammation, which was evident from the significant loss of inflammatory mediators like TNF-α, IL-6 and a shunted NF-κB nuclear translocation. Additionally, baicalein treatment resulted in a significant downregulation of active β-catenin, Wnt5b and Wnt3a proteins. In line with the in vivo evidences, baicaleintreatment in Neuro2a cells attenuated oxaliplatin-induced ROS, mitochondrial superoxide levels and improved neuritogenesis.Additionally, baicalein did not alter the cell viability of oxaliplatin in HCT-116 cell line. Collectively, these results suggest that baicalein may be useful for management of peripheral neuropathy associated with oxaliplatin.

Inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced blood\u2013brain barrier injury through the Wnt/\u03b2-catenin signalling pathway

BackgroundDisruption of the blood–brain barrier (BBB) after a stroke can lead to brain injury and neurological impairment. Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2 (LMP2) in the pathophysiology of ischemia stroke. However, the relationship between the immunoproteasome LMP2 and the BBB remains unclear.MethodsAdult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion (MCAO/R). Three days before MCAO, the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region. The rat brain microvascular endothelial cell (RBMVEC) line was exposed to oxygen–glucose deprivation/reperfusion (OGD/R) to mimic ischemic conditions in vitro. The RNA interference-mediated knockdown of LMP2 or β-catenin was analysed in vivo and in vitro. Analysis of the quantity of extravasated Evans blue (EB) and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB. Immunofluorescence and Western blotting were employed to detect the expression of target proteins. Cell migration was evaluated using a scratch migration assay. The results of immunofluorescence, Western blotting and cell migration were quantified using the software ImageJ (Version 1.53m). Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference (LSD) test.ResultsCerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins (occludin, claudin-1 and ZO-1) in the MCAO/R group compared with the sham group (P < 0.001). However, inhibition of the immunoproteasome LMP2 restored the expression of these proteins, resulting in higher levels of occludin, claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group (P < 0.001). In addition, inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability [e.g., the quantity of extravasated EB: LMP2-shRNA group (58.54 ± 7.37) µg/g vs. control-shRNA group (103.74 ± 4.32) µg/g, P < 0.001], and promoted the upregulation of Wnt-3a and β-catenin proteins in rats following MCAO/R. In vitro experiments, OGD/R induced marked upregulation of LMP2, proapoptotic protein Bax and cleaved caspase-3, and downregulation of occludin, claudin-1, ZO-1 and Bcl-2, as well as inhibition of the Wnt/β-catenin pathway Wnt-3a and β-catenin proteins in RBMVECs, compared with the control group under normal culture conditions (P < 0.001). However, silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R. Silencing of β-catenin by transfection of RBMVECs with β-catenin-siRNA aggravated the downregulation of tight junction proteins, and reduced the proliferation and migration of RBMVECs following OGD/R, compared with the control-siRNA group (P < 0.001). LMP2-siRNA and β-catenin-siRNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.ConclusionThis study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia-induced BBB injury, and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.

The Expression and Significance of Matrix Metalloproteinase, Wnt5a and β-Catenin in Placental Tissue of Preeclampsia

In order to investigate the expression and clinical significance of MMPs (Matrix Metalloproteinase) (MPP-2 and MPP-9), Wnt5a as well as β-catenin in placental tissues of preeclampsia (PE) patients, 36 PE patients (PE group) and 25 pregnant women (control group) with normal pregnancy were selected for cesarean delivery. The expression and localization of MPP-2, MPP-9, Wnt5a as well as β-catenin proteins in placental tissue were detected by Western blot method and immunohistochemistry method. The correlation between the poor prognosis of mother and child and the expression level of MMPs, Wnt5a as well as β-catenin is analyzed. The results showed that Western blot results showed that MPP-2, MPP-9, Wnt5a as well as β-catenin proteins were expressed in placental tissue, and the expression level of them of placental tissue in PE group was significantly lower than that in control group (P &lt; 0.01). Immunohistochemistry showed that MMPs, Wnt5a, as well as β-catenin proteins were expressed in the cytotrophoblast cell (CTB) and syncytiotrophoblast cell (STB) of placental tissue, among which Wnt5a proteins as well as β-catenin proteins were mainly expressed in the cytoplasm of STB and CTB respectively. MMPs proteins were mainly expressed in the cytoplasm of these two types of cells. Low expression of MPPs, Wnt5a, β-catenin proteins may be related to blood pressure as well as 24 h urine protein of pregnant women at admission. In conclusion, Low expression of MPPs, Wnt5a, β-catenin proteins may be involved in the PE pathogenesis.