Wnt Expression Research Articles

Angiotensin II receptor 1 controls profibrotic Wnt/β-catenin signalling in experimental autoimmune myocarditis.

AimsAngiotensin (Ang) II signalling has been suggested to promote cardiac fibrosis in inflammatory heart diseases; however, the underlying mechanisms remain obscure. Using Agtr1a-/- mice with genetic deletion of angiotensin receptor type 1 (ATR1) and the experimental autoimmune myocarditis (EAM) model, we aimed to elucidate the role of Ang II-ATR1 pathway in development of heart-specific autoimmunity and post-inflammatory fibrosis.Methods and resultsEAM was induced in wild-type (WT) and Agtr1a-/- mice by subcutaneous injections with alpha myosin heavy chain peptide emulsified in complete Freund’s adjuvant. Agtr1a-/- mice developed myocarditis to a similar extent as WT controls at day 21 but showed reduced fibrosis and better systolic function at day 40. Crisscross bone marrow chimaera experiments proved that ATR1 signalling in the bone marrow compartment was critical for cardiac fibrosis. Heart infiltrating, bone-marrow-derived cells produced Ang II, but lack of ATR1 in these cells reduced transforming growth factor beta (TGF-β)-mediated fibrotic responses. At the molecular level, Agtr1a-/- heart-inflammatory cells showed impaired TGF-β-mediated phosphorylation of Smad2 and TAK1. In WT cells, TGF-β induced formation of RhoA-GTP and RhoA-A-kinase anchoring protein-Lbc (AKAP-Lbc) complex. In Agtr1a-/- cells, stabilization of RhoA-GTP and interaction of RhoA with AKAP-Lbc were largely impaired. Furthermore, in contrast to WT cells, Agtr1a-/- cells stimulated with TGF-β failed to activate canonical Wnt pathway indicated by suppressed activity of glycogen synthase kinase-3 (GSK-3)β and nuclear β-catenin translocation and showed reduced expression of Wnts. In line with these in vitro findings, β-catenin was detected in inflammatory regions of hearts of WT, but not Agtr1a-/- mice and expression of canonical Wnt1 and Wnt10b were lower in Agtr1a-/- hearts.ConclusionAng II-ATR1 signalling is critical for development of post-inflammatory fibrotic remodelling and dilated cardiomyopathy. Our data underpin the importance of Ang II-ATR1 in effective TGF-β downstream signalling response including activation of profibrotic Wnt/β-catenin pathway.

Wnt16 Elicits a Protective Effect Against Fractures and Supports Bone Repair in Zebrafish

ABSTRACTBone homeostasis is a dynamic, multicellular process that is required throughout life to maintain bone integrity, prevent fracture, and respond to skeletal damage. WNT16 has been linked to bone fragility and osteoporosis in human genome wide‐association studies, as well as the functional hematopoiesis of leukocytes in vivo. However, the mechanisms by which WNT16 promotes bone health and repair are not fully understood. In this study, CRISPR‐Cas9 was used to generate mutant zebrafish lacking Wnt16 (wnt16 −/−) to study its effect on bone dynamically. The wnt16 mutants displayed variable tissue mineral density (TMD) and were susceptible to spontaneous fractures and the accumulation of bone calluses at an early age. Fractures were induced in the lepidotrichia of the caudal fins of wnt16 −/− and WT zebrafish; this model was used to probe the mechanisms by which Wnt16 regulates skeletal and immune cell dynamics in vivo. In WT fins, wnt16 expression increased significantly during the early stages for bone repair. Mineralization of bone during fracture repair was significantly delayed in wnt16 mutants compared with WT zebrafish. Surprisingly, there was no evidence that the recruitment of innate immune cells to fractures or soft callus formation was altered in wnt16 mutants. However, osteoblast recruitment was significantly delayed in wnt16 mutants postfracture, coinciding with precocious activation of the canonical Wnt signaling pathway. In situ hybridization suggests that canonical Wnt‐responsive cells within fractures are osteoblast progenitors, and that osteoblast differentiation during bone repair is coordinated by the dynamic expression of runx2a and wnt16. This study highlights zebrafish as an emerging model for functionally validating osteoporosis–associated genes and investigating fracture repair dynamically in vivo. Using this model, it was found that Wnt16 protects against fracture and supports bone repair, likely by modulating canonical Wnt activity via runx2a to facilitate osteoblast differentiation and bone matrix deposition. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC. on behalf of American Society for Bone and Mineral Research.

The microRNA miR-19a-3p suppresses cell growth, migration, and invasion in multiple myeloma via the Wnt/β-catenin pathway.

BackgroundMicroRNAs have been suggested as potential regulators in the development of multiple myeloma (MM) through affecting the expression of their target genes. This study aimed to investigate the effects of miR-19a-3p in MM, and its underlying mechanisms in regulating cell proliferation and invasion.MethodsBone marrow samples from 25 MM patients and 12 healthy donors were collected and miR-19a-3p and Wnt1 mRNA expression was assessed. The effects of miR-19a-3p on cell proliferation, migration, and invasion in U226 and RPMI-8226 MM cells were evaluated by miR-19a-3p overexpression. Luciferase assays were performed to explore the potential target genes. Knock down or overexpression of Wnt1 was used to explore the effects of miR-19a-3p on cell growth, migration, and invasion.ResultsThe expression of miR-19a-3p was downregulated in MM and cell lines, while Wnt1 mRNA levels were increased. Overexpression of miR-19a-3p inhibited cell proliferation, migration, and invasion in U226 and RPMI-8226 cells. Additionally, western blot assays revealed that miR-19a-3p could suppress Wnt1, β-catenin, cyclin D1, and c-Myc expression. Knockdown of Wnt1 also inhibited cell growth, migration, and invasion. Moreover, luciferase reporter assay revealed direct binding between Wnt1 and miR-19a-3p. Wnt1 overexpression partially reversed the suppressive effects of miR-19a-3p on cell proliferation, migration, and invasion in U266 cells.ConclusionsThe expression of miR-19a-3p was downregulated in MM patients and MM cell lines. Overexpression of miR-19a-3p inhibited proliferation, migration, and invasion by targeting Wnt1 via the Wnt/β-catenin signaling pathway.

Baicalein inhibits inflammatory response and promotes osteogenic activity in periodontal ligament cells challenged with lipopolysaccharides

BackgroundPeriodontitis is a chronic infection initiated by oral bacterial and their virulence factors, yet the severity of periodontitis is largely determined by the dysregulated host immuno-inflammatory response. Baicalein is a flavonoid extracted from Scutellaria baicalensis with promising anti-inflammatory properties. This study aims to clarify the anti-inflammatory and osteogenic effects of baicalein in periodontal ligament cells (PDLCs) treated with lipopolysaccharides (LPS).MethodsHuman PDLCs were incubated with baicalein (0–100 μM) for 2 h prior to LPS challenge for 24 h. MTT analysis was adopted to assess the cytoxicity of baicalein. The mRNA and protein expression of inflammatory and osteogenic markers were measured by real-time polymerase chain reaction (PCR), western blot and enzyme-linked immunosorbent assay (ELISA) as appropriate. Alkaline phosphatase (ALP) and Alizarin red S (ARS) staining were performed to evaluate the osteogenic differentiation of PDLCs. The expression of Wnt/β-catenin and mitogen-activated protein kinase (MAPK) signaling related proteins was assessed by western blot.ResultsMTT results showed that baicalein up to 100 μM had no cytotoxicity on PDLCs. Baicalein significantly attenuated the inflammatory factors induced by LPS, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), matrix metalloprotein-1 (MMP-1), MMP-2 and monocyte chemoattractant protein 1 (MCP-1) at both mRNA and protein level. Moreover, MAPK signaling (ERK, JNK and p38) was significantly inhibited by baicalein, which may account for the mitigated inflammatory response. Next, we found that baicalein effectively restored the osteogenic differentiation of LPS-treated PDLCs, as shown by the increased ALP and ARS staining. Accordingly, the protein and gene expression of osteogenic markers, namely runt-related transcription factor 2 (RUNX2), collagen-I, and osterix were markedly upregulated. Importantly, baicalein could function as the Wnt/β-catenin signaling activator, which may lead to the increased osteoblastic differentiation of PDLCs.ConclusionsWith the limitation of the study, we provide in vitro evidence that baicalein ameliorates inflammatory response and restores osteogenesis in PDLCs challenged with LPS, indicating its potential use as the host response modulator for the management of periodontitis.

Enhancer of zeste homolog 2-mediated paired box 8 methylation promotes gastrointestinal stromal tumor progression through Wnt4 downregulation.

Gastrointestinal stromal tumor (GIST) is a refractory malignant tumor without satisfactory therapy. In recent years, aberrant gene methylation has been highlighted as an inducer for tumor progression. In this study, we explored whether enhancer of zeste homolog 2 (EZH2)-mediated paired box 8 (PAX8) methylation affects GIST development through regulation of Wnt4. A total of 50 cases of GIST tissues were collected and the human GIST cell lines were cultured. PAX8 methylation was examined using MS-PCR. Following loss- and gain-function approaches, GIST cell proliferation, migration, invasion, and apoptosis were examined by CCK-8 assay, Transwell assay and flow cytometry. The expression of proliferation related factors and apoptosis related factors was determined. Finally, xenograft tumors in nude mice were observed to examine in vivo tumorigenicity of GIST cells. Downregulated PAX8 and upregulated EZH2 expression was found in GIST tissues. Overexpression of PAX8 or suppression of PAX8 methylation using DNA methyltransferase inhibitor 5-Aza-dC inhibited the proliferation, migration, and invasion of GIST cells while promoting their apoptosis (diminished PCNA, Ki67 and Bcl-2, elevated Bax, and cleaved caspase-3). EZH2 promoted PAX8 methylation to inhibit its expression. Downregulated PAX8 decreased Wnt4 expression to accelerate GIST progression both in vitro and in vivo. Collectively, EZH2 inhibits PAX8 expression by promoting its methylation, which thus downregulates Wnt4 expression, thereby promoting the development of GIST.

ODAM promotes junctional epithelium‐related gene expression via activation of WNT1 signaling pathway in an ameloblast‐like cell line ALC

In this study, we investigated the potential and mechanism of odontogenic ameloblast-associated protein (ODAM) in the promoting junctional epithelium-related gene expression in an ameloblast-like cell line ALC. ODAM is expressed in ameloblasts and JE and acts as a component of the inner basal lamina (IBL) and intercellular matrix of JE. ODAM KO mice showed destruction of the integrity of the JE, which detaches from teeth. ODAM was confirmed to regulate the cytoskeleton through the ODAM-ARHGEF5-RhoA signaling pathway of the JE. Whether ODAM contributes to the regulation of ameloblast differentiation in JE remains unclear. After the formation of enamel, the ameloblast undergoes a series of morphological changes. Whether ODAM will affect the biological behavior of ameloblasts making them have the characteristics of JE is unclear. A murine ameloblast-like cell line, ALC, was used to investigate the effects of ODAM on the JE-like changes of ALC cells in an epithelium-induced environment by generating ODAM overexpression and ODAM knockdown cells through a lentivirus transduction approach. The biomarkers of junctional epithelium CK19, SLPI, and ODAM and the potential regulatory gene WNT1 were investigated by real-time PCR, western blot, immunocytochemistry, immunostaining, luciferase reporter, and rescue assays. ODAM, CK19, and SLPI were significantly upregulated after epithelial induction. Overexpression of ODAM in ALC cells markedly increased CK19 and SLPI expression, while knockdown of ODAM in ALC cells clearly decreased CK19 and SLPI expression. A reporter luciferase assay showed that ODAM activated the WNT signaling pathway, especially through WNT1. Exogenous overexpression of ODAM upregulated WNT1 expression, while knockdown of ODAM reversed this effect. The WNT1 inhibition assay further confirmed the above results and showed that the WNT1 pathway was positively correlated with biomarkers of junctional epithelium CK19 and SLPI expression. Rescue studies showed that knocking down WNT1 in the ODAM-overexpressing ALC cells decreased the expression of CK19 and SLPI. Immunocytochemistry showed that ODAM colocalized with CK19, SLPI, and WNT1 in the cells. In conclusion, the research work showed that ODAM promotes junctional epithelium-related gene expression in ALC via the ODAM-WNT1 axis, which may provide new insight into the function of ODAM and JE formation.

FoxM1 promotes Wnt/β-catenin pathway activation and renal fibrosis via transcriptionally regulating multi-Wnts expressions.

The activation of Wnt/β‐catenin pathway plays a pivotal role in promoting renal fibrosis. The activation of Wnt/β‐catenin pathway relies on the binding of Wnts to Frizzled receptors on cell membrane. However, the factor regulating Wnts production remains unclear. Here, we demonstrated that transcriptional factor FoxM1 was significantly increased in obstructed kidneys and patients' kidneys with fibrosis. The up‐regulation of FoxM1 mainly distributed in tubular epithelial cells. Pharmacological inhibition of FoxM1 down‐regulated multi‐Wnts elevation in UUO mice and attenuated renal fibrosis. In cultured renal tubular epithelial cells, overexpression of FoxM1 promoted 8 Wnts expression, while knock‐down on FoxM1‐suppressed multi‐Wnts including Wnt1, Wnt2b and Wnt3 expression induced by Ang II. Chromatin immunoprecipitation PCR confirmed that FoxM1 bound to Wnt1, Wnt2b, Wnt3 promoters and luciferase assay further identified that the transcriptions of Wnt1, Wnt2b and Wnt3 were regulated by FoxM1. Thus, our findings show that multi‐Wnt family members were regulated by transcriptional factor FoxM1. FoxM1 might be a key switch for activating β‐catenin pathway and renal fibrosis. Therefore, FoxM1 might be a potential therapeutic target in manipulating renal fibrosis.

LncRNAS56464.1 as a ceRNA promotes the proliferation of fibroblast‑like synoviocytes in experimental arthritis via the Wnt signaling pathway and sponges miR‑152‑3p.

Rheumatoid arthritis (RA) is an autoimmune disease that occurs in approximately 1.0% of the general population. In RA patients, physical disability and joint damage are the major prognostic factors, which are associated with a reduction in the quality of life and early mortality. At present, the exact molecular mechanism of RA remains elusive. Long noncoding RNAs (lncRNAs) have been revealed to play a regulatory role in the pathogenesis of RA. To reveal the function of lncRNAs in rheumatoid arthritis, lncRNAS56464.1 was screened to verify its targeting of the microRNA (miR)-152-3p/Wnt pathway and its effect on the proliferation of fibroblast-like synoviocytes (FLS). In the present study, based on the competing endogenous RNA (ceRNA) theory, siRNA was designed for transfection into FLS to calculate the lncRNAS56464.1 interference efficiency and then the effect of lncRNAS56464.1 interference on FLS proliferation was detected by MTT assay. Then, lncRNAS56464.1 targeting of the miR-152-3p/Wnt pathway was detected by a dual-luciferase reporter assay. In addition, RT-qPCR, immunofluorescence and western blotting techniques were employed to detect the expression of lncRNAS56464.1, miR-152-3p and some key genes of the Wnt signaling pathway in FLS after lncRNAS56464.1 interference. The results revealed that lncRNAS56464.1 could combine with miR-152-3p and promoted the proliferation of FLS. In addition, lncRNAS56464.1 interference could not only decrease the proliferation of FLS and the expression of Wnt1, β-catenin, c-Myc, cyclin D1, and p-GSK-3β/GSK-3β, but it also increased the expression of SFRP4. The present data indicated that lncRNAS56464.1 could target the miR-152-3p/Wnt pathway to induce synovial cell proliferation and then participate in the pathogenesis of RA.

Prognostic role of Wnt and Fzd gene families in acute myeloid leukaemia.

Wnt‐Fzd signalling pathway plays a critical role in acute myeloid leukaemia (AML) progression and oncogenicity. There is no study to investigate the prognostic value of Wnt and Fzd gene families in AML. Our study screened 84 AML patients receiving chemotherapy only and 71 also undergoing allogeneic haematopoietic stem cell transplantation (allo‐HSCT) from the Cancer Genome Atlas (TCGA) database. We found that some Wnt and Fzd genes had significant positive correlations. The expression levels of Fzd gene family were independent of survival in AML patients. In the chemotherapy group, AML patients with high Wnt2B or Wnt11 expression had significantly shorter event‐free survival (EFS) and overall survival (OS); high Wnt10A expressers had significantly longer OS than the low expressers (all P < .05), whereas, in the allo‐HSCT group, the expression levels of Wnt gene family were independent of survival. We further found that high expression of Wnt10A and Wnt11 had independent prognostic value, and the patients with high Wnt10A and low Wnt11 expression had the longest EFS and OS in the chemotherapy group. Pathway enrichment analysis showed that genes related to Wnt10A, Wnt11 and Wnt 2B were mainly enriched in ‘cell morphogenesis involved in differentiation’, ‘haematopoietic cell lineage’, ‘platelet activation, signalling and aggregation’ and ‘mitochondrial RNA metabolic process’ signalling pathways. Our results indicate that high Wnt2B and Wnt11 expression predict poor prognosis, and high Wnt10A expression predicts favourable prognosis in AML, but their prognostic effects could be neutralized by allo‐HSCT. Combined Wnt10A and Wnt11 may be a novel prognostic marker in AML.

Regulation of complexed and free catenin pools by distinct mechanisms. Differential effects of Wnt-1 and v-Src.

Cadherins are transmembrane receptors with an extracellular domain that participates in homophilic cell to cell adhesion and a cytoplasmic domain that associates with proteins called catenins. Cadherin-mediated adhesion as well as adhesion-independent functions for catenins play important roles in differentiation, development, and malignant transformation. Mechanisms that regulate steady-state catenin levels and cadherin-catenin complex stability are poorly understood, but activities of both the Wnt-1 proto-oncogene and tyrosine kinases are implicated. Here I define, at the biochemical level, distinct mechanisms that modulate steady-state catenin levels. Increased cadherin expression, providing more catenin binding sites, leads to selective stabilization of the cadherin-associated population of alpha- and beta-catenin, but not p120(cas). In contrast, expression of Wnt-1 leads primarily to increased stability of the uncomplexed pool of beta-catenin without effect on p120(cas). Significantly, the Wnt-1-induced stabilization of uncomplexed beta-catenin is independent of cadherin expression. Transformation by v-Src does not disrupt the catenin-cadherin complex despite the phosphorylation of E-cadherin and beta-catenin on tyrosine. In contrast to the effects of Wnt-1, v-Src does not modulate the uncomplexed population of beta-catenin. p120(cas) is phosphorylated on tyrosine by v-Src, and this is accompanied by a significant decrease in the level of uncomplexed p120(cas) as well as a change in behavior of p120(cas) upon biochemical fractionation. Taken together these data suggest that p120(cas) and beta-catenin are regulated independently.

Low-density lipoprotein receptor-related protein 6 regulates cardiomyocyte-derived paracrine signaling to ameliorate cardiac fibrosis.

Rationale: Maladaptive cardiac remodeling is a critical step in the progression of heart failure. Low-density lipoprotein receptor-related protein 6 (LRP6), a co-receptor of Wnt, has been implicated in cardiac protection. We aimed to study the role of cardiomyocyte-expressed LRP6 in cardiac remodeling under chronic pressure overload.Methods: Cardiac parameters were analyzed in inducible cardiac-specific LRP6 overexpressing and control mice subjected to transverse aortic constriction (TAC).Results: Cardiac LRP6 was increased at an early phase after TAC. Cardiomyocyte-specific LRP6 overexpression improved cardiac function and inhibited cardiac hypertrophy and fibrosis four weeks after TAC. The overexpression significantly inhibited β-catenin activation, likely contributing to the inhibitory effect on cardiac hypertrophy after TAC. LRP6 overexpression reduced the expression and secretion of Wnt5a and Wnt11 by cardiomyocytes, and knockdown of Wnt5a and Wnt11 greatly inhibited cardiac fibrosis and dysfunction under pressure overload in vitro and in vivo. Cardiomyocyte-expressed LRP6 interacted with cathepsin D (CTSD, a protease) and promoted the degradation of Wnt5a and Wnt11, inhibiting cardiac fibrosis and dysfunction induced by TAC. The protease inhibitor leupeptin attenuated the interaction between LRP6 and CTSD, enhanced the expression of Wnt5a and Wnt11, and deteriorated cardiac function and fibrosis in cardiomyocyte-specific LRP6-overexpressing mice under pressure overload. Mutants from human patients, P1427Q of LRP6 and G316R of CTSD significantly inhibited the interaction between LRP6 and CTSD and increased Wnt5a and Wnt11 expression.Conclusion: Cardiomyocyte-expressed LRP6 promoted the degradation of Wnt5a and Wnt11 by regulating CTSD and inhibited cardiac fibrosis under pressure overload. Our study demonstrated a novel role of LRP6 as an anti-fibrosis regulator.

Platelet Rich Plasma Regulates Wnt/Beta-Catenin Signal Pathway in Rabbit Chondrocytes by Mediating Interleukin 1 Beta

It was reported that platelet rich plasma can reduce the degeneration of articular cartilage. The purpose of this study was to investigate the effect of platelet rich plasma on Wnt/beta-catenin signal transduction in rabbit chondrocytes. Platelet rich plasma was prepared from the ear vein blood of 3-mo old New Zealand white rabbits. Chondrocytes were isolated from knee cartilage and cultured. The cell identification and the effect of platelet rich plasma on the viability of chondrocytes were measured by the C-terminal telopeptide II and proteoglycan staining. Chondrocytes were divided into 5 groups, control group, interleukin-1 beta group, platelet rich plasma (100 x dilutions) group, Dickkopf WNT signaling pathway inhibitor 1 (100 ng/mL) group and Dickkopf WNT signaling pathway inhibitor 1+platelet rich plasma group. After being treated with interleukin-1 beta (50 μL, 10 μg/mL) for 24 h, the morphology of chondrocytes was observed by an electron microscope. The levels of C-terminal telopeptide II and cartilage oligomeric matrix protein in the culture medium were determined by enzyme linked immunosorbent assay. The messenger RNA and protein expression levels of Wnt-1, beta-catenin and glycogen synthase kinase 3 beta were detected by Real-time polymerase chain reaction and Western blot respectively. Platelet rich plasma promotes the proliferation of chondrocytes. The chondrocytes in the interleukin-1 beta group showed an ultrastructural abnormality, which was not obvious in platelet rich plasma group, Dickkopf WNT signaling pathway inhibitor 1group, and Dickkopf WNT signaling pathway inhibitor 1+platelet rich plasma group. The concentrations of C-terminal telopeptide II and cartilage oligomeric matrix protein in the interleukin-1 beta group were higher than those in the control group, platelet rich plasma group, Dickkopf WNT signaling pathway inhibitor 1group and Dickkopf WNT signaling pathway inhibitor 1+platelet rich plasma group. Compared with the control group, platelet rich plasma group, Dickkopf WNT signaling pathway inhibitor 1group and Dickkopf WNT signaling pathway inhibitor 1+platelet rich plasma group, the expression of Wnt1 and beta-catenin was higher and the level of glycogen synthase kinase 3 beta was lower in the interleukin-1 beta group. Platelet rich plasma may protect interleukin-1 beta activated chondrocytes by inhibiting Wnt/beta-catenin signal.

Elevated Wnt2 and Wnt4 Activate NF-κB Signaling to Promote Cardiac Fibrosis by Cooperation of Fzd4/2 and LRP6 Following Myocardial Infarction

Background: Acute myocardial infarction (AMI)-induced excessive cardiac fibrosis exaggerates infarcted area and cardiac dysfunction, which is a significant clinical problem. Here, we investigated Wnt2 and Wnt4 levels in AMI patients or MI mouse model, and delineated the potential mechanism in cardiac fibrosis following MI. Methods: We examined serum Wnt2 and Wnt4 levels in AMI and non-AMI patients by ELISA analysis. MI mouse model was built by ligation of left anterior descending branch (LAD). Findings: Serum Wnt2 or Wnt4 level was increased in patients with AMI, and the high serum Wnt2 and Wnt4 levels correlated to adverse outcome of these patients. Elevated serum and cardiac Wnt2 or Wnt4 levels were observed at early-phase after experimental MI. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial fibrosis and cardiac dysfunction following MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in cardiomyocytes (CMs) or cardiac fibroblasts (CFs). Si-Wnt2 or si-Wnt4 partly abolished the pro-fibrotic effect in cardiac fibroblasts in response to hypoxia. Mechanistically, exogenous Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured CFs. In cultured CFs, Wnt2 or Wnt4 upregulated Frizzled (Fzd) 2 or Fzd4 and LRP6 expression. Si-Fzd4 or si-Fzd2 significantly attenuated NF-κB activation and the pro-fibrotic effects in CFs treated with Wnt2 or Wnt4, respectively. Knockdown of LRP6 showed the similar results. However, Wnt2 or Wnt4 induced the activation of β-catenin dependently on LRP6 not Fzd4 or Fzd2. Interpretation: Elevated Wnt2 and Wnt4 activate NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which may contribute to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI. Clinical Trial Registration Details: ClinicalTrials.gov Identifier: NCT03342131. Funding Information: This study was supported by National Natural Science Foundation of China (No. 81770239, 81730009, 82070232). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: This study was approved by the Ethics Committee of Dalian Medical University/First Affiliated Hospital of Dalian Medical University (PJ-KS-KY-2017-104(x)). Before inclusion in the study, written informed consent was obtained from the patient's family/patient. The investigation conformed to the principles outlined in the Declaration of Helsinki. All experiment protocols about animal in this study were approved by the Animal Care and Use Committee of Zhongshan Hospital, Fudan University, and conformed to the ‘Guidelines for the Care and Use of Laboratory Animals’ published by the National Institutes of Health (No. 85-23, revised 1996).

Effect and mechanism of miR-217 on drug resistance, invasion and metastasis of ovarian cancer cells through a regulatory axis of CUL4B gene silencing/inhibited Wnt/β-catenin signaling pathway activation.

To explore the effect and mechanism of miR-217 in cisplatin resistance, as well as invasion and metastasis of ovarian cancer by inhibiting the expression of Cullin 4B (CUL4B) and the activation of Wnt/β-catenin signaling pathway. Human ovarian cancer cell lines COC1 (cisplatin sensitive) and COC1/DDP (cisplatin resistant) were cultured and were used to construct the COC1 group and COC1/DDP group, respectively. COC1/DDP cells were divided into blank group, NC group, miR-217 mimic group, miR-217 mimic NC group, miR-217 inhibitor group, miR-217 inhibitor NC group, si-CUL4B group, si-CUL4B NC group, overexpressed (oe) oe-CUL4B group, oe-CUL4B NC group, and miR-217 mimic +oe-CUL4B group, with the identification of cell transfection simultaneously. Bioinformatics prediction and Dual-Luciferase reporter gene assay of the targeting effect of miR-217 on CUL4B were performed, followed by MTT assay for cell proliferation, associated with the measurement of median inhibitory concentration (IC50). Real-time quantitative PCR (qRT-PCR) detected the mRNA expression of miR-217 and CUL4B, and Western blotting for detecting CUL4B, Wnt1, Wnt3, Wnt3a and β-catenin protein expression. The cell invasion of cells was detected by transwell assay, cell migration by cell scratch assay and cell apoptosis by flow cytometry. Bioinformatics prediction and Dual-Luciferase reporter gene assay verified that CUL4B was a target gene of miR-217, and the latter could silence the expression of the former gene. Compared with COC1 group, the relative expression of miR-217 was significantly decreased, while CUL4B mRNA and protein expression, as well as Wnt1, Wnt3, Wnt3a and β-catenin protein expression were increased significantly, with evidently increased cell proliferation, IC50, invasion and migration, and decreased apoptosis rate in COC1/DDP group (all p<0.05). Compared with blank group and corresponding NC groups, miR-217 mimic group had increased expression of miR-217 and decreased expression of CUL4B; miR-217 inhibitor group showed decreased miR-217 expression while increased CUL4B expression; si-CUL4B group indicated no significant change of miR-217 expression but decreased CUL4B expression; oe-CUL4B group showed no difference in miR-217 expression but increased CUL4B expression; miR-217 mimic +oe-CUL4B had increased miR-217 expression and no change of CUL4B expression. Meanwhile, miR-217 mimic group and si-CUL4B group exhibited decreased Wnt/β-catenin, Wnt1, Wnt3, Wnt3a and β-catenin expression, decreased cell proliferation, IC50, invasion and migration, and increased apoptosis (all p<0.05). Furthermore, miR-217 inhibitor group and oe-CUL4B group revealed increased Wnt/β-catenin, Wnt1, Wnt3, Wnt3a and β-catenin expression, increased cell proliferation, IC50, invasion and migration, and decreased apoptosis (all p<0.05). NC group and miR-217 mimic +oe-CUL4B group showed no significant difference in the above indexes (all p>0.05). While compared with miR-217 mimic group, miR-217 mimic +oe-CUL4B group showed increased cell proliferation, IC50, invasion and migration, and decreased apoptosis (all p<0.05). CUL4B gene is the target gene of miR-217. MiR-217 can silence the expression of this gene, and inhibit the activation of Wnt/β-catenin signaling pathway to enhance the cisplatin sensitivity and reverse drug resistance, inhibit cell invasion and migration, and promote cell apoptosis.

Effects of Dexamethasone Exposure on Neural Crest Cells and Primary and Secondary Neurulation in Chick Embryos.

To evaluate the effects of dexamethasone (Dex) treatment on neural crest cells and primary and secondary neurulation in chick embryos. Sixty fertilized eggs with an average weight of 65 ± 2 g were incubated in 60%?70% humidity at 37.2°C ± 0.1°C. After 26 hours of incubation, the control group (n=12) received 0.1 mg/kg physiologic saline (S), group 1 (n=12) received 0.1 mg/kg Dex, group 2 (n=12) received 1 mg/kg Dex, and group 3 (n=12) received 5 mg/kg Dex into each embryonic disc. The eggs were incubated until Hamburger?Hamilton stage (HH) 15, HH18, and HH20. Then, the embryos were dissected and evaluated both macroscopically and microscopically. The mortality rate in the control group, group 1, and groups 2 and 3 was 27%, 48%, and 100%, respectively. The neural tube thicknesses in group 1 significantly increased in HH 15 and HH20 (p < 0.05). The mitosis number in group 1 significantly decreased in each stage (p < 0.05). Wnt-1 expression was significantly lower in group 1 in HH15 (p < 0.05) and HH18 (p < 0.05), but there was no significant difference in HH20 (p > 0.05). Fibroblast growth factor (FGF) expression was significantly lower in group 1 in HH15 (p < 0.05). The expression of N-cadherin was significantly higher in group 1 in HH20 (p < 0.05). Fibronectin expression decreased in group 1 in HH18 (p < 0.01). Although the Dex treatment did not result in neural tube closure defect, the mortality rates and neural tube thicknesses increased, whereas mitotic activation and Wnt-1 and FGF signal pathways reduced in some stages.

Abnormal proinflammatory and stressor environmental with increased the regulatory cellular IGF-1/PAPP-A/STC and Wnt-1/β-Catenin canonical pathway in placenta of women with Chronic venous Disease during Pregnancy.

Lower limbs venous insufficiency refers to a wide variety of venous disorders grouped by the term of chronic venous disease (CVD). Hemodynamic and hormonal changes related to pregnancy period, may promote the development of CVD affecting approximately 1 in 3 women. It has been shown that the presence of this condition is associated with damage and placental suffering. Thus, taking IGF-1/PAPP-A/STC-2, inflammatory cytokines production, PI3K/Akt and Wnt/ β-catenin pathways as a part of the alterations that occurs in the placenta due to CVD, the aim of this study will be to examine the main components of these pathways. Genic and protein expression of PAPP-A, STC-2, IGF-1, IRS-4 Wnt-1, β-catenin, c-myc, Cyclin D1, IL-4/IL-6 and PI3K/Akt/mTOR pathway will be analysed through RT-qPCR and immunohistochemical techniques in women with CVD (n=62) and pregnant women without this condition (HC) (n=52). PAPP-A, IGF-1, IL-4, IL-6, IRS-4, PI3K, Akt, mTOR, Wnt-1, β-catenin, c-myc and Cyclin D1 expression were found to be increased in women with CVD, whereas STC-2 were decreased in this group, compared to non-affected women. Our study has demonstrated that IGF-1/PAPP-A/STC-2 axis, PI3K/Akt and Wnt/β-catenin pathways, along with c-myc, Cyclin D1 and inflammatory cytokines are altered in placenta women with CVD. These results extent the knowledge that CVD is associated to a placenta damage with abnormal tissue environment and cellular regulation.

E2F1 regulates testicular descent and controls spermatogenesis by influencing WNT4 signaling.

Cryptorchidism is the most common urologic birth defect in men and is a predisposing factor of male infertility and testicular cancer, yet the etiology remains largely unknown. E2F1 microdeletions and microduplications contribute to cryptorchidism, infertility and testicular tumors. Although E2f1 deletion or overexpression in mice causes spermatogenic failure, the mechanism by which E2f1 influences testicular function is unknown. This investigation revealed that E2f1-null mice develop cryptorchidism with severe gubernacular defects and progressive loss of germ cells resulting in infertility and, in rare cases, testicular tumors. It was hypothesized that germ cell depletion resulted from an increase in WNT4 levels. To test this hypothesis, the phenotype of a double-null mouse model lacking both Wnt4 and E2f1 in germ cells was analyzed. Double-null mice are fertile. This finding indicates that germ cell maintenance is dependent on E2f1 repression of Wnt4, supporting a role for Wnt4 in germ cell survival. In the future, modulation of WNT4 expression in men with cryptorchidism and spermatogenic failure due to E2F1 copy number variations may provide a novel approach to improve their spermatogenesis and perhaps their fertility potential after orchidopexy.

MiR-634 inhibits human vascular smooth muscle cell proliferation and migration in hypertension through Wnt4/β-catenin pathway.

MicroRNAs (miRNAs) have been regarded as modulators in vascular pathologies, including hypertension. Dysregulated proliferation and migration of VSMCs (vascular smooth muscle cells) contributes to vascular remodeling during hypertension. miR-634 was reported to be dysregulated in hypertensive patients. The involvement of miR-634 in hypertension and the role of miR-634 on VSMCs proliferation and migration were then evaluated. Firstly, HASMCs (human aortic smooth muscle cells) were incubated with 2 μM angiotensin (Ang) II for 12 hours to establish the cell model of Ang II-induced hypertension. Results showed that Ang II treatment promoted proliferation and migration of HASMCs. Secondly, miR-634 was down-regulated in the hypertensive patients, and reduced in Ang II-induced HASMCs in a time dependent manner. Functional assays revealed that Ang II promoted proliferation and migration of HASMCs were suppressed by miR-634 mimic. Lastly, miR-634 targeted 3' untranslated region (UTR) of Wnt4, and reduced Wnt4 expression in HASMCs. miR-634 inhibited β-catenin nuclear translocation. Over-expression of Wnt4 counteracted the suppressive effects of miR-634 on Ang II-induced proliferation and migration of HASMCs. In conclusion, miR-634 inhibited HASMCs proliferation and migration through inactivation of Wnt4/β-catenin pathway.

A circular RNA, circUSP36, accelerates endothelial cell dysfunction in atherosclerosis by adsorbing miR-637 to enhance WNT4 expression

ABSTRACT Atherosclerosis is a fatal disorder that is fundamental to various cardiovascular diseases and severely threatens people’s health worldwide. Several studies have demonstrated the role of circular RNAs (circRNAs) in the pathogenesis of atherosclerosis. circUSP36 acts as a key modulator in the progression of atherosclerosis, but the molecular mechanism underlying this role is as yet unclear. This study aimed to elucidate the mechanism by which circUSP36 exerts its function in an in vitro cell model of endothelial cell dysfunction, which is one of pathological features of atherosclerosis. The circRNA traits of circUSP36 were confirmed, and we observed high expression of circUSP36 in endothelial cells exposed to oxidized low-density lipoprotein (ox-LDL). Functional assays revealed that overexpression of circUSP36 suppressed proliferation and migration of ox-LDL-treated endothelial cells. In terms of its mechanism, circUSP36 adsorbed miR-637 by acting as an miRNA sponge. Moreover, enhanced expression of miR-637 abated the impact of circUSP36 on ox-LDL-treated endothelial cell dysregulation. Subsequently, the targeting relationship between miR-637 and WNT4 was predicted using bioinformatics tools and was confirmed via luciferase reporter and RNA pull-down assays. Notably, depletion of WNT4 rescued circUSP36-mediated inhibition of endothelial cell proliferation and migration. In conclusion, circUSP36 regulated WNT4 to aggravate endothelial cell injury caused by ox-LDL by competitively binding to miR-637; this finding indicates circUSP36 to be a promising biomarker for the diagnosis and therapy of atherosclerosis.

Evaluating the Expression of Wnt Pathway Related Genes in Mouse Vitrified Preantral Follicles: An Experimental Study

Background:Wnt signaling pathway plays critical role in ovarian follicle development. Therefore, the aim of this study was to evaluate the effects of vitrification on the expression of Wnt pathway related genes in preantral follicles (PFs).Methods:Isolated PFs (n=982) of 14–16 day old female mice (n=45: 15 for each group) were divided into fresh (n=265), toxicity (n=272), and vitrified (n=265). The mRNA levels of Wnt2, Wnt4, Lrp5 and Fzd3 were evaluated by real-time PCR on the 2nd and 6th days of culture period. One-way ANOVA was conducted to analyze the data. Post hoc Tukey’s HSD was used for multiple comparisons and p-value less than 0.05 was considered statistically significant.Results:The developmental parameters of fresh PFs were significantly higher than those of vitrified (p<0.001). There were no differences between fresh and vitrified PFs on the 2nd day of culture (p<0.001). Wnt4 expression levels decreased significantly in vitrified groups compared with fresh ones (p<0.001). Fzd3 and Lrp expression levels increased significantly in vitrified groups compared with those in the fresh group on the 2nd day (p<0.001). On the 6th day of culture period, the expression levels of Wnt2 and Fzd3 increased significantly in vitrified group compared to those of fresh group (p<0.001). Moreover, the expression levels of Wnt4 and Lrp increased significantly in toxicity groups compared to those of the control group (p<0.001).Conclusion:Vitrification increase the expression levels of Wnt2, Lrp and Fzd3 genes of PFs during in vitro culture.