Properties of biomolecular condensates defined by Activator of G-protein Signaling 3.

Activator of G-protein signaling 3 (AGS3; also known as GPSM1), a receptor-independent activator of G-protein signaling, oscillates among defined subcellular compartments and biomolecular condensates (BMCs) in a regulated manner that is likely related to the functional diversity of the protein. We determined the influence of cell stress on the cellular distribution of AGS3 and core material properties of AGS3 BMCs. Cellular stress (oxidative, pHi and thermal) induced the formation of AGS3 BMCs in HeLa and COS-7 cells, as determined by fluorescent microscopy. Oxidative stress-induced AGS3 BMCs were distinct from G3BP1 stress granules and from RNA processing BMCs defined by the P-body protein Dcp1a. Immunoblots indicated that cellular stress shifted AGS3, but not the stress granule protein G3BP1 to a membrane pellet fraction following cell lysis. The stress-induced generation of AGS3 BMCs was reduced by co-expression of the signaling protein Gαi3, but not the AGS3-binding partner DVL2. Fluorescent recovery following photobleaching of individual AGS3 BMCs indicated that there are distinct diffusion kinetics and restricted fluidity for AGS3 BMCs. These data suggest that AGS3 BMCs represent a distinct class of stress granules that serve as a previously unrecognized signal processing node.

Aquaporin 9 is involved in CRC metastasis through DVL2-dependent Wnt/ß-catenin signaling activation.

Background: Aquaporin 9 (AQP9) is permeable to water or other small molecules, and plays an important role in various cancers. We previously found that AQP9 was related to the efficacy of chemotherapy in patients with colorectal cancer (CRC). This study aimed to identify the role and regulatory mechanism of AQP9 in CRC metastasis. Methods: The clinical significance of AQP9 was analysed by using bioinformatics and tissue microarray. Transcriptome sequencing, Dual-Luciferase Reporter Assay, Biacore, and co-immunoprecipitation were employed to demonstrate the regulatory mechanism of AQP9 in CRC. The relationship between AQP9 and CRC metastasis was verified in vitro and in vivo by using real-time cell analysis assay, high content screening, and liver metastasis models of nude mice. Results: We found that AQP9 was highly expressed in metastatic CRC. AQP9 overexpression reduced cell roundness and enhanced cell motility in CRC. We further showed that AQP9 interacted with Dishevelled 2 (DVL2) via the C-terminal SVIM motif, resulting in DVL2 stabilization and the Wnt/ß-catenin pathway activation. Additionally, we identified the E3 ligase neural precursor cell expressed developmentally downregulated 4-like (NEDD4L) as a modulator regulating the ubiquitination and degradation of AQP9. Conclusions: Collectively, our study revealed the important role of AQP9 in regulating DVL2 stabilization and Wnt/ß-catenin signaling to promote CRC metastasis. Targeting the NEDD4L-AQP9-DVL2 axis might have therapeutic usefulness in metastatic CRC treatment.

Inhibiting von Hippel‒Lindau protein-mediated Dishevelled ubiquitination protects against experimental parkinsonism.

Dopaminergic neuron degeneration is a hallmark of Parkinson's disease (PD). We previously reported that the inactivation of von Hippel‒Lindau (VHL) alleviated dopaminergic neuron degeneration in a C. elegans model. In this study, we investigated the specific effects of VHL loss and the underlying mechanisms in mammalian PD models. For in vivo genetic inhibition of VHL, AAV-Vhl-shRNA was injected into mouse lateral ventricles. Thirty days later, the mice received MPTP for 5 days to induce PD. Behavioral experiments were conducted on D1, D3, D7, D14 and D21 after the last injection, and the mice were sacrificed on D22. We showed that knockdown of VHL in mice significantly alleviated PD-like syndromes detected in behavioral and biochemical assays. Inhibiting VHL exerted similar protective effects in MPP+-treated differentiated SH-SY5Y cells and the MPP+-induced C. elegans PD model. We further demonstrated that VHL loss-induced protection against experimental parkinsonism was independent of hypoxia-inducible factor and identified the Dishevelled-2 (DVL-2)/ß-catenin axis as the target of VHL, which was evolutionarily conserved in both C. elegans and mammals. Inhibiting the function of VHL promoted the stability of ß-catenin by reducing the ubiquitination and degradation of DVL-2. Thus, in vivo overexpression of DVL-2, mimicking VHL inactivation, protected against PD. We designed a competing peptide, Tat-DDF-2, to inhibit the interaction between VHL and DVL-2, which exhibited pharmacological potential for protection against PD in vitro and in vivo. We propose the therapeutic potential of targeting the interaction between VHL and DVL-2, which may represent a strategy to alleviate neurodegeneration associated with PD.

Filamin C regulates skeletal muscle atrophy by stabilizing dishevelled-2 to inhibit autophagy and mitophagy.

FilaminC (Flnc) is a member of the actin binding protein family, which is preferentially expressed in the cardiac and skeletal muscle tissues. Although it is known to interact with proteins associated with myofibrillar myopathy, its unique role in skeletal muscle remains largely unknown. In this study, we identify the biological functions of Flnc in vitro and in vivo using chicken primary myoblast cells and animal models, respectively. From the results, we observe that the growth rate and mass of the skeletal muscle of fast-growing chickens (broilers) were significantly higher than those in slow-growing chickens (layers). Furthermore, we find that the expression of Flnc in the skeletal muscle of broilers was higher than that in the layers. Our results indicated that Flnc was highly expressed in the skeletal muscle, especially in the skeletal muscle of broilers than in layers. This suggests that Flnc plays a positive regulatory role in myoblast development. Flnc knockdown resulted in muscle atrophy, whereas the overexpression of Flnc promotes muscle hypertrophy in vivo in an animal model. We also found that Flnc interacted with dishevelled-2 (Dvl2), activated the wnt/ß-catenin signaling pathway, and controlled skeletal muscle development. Flnc also antagonized the LC3-mediated autophagy system by decreasing Dvl2 ubiquitination. Moreover, Flnc knockdown activated and significantly increased mitophagy. In summary, these results indicate that the absence of Flnc induces autophagy or mitophagy and regulates muscle atrophy.

Insulin receptor substrate-1 and dishevelled 2 are negatively regulated by microRNA-144 and inhibit nasopharyngeal carcinoma cell malignancy.

Insulin receptor substrate-1 (IRS-1) is reported to play a critical role in the development, progression, invasion and metastasis of several types of tumors and is abnormally expressed in nasopharyngeal carcinoma (NPC). Although IRS-1 is predicted to be targeted by microRNA (miR)-144, the biological roles and potential mechanisms of miR-144 in NPC remain unclear. In the present study, the expression levels of miR-144 and IRS-1 in several NPC cell lines were first examined, and found that they were negatively correlated. Following the introduction of the miR-144 mimic, IRS-1 was downregulated at the protein level without affecting the mRNA level. The Cell Counting Kit-8 assay showed that the miR-144 mimic and siRNA targeting IRS-1 mRNA significantly decreased cell proliferation by arresting the cell cycle at the G1/G0 phase. The malignant behaviours of NPC cell lines, including migration, invasion and tumour formation in soft agar, were then analyzed after regulating miR-144 levels; as expected, the results showed that both the miR-144 mimic and siIRS-1 decreased these malignant behaviours. Furthermore, the downregulation of IRS-1 by miR-144 decreased the expression level of dishevelled 2 (Dvl2) protein without affecting its mRNA level, and Dvl2 overexpression abolished the inhibitory effect of the miR-144 mimic in NPC, indicating that miR-144 potentially regulates NPC by indirectly regulating Dvl2. Taken together, the present study results suggest that miR-144 acts as a tumour suppressor in NPC cell lines by regulating IRS-1 and Dvl2, which indicates that it is a potential therapeutic target for NPC treatment.

Inhibition of Dishevelled-2 suppresses the biological behavior of pancreatic cancer by downregulating Wnt/ß-catenin signaling.

Dishevelled-2 (DVL2) has been proven to be involved in the tumorigenesis of several human cancers, such as colorectal cancer, lung cancer, prostate cancer, etc. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. The present study investigated the effects of aberrantly expressed DVL2 on PDAC. A total of 97 pancreatic cancer (PC) samples and 85 adjacent normal samples were obtained from patients who were histopathologically diagnosed with primary PDAC. The present study demonstrated that DVL2 expression was upregulated in PDAC tissues and was positively associated with advanced clinical stage and lymph node metastasis in patients with PDAC. In addition, patients with high expression of DVL2 had a shorter overall survival rate compared with those with low expression. To elucidate the role of DVL2 in PDAC, lentivirus-mediated short hairpin RNA was used to silence DVL2 and its physiological function was analyzed in CFPAC-1 and PANC-1 cells. The results indicated that DVL2 downregulation significantly impaired its oncogenic functions including cell proliferation, migration, invasion and epithelial-mesenchymal transition. Furthermore, DVL2 knockdown inhibits the proliferation and invasion of PC cells in vivo. In addition, co-immunoprecipitation assays revealed that DVL2 interacted with ß-catenin; knockdown of DVL2 reduced the expression level of ß-catenin and inhibited ß-catenin translocation into the nucleus. In conclusion the findings of the present study suggested that DVL2 may be a potential therapeutic target in the treatment of PDAC.

Plectin regulates Wnt signaling mediated-skeletal muscle development by interacting with Dishevelled-2 and antagonizing autophagy.

Skeletal muscle is the most abundant tissue in the human and animal body, loss of its function can lead to muscle aging and various myogenic diseases. The skeletal muscle development is a complex and tightly regulated process, which is driven by a variety of many factors, signaling pathways and regulatory mechanisms. Plectin (Plec), a cytolinker protein, is ubiquitously expressed in various tissues such as skin, muscle, plasma membrane, and most types of cells. Although known isoforms of Plec is well-characterized in muscle dystrophy, very little is known on the function of Plec in the skeletal muscle development. Here, we found that Plec plays a vital role in promoting C2C12 myoblasts differentiation and proliferation, but inhibits their apoptosis. Also, Plec regulates the expression of atrophy-related genes (atrogin-1 and muRF-1) to rescue muscle atrophy. Furthermore, we have demonstrated that Plec binds to Dishevelled-2 (Dvl-2) and forms a protein complex, which is then activate the canonical Wnt signaling. We also observed that Plec resists ubiquitination by stabilizing Dvl-2 and reduces the level of LC3-labeled Dvl-2 and antagonizes the autophagy system. In conclusion, our findings suggest that Plec regulates canonical Wnt signaling mediated skeletal development by stabilizing Dvl-2 and downregulating the cellular autophagic degradation system.

Intersection of two key signal integrators in the cell: activator of G-protein signaling 3 and dishevelled-2.

Activator of G-protein signaling 3 (AGS3, encoded by GPSM1) was discovered as a one of several receptor-independent activators of G-protein signaling, which are postulated to provide a platform for divergence between canonical and noncanonical G-protein signaling pathways. Similarly, Dishevelled (DVL) proteins serve as a point of divergence for ß-catenin-dependent and -independent signaling pathways involving the family of Frizzled (FZD) ligands and cell-surface WNT receptors. We recently discovered the apparent regulated localization of dishevelled-2 (DVL2) and AGS3 to distinct cellular puncta, suggesting that the two proteins interact as part of various cell signaling systems. To address this hypothesis, we asked the following questions: (1) do AGS3 signaling pathways influence the activation of ß-catenin (CTNNB1)-regulated transcription through the WNT-Frizzled-Dishevelled axis, and (2) is the AGS3 and DVL2 interaction regulated? The interaction of AGS3 and DVL2 was regulated by protein phosphorylation, subcellular distribution, and a cell-surface G-protein-coupled receptor. These data, and the commonality of functional system impacts observed for AGS3 and DVL2, suggest that the AGS3-DVL2 complex presents an unexpected path for functional integration within the cell.This article has an associated First Person interview with the first author of the paper.

Myoferlin Regulates Wnt/ß-Catenin Signaling-Mediated Skeletal Muscle Development by Stabilizing Dishevelled-2 Against Autophagy.

Myoferlin (MyoF), which is a calcium/phospholipid-binding protein expressed in cardiac and muscle tissues, belongs to the ferlin family. While MyoF promotes myoblast differentiation, the underlying mechanisms remain poorly understood. Here, we found that MyoF not only promotes C2C12 myoblast differentiation, but also inhibits muscle atrophy and autophagy. In the present study, we found that myoblasts fail to develop into mature myotubes due to defective differentiation in the absence of MyoF. Meanwhile, MyoF regulates the expression of atrophy-related genes (Atrogin-1 and MuRF1) to rescue muscle atrophy. Furthermore, MyoF interacts with Dishevelled-2 (Dvl-2) to activate canonical Wnt signaling. MyoF facilitates Dvl-2 ubiquitination resistance by reducing LC3-labeled Dvl-2 levels and antagonizing the autophagy system. In conclusion, we found that MyoF plays an important role in myoblast differentiation during skeletal muscle atrophy. At the molecular level, MyoF protects Dvl-2 against autophagy-mediated degradation, thus promoting activation of the Wnt/ß-catenin signaling pathway. Together, our findings suggest that MyoF, through stabilizing Dvl-2 and preventing autophagy, regulates Wnt/ß-catenin signaling-mediated skeletal muscle development.

MiR-495 targeting dvl-2 represses the inflammatory response of ankylosing spondylitis.

Ankylosing spondylitis (AS) is a type of rheumatic inflammatory disease. miRNAs participate in the process of regulating inflammatory response and bone differentiation. Herein, we aimed to test the effect of miR-495 on AS. The serum and tissues were obtained from traumatic fracture (health) and AS patients. The human fibroblast-like synovial (HFLS) cells were extracted from AS tissues. The contents of inflammatory factors and dishevelled 2 (DVL-2) were examined using enzyme-linked immunosorbent assay (ELISA). The ossification factors were detected by immunohistochemistry assay. Osteoclast was assessed by tartaric acid acid phosphatase (TRAP) assay. The cell viability and luciferase activity were measured using cell counting kit-8 (CCK-8) and dual-luciferase reporter system. The levels of factors were evaluated using quantitative real-time PCR (qRT-PCR) and western blotting. DVL-2 was a target gene for miR-495, according to the MicroRNA.org website and luciferase activity assay. The expressions of miR-495 and DVL-2 were negative corrected in AS. miR-495 and si-DVL-2 did not affect the cell viability. miR-495 and si-DVL-2 obviously inhibited inflammatory response by down-regulating tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and IL-6 levels, and facilitated bone differentiation by up-regulating osteoprotegerin (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) levels in HFLS cells. Besides, miR-495 and si-DVL-2 increased the expression of wnt3a, runt-related transcription factor 2 (RUNX-2) and ß-catenin and reduced the phosphorylation of ß-catenin. Collectively, miR-495 depressed inflammatory response and promoted bone differentiation of HFLS cells, and this was accompanied by mediating wnt/ß-catenin/Runx-2 pathway by targeting DVL-2.

[miR-29c-3p targeted dishevelled 2 on osteogenesis differentiation of rat bone marrow mesenchymal stem cells in high-fat environment].

Objective: To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation. Methods: BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 µl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured. Results: On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups. Conclusions: miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.

[Expression of dishevelled-2 in cartilage of rheumatoid arthritis and its effect on cartilage destruction].

Objective: To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction. Methods: Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA), osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting. DVL2 overexpressed lentivirus was transfected into the knee of CIA rats. Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus. Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR) . Results: Compared with knee articular cartilage in OA patients and normal rats, DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032, respectively). DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885). DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2), inducible nitric oxide synthase (NOS), matrix metalloproteinase (MMP) 2, MMP-3, and MMP-9, which could be more pronounced when tumor necrosis factor alpha was added. Conclusions: DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/ß-catenin pathway, which involves in the destruction of articular cartilage in RA.

Down-regulation of dishevelled-2 inhibits cell proliferation and invasion in hepatoblastoma.

BACKGROUND: Hepatoblastoma (HB) is the most common liver cancer found in early childhood. These patients suffer poor outcomes and need novel therapies. An abnormal activation of Wnt signaling is the hallmark of HB tumorigenesis, and its pathway is a potential candidate for a pharmacological intervention. PROCEDURE: Tissue samples of patients with HB were collected for RNA-seq, quantitative real-time PCR, and immunohistochemistry to identify if disheveled-2 (Dvl-2) was a target gene. The correlation between Dvl-2 expression and different clinicopathological features was analyzed using statistical methods. Proliferation and invasion assays were applied after knocking down Dvl-2 by shRNA in HepG2 and Huh6 HB cell lines. The antitumor effect of niclosamide on HB was ascertained in vitro and in vivo. RESULTS: Dvl-2 was overexpressed in 90% of patients with HB, and Dvl-2 expression was positively correlated with the age of patients with HB. Knockdown of Dvl-2 could inhibit proliferation and invasion of HB cell lines. Also, niclosamide, a Food and Drug Administration approved antihelminth compound, could effectively inhibit HB cell growth in vitro and in vivo via downregulation of Dvl-2 and ß-catenin expression. CONCLUSIONS: Our results implicate that Dvl-2 is a potential therapeutic target in HB, and niclosamide could have clinical potential to treat patients with HB.

miR-29c-3p targeted dishevelled 2 on osteogenesis differentiation of rat bone marrow mesenchymal stem cells in high-fat environment / 中华口腔医学杂志

Objective@#To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation.@*Methods@#BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 μl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured.@*Results@#On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups.@*Conclusions@#miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.

Expression of dishevelled-2 in cartilage of rheumatoid arthritis and its effect on cartilage destruction / 中华内科杂志

Objective To study the expression of dishevelled-2 (DVL2) in rheumatoid arthritis cartilage and its effect on cartilage destruction.Methods Cartilage DVL2 expression in rat models of rheumatoid arthritis (RA),osteoarthritis(OA) and collagen-induced arthritis(CIA) were tested by Western blotting.DVL2 overexpressed lentivirus was transfected into the knee of CIA rats.Primary chondrocytes were extracted from RA patients by knee arthroplasty and transfected with DVL2 overexpressed lentivirus.Gene expression of related inflammation related cytokines was detected by real-time polymerase chain reaction (PCR).Results Compared with knee articular cartilage in OA patients and normal rats,DVL2 protein was highly expressed in knee cartilage of RA patients and CIA rats (P values 0.041 and 0.032,respectively).DVL2 did not significantly affect the destruction of knee cartilage in CIA rats (P=0.885).DVL2 overexpression in chondrocytes enhanced gene expression of cyclo-oxygenase-2 (COX-2),inducible nitric oxide synthase (NOS),matrix metalloproteinase (MMP) 2,MMP-3,and MMP-9,which could be more pronounced when tumor necrosis factor alpha was added.Conclusions DVL2 is highly expressed in RA articular cartilage and promotes the expression of inflammatory cytokines and MMP gene in chondrocytes by activating Wnt/β-catenin pathway,which involves in the destruction of articular cartilage in RA.

Identification of Lipid Binding Modulators Using the Protein-Lipid Overlay Assay.

The protein-lipid overlay assay is an inexpensive, easy-to-implement, and high-throughput methodology that employs nitrocellulose membranes to immobilize lipids in order to rapid screen and identify protein-lipid interactions. In this chapter, we show how this methodology can identify potential modulators of protein-lipid interactions by screening water-soluble lipid competitors or even the introduction of pH changes during the binding assay to identify pH-dependent lipid binding events.

Overexpression of dishevelled 2 is involved in tumor metastasis and is associated with poor prognosis in hepatocellular carcinoma.

PURPOSE: Although hepatocellular carcinoma (HCC) is one of the most common malignant tumors, its molecular mechanism is still unknown. Dishevelled 2 (Dvl2) is one of the downstream targets of non-canonical Wnt signaling, which has been demonstrated to be of great importance in the progression of cancers. Nevertheless, the expression mechanisms and physiological significance of Dvl2 in HCC remain unclear. METHODS: Western blotting and immunohistochemistry were used to measure Dvl2 protein expression in HCC and adjacent normal tissues of 101 patients. Wound healing and transwell assays were used to determine cell migration and invasion. RESULTS: Dvl2 expression was upregulated in HCC tissues compared to the adjacent normal tissues. Moreover, its expression level was significantly correlated with histological grade (P = 0.042), metastasis (P = 0.005) and vein invasion (P = 0.009) in patients with HCC. Wound healing and transwell assays showed that knockdown of Dvl2 reduced cell migration and invasion in HepG2 cells. Finally, we confirmed that Dvl2 could regulate the migration and invasion of HCC cells by interacting with P62 in non-canonical Wnt signaling. CONCLUSIONS: Our data showed that Dvl2 was overexpressed in HCC tissues and was also correlated with poor prognosis, suggesting that Dvl2 is a novel therapeutic target for HCC.

Potential role and prognostic importance of dishevelled-2 in epithelial ovarian cancer.

OBJECTIVE: To investigate the role and prognostic importance of Dvl2 in human epithelial ovarian cancer (EOC). METHODS: A multimethod study was undertaken including patients with pathologically confirmed non-metastatic EOC who underwent surgery for maximum tumor resection at a center in China. Dvl2 expression was assessed by western blot using fresh EOC tissues and normal ovarian tissues obtained between June 2014 and January 2015. Additionally, retrospective data were obtained for patients treated between April 2004 and September 2009. Their tumor specimens were used in immunohistochemistry analysis. Kaplan-Meier survival plots were constructed to estimate the overall survival by Dvl2 expression, and a Cox proportional hazards model was used to analyze prognostic factors. Alterations in Dvl2 expression during the cell cycle were assessed by a starvation and refeeding assay. RESULTS: Dvl2 expression was higher in EOC samples than in normal tissues on western blot. Overall, 124 patients were included in immunohistochemistry analysis, and Dvl2 expression level was significantly associated with the tumor grade and Ki-67 expression. Overexpression of Dvl2 was correlated with poor prognosis. The pattern of Dvl2 expression throughout the cell cycle matched that of the cell proliferation marker cyclin D1. CONCLUSION: Dvl2 could play a part in EOC progression and might be an independent prognostic factor. Additionally, it might be a prospective therapeutic target in the treatment of EOC.

Dishevelled2 promotes apoptosis and inhibits inflammatory cytokine secretion in rheumatoid arthritis fibroblast-like synoviocytes through crosstalk with the NF-κB pathway.

Dishevelled (Dvl) not only links the canonical Wnt and non-canonical Wnt pathways but can also crosstalk with other pathways. As there is no systematic study to date on Dvl in rheumatoid arthritis (RA), we explored the impact of Dvl2 on proliferation and inflammatory cytokine secretion in RA fibroblast-like synoviocytes (FLSs). Expression of Dvl2 in RA synovial tissue and RA-FLSs was measured. Dvl2 was overexpressed in collagen-induced arthritis rats and human RA-FLSs,. the apoptosis and secretion of inflammatory cytokines were observed. Genetic changes and corresponding mechanisms caused by overexpressing Dvl2 in RA-FLSs were assessed. Dvl2 was found to be overexpressed in RA synovial tissue and RA-FLSs. Overexpression of Dvl2 increased apoptosis and inhibited inflammatory cytokine secretion by RA-FLSs in vivo and in vitro, and Dvl2 inhibited expression of anti-apoptotic and inflammatory genes. One possible mechanism is that Dvl2 decreases the nuclear translocation of P65 and inhibits its ability to bind to the promoters of NF-κB target genes. Our findings reveal an underappreciated role of Dvl2 in regulating inflammation and RA-FLS apoptosis and provide insight into crosstalk between the Wnt and nuclear factor-κB (NF-κB) pathways.

Effect of Dishevelled 2 on the secretion of vascular endothelial growth factor in rheumatoid arthritis fibroblast-like synoviocytes / 中华风湿病学杂志

Objective To optimize the culture method for rheumatoid arthritis fibroblast-like synoviocytes in vitro,and observe the effect of Dishevelled (Dvl) 2 on vascular endothelial growth factor (VEGF) in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS).Methods Synovium from RA patients who underwent knee arthroplasties were cut into small piece,and RA-FLS were isolated and cultured in vitro using tissue block method.Dvl 2 lentivirus overexpressing plasmid was constructed and transfected into RAFLS.Q-polymerase chain reaction (PCR) and enzyme linked immunosorbent assay (ELISA) were used to detect the mRNA and protein expression levels of VEGF.Then we used 10 ng/ml tumor necrosis factor (TNF)-α recombinant protein to stimulate the transfected RA-FLS.24 h after stimulation,mRNA and protein expression of VEGF were detected again.Student's t test was used for two group analyses.Results RA-FLS was successfully isolated and cultured in vitro.The multiplicity of infection was 30 and was in conjunction with appropriate concentration of polybrene to promote transfection.Transfection efficiency could meet the test requirements.The mRNA of Dvl 2 increased for 79-fold than the control group.Compared with the control group,Dvl 2 could mildly inhibit RA-FLS secretion of VEGF.After TNF-α stimulation,Dvl 2 could significantly inhibit the VEGF's mRNA (2.15±0.10,2.92±0.47 fold,t=-3.924,P=0.003) and protein [(285±100) pg/ml,(155±61) pg/ml,t=-2.714,P=0.022] expression compared with the control group.Conclusion Dvl 2 can inhibit the effect of TNF-α induced secretion of VEGF in RA-FLS.The specific mechanism needs further study.