Overexpression Of Wnt5a Research Articles

Overexpression of Wnt5a promoted the protective effect of mesenchymal stem cells on Lipopolysaccharide-induced endothelial cell injury via activating PI3K/AKT signaling pathway.

Lung endothelial barrier injury plays an important role in the pathophysiology ofacute lung injury/acute respiratory distress syndrome (ALI/ARDS). Mesenchymal stem cells (MSCs) therapy has shown promise in ARDS treatment and restoration of the impaired barrier function. It has been reported that Wnt5a shows protective effects on endothelial cells. Therefore, the study aimed to investigate whether overexpression of Wnt5a could promote the protective effects of MSCs on Lipopolysaccharide (LPS)-induced endothelial cell injury. To evaluate the protective effects of MSCs overexpressing Wnt5a, we assessed the migration, proliferation, apoptosis, and angiogenic ability of endothelial cells. We assessed the transcription of protective cellular factors using qPCR and determined the molecular mechanism using Western blot analysis. Overexpression of Wnt5a upregulated the transcription of protective cellular factors in MSCs. Co-culture of MSCWnt5a promoted endothelial migration, proliferation and angiogenesis, and inhibited endothelial cell apoptosis through the PI3K/AKT pathway. Overexpression of Wnt5a promoted the therapeutic effect of MSCs on endothelial cell injury through the PI3K/AKT signaling. Our study provides a novel approach for utilizing genetically modified MSCs in the transplantation therapy for ARDS.

Exploring the Role of Wnt Ligands in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

This study aimed to investigate the functions of 19types of Wnt ligands during the process of osteogenic differentiation inhuman periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4. The expression levels of 19 types ofWnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability. During hPDLSCs osteogenic differentiation, the expression of 19types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly. Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis. Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

Haspin balances the ratio of asymmetric cell division through Wnt5a and regulates cell fate decisions in mouse embryonic stem cells

Many different types of stem cells utilize asymmetric cell division (ACD) to produce two daughter cells with distinct fates. Haspin-catalyzed phosphorylation of histone H3 at Thr3 (H3T3ph) plays important roles during mitosis, including ACD in stem cells. However, whether and how Haspin functions in ACD regulation remains unclear. Here, we report that Haspin knockout (Haspin-KO) mouse embryonic stem cells (mESCs) had increased ratio of ACD, which cumulatively regulates cell fate decisions. Furthermore, Wnt5a is significantly downregulated due to decreased Pax2 in Haspin-KO mESCs. Wnt5a knockdown mESCs phenocopied Haspin-KO cells while overexpression of Wnt5a in Haspin-KO cells rescued disproportionated ACD. Collectively, Haspin is indispensable for mESCs to maintain a balanced ratio of ACD, which is essential for normal development and homeostasis.

MiR-139-3p/Wnt5A Axis Inhibits Metastasis in Hepatoblastoma.

In order to examine new potential treatment options for the treatment of hepatoblastoma (HB), we identified the differential expression of five-candidate tumor suppressor miRNAs in HB and explored possible regulatory mechanisms of target miRNA molecule. By using real-time quantitative polymerase chain reaction (qPCR), we examined the expression of miRNAs in HB tissues and cells. The effect of has-miR-139-3p mimics on the invasion and migration ability was assessed by transwell assay and scratch-wound assay in HepG2 cells. Subsequently, we analyzed the target genes of miR-139-3p and their enrichment signaling pathways through bioinformatics. qPCR, Western-blot and dual-luciferase assays were further used to assess whether has-miR-139-3p targets Wnt5A. The results showed that hsa-miR-139-3p was significantly decreased in HB cells. Upregulation of hsa-miR-139-3p inhibited the invasive and migratory ability of HepG2. Bioinformatics analysis showed that hsa-miR-139-3p may target Wnt5A to regulate the WNT pathway, which was further confirmed by Western-blot and dual-luciferase assays. Overexpression of Wnt5A can reverse the miR-139-3p mimic-induced declines in the expression of WNT pathway-related proteins and restore the invasion and migration of HepG2. These data indicated that the hsa-miR-139-3p/Wnt5A axis inhibited HB metastasis, suggesting that miR-139-3p and Wnt5A may be potential targets for the treatment of HB.

Overactive Wnt5a signaling disrupts hair follicle polarity during mouse skin development.

The polarity of mouse hair follicles is controlled by the Frizzled (Fzd) receptors and other membrane planar cell polarity (PCP) proteins. Whether Wnt proteins can act as PCP ligands in the skin remains unknown. Here, we show that overexpression of Wnt5a in the posterior part of mouse embryos causes a local disruption of hair follicle orientation. The misoriented hair follicle phenotype in Wnt5a overexpressing mice can be rescued by a heterozygous loss of Fzd6, suggesting Wnt5a is likely to signal through Fzd6. Although the membrane distribution of PCP proteins seems unaffected by Wnt5a overexpression, transcriptional profiling analyses identify a set of genes as potential targets of the skin polarization program controlled by Wnt5a/Fzd6 signaling. Surprisingly, deletion of Wnt5a globally or in the posterior part of the mouse embryos does not affect hair follicle orientation. We show that many other Wnts are highly expressed in the developing skin. They can activate the Fzd6 signaling pathway in vitro and may act together with Wnt5a to regulate the Fzd6-mediated skin polarization. Our experiments demonstrate for the first time that Wnt5a can function as an orienting cue for mouse skin PCP.

Exosomes Derived from Dermal Papilla Cells Mediate Hair Follicle Stem Cell Proliferation through the Wnt3a/β-Catenin Signaling Pathway.

Both hair follicle stem cells (HFSC) and dermal papilla cells (DPC) are essential for hair follicle growth and proliferation. In this study, HFSCs and DPCs that made signature proteins like KRT14, KRT15, KRT19, α-SMA, and Versican were obtained. Cell coculture systems between HFSCs and DPCs were used to measure the increased PCNA protein content in HFSCs. Additionally, exosomes from dermal papilla cells (DPC-Exos), the overexpression and silencing of Wnt3a, could regulate the Wnt/β-catenin signaling pathway downstream genes. After collecting DPC-ExosOE-Wnt3a, the treatment of HFSC with DPC-ExosOE-Wnt3a showed that DPC-ExosOE-Wnt3a could upregulate the mRNA expression of downstream genes in the Wnt/β-catenin signaling pathway and that DPC-ExosOE-Wnt3a enhanced the proliferation of HFSCs while inhibiting their apoptosis. These findings suggest that DPC-Exos could regulate HFSC cell proliferation via the Wnt3a/β-catenin signaling pathway. This research offers novel concepts for the molecular breeding and efficient production of Angora rabbits, as well as for the treatment of human hair problems.

Alteration of Wnt3a overexpression and its early monitoring value during hepatocellular carcinogenesis

Objective: To investigate the dynamic expression pattern of carcinoembryonic Wnt3a and its early monitoring value using a hepatocellular carcinoma model. Methods: Forty-eight Sprague Dawley (SD) rats were fed with pellet feed containing 2-acetylaminofluorene (2-AAF, 0.05%) to induce hepatocarcinogenesis, and control rats were fed a pellet diet. Liver tissue and blood samples were collected every two weeks. Liver tissues were pathologically examined using HE staining and grouped. The gene and Wnt3a mRNA expression were analyzed by genome-wide microarray. The expression and distribution of Wnt3a in liver tissue were analyzed by immunohistochemistry. Wnt3a concentration in liver tissue and serum was quantified by enzyme-linked immunosorbent assay. Statistical methods such as χ2 test, Mann-Whitney test and analysis of variance were used to analyze the differences between groups. Results: According to the pathological examination results, the rat livers were divided into four groups: control, hepatocyte degeneration, precancerous lesions and hepatocellular carcinoma. Genome-wide expression profiling analysis and comparison with the control group revealed that 268 and 312 genes were up-regulated and 57 and 201 genes were down-regulated in the precancerous and cancerous group when signal logarithm ratio (SLR) was >8 log2cy5/cy3, and these significantly altered genes mainly involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. The expression of Wnt3a at mRNA level was significantly increased in all stages of cancer induction, including degeneration group (1.15±0.24, q=8.227), precancerous group (1.85±0.18, q=12.361) and cancerous group (2.59±0.55, q=18.082). Compared with the control group (0.25±0.11, F=121.103, P<0.001), the degeneration group, the precancerous group and the liver cancer group were up-regulated by 4.6, 7.4 and 10.4-folds, respectively. Immunohistochemistry showed that compared with the control group, the positive rate of Wnt3a in the degeneration group was 66.7% (12/18, χ2=10.701, P=0.001), and both the precancerous and liver cancer groups were positive (9/9, χ2=17.115, P<0.001). Wnt3a expression was gradually increased in liver and blood samples during the process of carcinogenesis, and the difference between two groups was statistically significant (F=176.711, P<0.001). Wnt3a overexpression was secreted into blood stream via cancerous liver tissue, and there was a linear correlation between Wnt3a levels in blood and liver samples (r=0.732, P<0.001). Conclusions: Wnt3a overexpression is closely related with hepatocellular carcinogenesis, and thus may become a new monitoring marker.

Tumor suppressive functions of WNT5A in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly aggressive soft tissue malignancy that predominantly affects children. The main subtypes are alveolar RMS (ARMS) and embryonal RMS (ERMS) and the two show an impaired muscle differentiation phenotype. One pathway involved in muscle differentiation is WNT signaling. However, the role of this pathway in RMS is far from clear. Our recent data showed that the canonical WNT/β-Catenin pathway serves a subordinate role in RMS, whereas non-canonical WNT signaling probably is more important for this tumor entity. The present study investigated the role of WNT5A, which is the major ligand of non-canonical WNT signaling, in ERMS and ARMS. Gene expression analysis showed that WNT5A was expressed in human RMS samples and that its expression is more pronounced in ERMS. When stably overexpressed in RMS cell lines, WNT5A decreased proliferation and migration of the cells as demonstrated by BrdU incorporation and Transwell migration or scratch assay, respectively. WNT5A also decreased the self-renewal capacity and the expression of stem cell markers and modulates the levels of muscle differentiation markers as shown by sphere assay and western blot analysis, respectively. Finally, overexpression of WNT5A can destabilize active β-Catenin of RMS cells. A WNT5A knockdown has opposite effects. Together, the results suggest that WNT5A has tumor suppressive functions in RMS, which accompanies downregulation of β-Catenin.

Carbon Monoxide-Releasing Molecule-3 Enhances Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells via miR-195-5p/Wnt3a Pathway.

PurposeBone marrow-derived mesenchymal stem cells (BMSCs) are hopeful in promoting bone regeneration as their pluripotency in differentiation. Our previous study showed that carbon monoxide-releasing molecule-3 (CORM-3) increased the osteogenic differentiation of rat BMSCs in vitro. However, the mechanism remained unclear. MicroRNAs (miRNAs) play a very important role in modulating the osteogenic differentiation of BMSCs. Therefore, we researched the miRNAs involved in CORM-3-stimulated osteogenic differentiation.MethodsThe CORM-3-stimulated osteogenic differentiation of rat BMSCs was further studied in vivo. Based on the gene sequencing experiment, the rat BMSCs were transfected with miR-195-5p mimics and inhibitor, pcDNA3.1-Wnt3a and Wnt3a siRNA. The osteogenic differentiation of rat BMSCs was measured by quantitative real-time polymerase chain reaction, Western blot and alizarin red staining. Additionally, the targeting relationship between miR-195-5p and Wnt3a was confirmed by the dual-luciferase assay.ResultsMiR-195-5p was down-expressed during the CORM-3-stimulated osteogenic differentiation of rat BMSCs. CORM-3-stimulated osteogenic differentiation of rat BMSCs was inhibited with miR-195-5p overexpression, evidenced by significantly reduced mRNA and protein expressions of runt-related transcription factor 2 and osteopontin, and matrix mineralization demonstrated. On the contrary, the osteogenic differentiation was enhanced with inhibition of miR-195-5p. CORM-3-stimulated osteogenic differentiation of rat BMSCs was increased by overexpression of Wnt3a, while the opposite was observed in the Wnt3a-deficient cells. Moreover, the decreased osteogenic differentiation capacity by increased expression of miR-195-5p was rescued by Wnt3a overexpression, showing miR-195-5p directly targeted Wnt3a.ConclusionThese results demonstrate that CORM-3 promoted osteogenic differentiation of rat BMSCs via miR-195-5p/Wnt3a, which bodes well for the application of CORM-3 in the treatment of periodontal disease and other bone-defect diseases.

DOCK9 antisense RNA2 interacts with LIN28B to stabilize Wnt5a and boosts proliferation and migration of oxidized low densitylipoprotein-induced vascular smooth muscle cells

ABSTRACT Study has suggested that long non-coding RNA DOCK9 antisense RNA2 (LncRNA DOCK9-AS2) may play an important role in atherosclerosis, but the specific role is unclear. In this article, we aim to explore the role and mechanism of DOCK9-AS2 in the proliferation and migration of vascular smooth muscle cells (VSMCs) in atherosclerosis. VSMCs were treated with oxidized low densitylipoprotein (ox-LDL) for 24 h to establish the model of atherosclerosis in vitro. Gain- and loss-of function experiments were conducted. Cell Counting Kit-8 (CCK-8) assay and Ki67 staining were used to evaluate the ability cell proliferation. Transwell assay and immunofluorescence staining of N-Cadherin and E-cadherin were carried out to detect cell migration. RNA immunoprecipitation (RIP) experiment, pull down assay and mRNA stability analysis were used to assess the relationship of DOCK9-AS2, Wnt5a and LIN28B. Western blot analysis was used to measure the protein expression levels. The results showed that DOCK9-AS2 knockdown inhibited the proliferation and migration of ox-LDL-induced VSMCs. Further study on the interaction between DOCK9-AS2, Wnt5a and LIN28B revealed that LIN28B could both directly interact with DOCK9-AS2 and Wnt5a, and DOCK9-AS2 regulated Wnt5a by targeting LIN28B. In addition, Overexpression of Wnt5a partly abolished the inhibitory effects of LIN28B knockdown or DOCK9-AS2 knockdown on cell proliferation and migration induced by in ox-LDL-induced proliferation and migration. In conclusion, the results showed that DOCK9-AS2 promoted the proliferation and migration of vascular smooth muscle cells in atherosclerosis through regulating Wnt5a by targeting LIN28B.

N7-methylguanosine tRNA modification promotes tumorigenesis and chemoresistance through WNT/β-catenin pathway in nasopharyngeal carcinoma.

Treatment selections are very limited for patients with advanced nasopharyngeal carcinoma (NPC) experiencing disease progression. Uncovering mechanisms underlying NPC progression is crucial for the development of novel treatments. Here we show that N7-methylguanosine (m7G) tRNA modification enzyme METTL1 and its partner WDR4 are significantly elevated in NPC and are associated with poor prognosis. Loss-of-function and gain-of-function assays demonstrated that METTL1/WDR4 promotes NPC growth and metastasis in vitro and in vivo. Mechanistically, ARNT was identified as an upstream transcription factor regulating METTL1 expression in NPC. METTL1 depletion resulted in decreased m7G tRNA modification and expression, which led to impaired codon recognition during mRNA translation, therefore reducing the translation efficiencies of mRNAs with higher m7G codons. METTL1 upregulated the WNT/β-catenin signaling pathway and promoted NPC cell epithelial-mesenchymal transition (EMT) and chemoresistance to cisplatin and docetaxel in vitro and in vivo. Overexpression of WNT3A bypassed the requirement of METTL1 for EMT and chemoresistance. This work uncovers novel insights into tRNA modification-mediated mRNA translation regulation and highlights the critical function of tRNA modification in cancer progression.

LncRNA AZIN1-AS1 ameliorates myocardial ischemia-reperfusion injury by targeting miR-6838-5p/WNT3A axis to activate Wnt-β/catenin signaling pathway.

Myocardial reperfusion, the effective therapy for acute myocardial infarction (AMI), commonly leads to myocardial ischemia/reperfusion (I/R) injury. The effects and functional mechanisms of LncRNA AZIN1-AS1 on myocardial I/R injury in vivo and vitro are not uncovered. In our present study, we established myocardial I/R injury model of mice and H/R injury model of cardiomyocytes and we discovered AZIN1-AS1 was decreased but miR-6838-5p was increased significantly in myocardial tissues injured by I/R treatment and H9c2 cells injured by hypoxia/reoxygenation (H/R) treatment. Silencing AZIN1-AS1 down-regulated cell viability but up-regulated apoptosis rate and CK-MB in addition LDH release of cardiomyocyte under H/R injury. However, overexpression of AZIN1-AS1 recovered abovementioned effects. Additionally, miR-6838-5p was found to be the direct target of AZIN1-AS1 and exhibited negative correlation with AZIN1-AS1. Moreover, miR-6838-5p inhibitor effectively eliminated the effects of AZIN1-AS1 knockdown on H/R-injured myocardial cells. Further experiments showed that WNT3A was the target of miR-6838-5p axis and overexpression of WNT3A also counteracted the roles of AZIN1-AS1 knockdown. Furthermore, knockdown of AZIN1-AS1 dramatically inhibited the activity of WNT-β/catenin signaling pathway, which was recovered effectively by plasmid with overexpressing WNT3A. Therefore, this study firstly revealed that LncRNA AZIN1-AS1/miR-6838 axis inhibited apoptosis by activating WNT/β-catenin pathway to protect mice or H9c2 cell from I/R-induced or H/R-induced injury respectively, which advised that AZIN1-AS1 could be regarded as a potential target for treating patients with AMI.

LncRNA-mRNA Co-expression Profiles Relative to Vascular Remodeling in Moyamoya Patients Without RNF213 Mutation

Moyamoya disease (MMD) is an idiopathic cerebrovascular disease with unknown etiology. Long noncoding RNA (lncRNA) and messenger RNA (mRNA) profiles in MMD remain unknown. In this current study, we aim to investigate lncRNA-mRNA co-expression pattern and their biological functions in superficial temporal artery (STA) of MMD. STA of 3 MMD patients without RNF213 mutation and 3 age-matched controls were obtained for transcriptomic RNA sequencing. Bioinformatics analysis was performed to investigate their molecular functions and interactions. Then, differentially expressed genes relative to vascular remodeling were further validated by quantitative real-time polymerase chain reaction and immunofluorescence. WNT5A functions were tested by tube formation assay and wound scratching assay in human microvascular endothelial cells (HMECs). We detected 6235 different lncRNAs and 2065 different mRNAs from the RNA-sequencing between MMD patients and controls (P < 0.05; fold change >2.0). Gene ontology showed that altered mRNAs were enriched for endothelial cell morphogenesis and positive regulation of angiogenesis, which were closely related with vascular remodeling. We then searched 76 altered genes related with vascular remodeling and applied Kyoto Encyclopedia of Genes and Genomes analysis. Integrated analysis of lncRNA-TF-mRNA co-expression networks and gene verifications indicated that molecular including WNT5A, TEK, and GATA2 may contribute to the vascular malformation of MMD. Overexpression of WNT5A in HMECs promoted tube formation and cell migration. In MMD patients, genes related to vascular remodeling including WNT5A and their regulators were aberrantly disrupted. These results will help elucidate the complicated pathogenic mechanism of MMD and develop potential therapeutic targets facilitating MMD angiogenesis in the future.

Altered Wnt5a expression affects radiosensitivity of non-small cell lung cancer via the Wnt/β-catenin pathway.

It has been reported that upregulation of wingless-type protein 5a (Wnt5a) is associated with poor prognosis in patients with non-small cell lung cancer (NSCLC). Wnt5a expression is often upregulated in radiation-resistant NSCLC cells. However, the biological functions or molecular mechanisms of radiosensitivity in NSCLC remain unknown. In the present study, MTT assay and flow cytometric analysis were performed to assess the effect of overexpression or knockdown of Wnt5a and/or radiation on the proliferation and apoptosis of NSCLC cells. Furthermore, western blot analysis was performed to detect canonical Wnt signaling (β-catenin) in H1650 and A549 cells. The results demonstrated that Wnt5a knockdown combined with irradiation inhibited proliferation and induced apoptosis in NSCLC cells compared with Wnt5a knockdown or radiotherapy alone. In addition, the combination of Wnt5a knockdown and irradiation decreased nuclear and increased cytoplasmic β-catenin expression in H1650 and A549 cells, the effects of which were reversed following overexpression of Wnt5a. The combination of overexpressing Wnt5a and irradiation resulted in significant tumor regression, while β-catenin knockdown reversed Wnt5a overexpression-induced NSCLC cell proliferation. Taken together, these results suggest that Wnt5a may be involved in the activation of β-catenin-dependent canonical Wnt signaling, and thus may influence the effectiveness of radiation therapy in NSCLC.

LncRNA HCP5 Induces Gastric Cancer Cell Proliferation, Invasion, and EMT Processes Through the miR-186-5p/WNT5A Axis Under Hypoxia.

ObjectiveTo experimentally determine the involvement and mechanism of long non-coding RNA (lncRNA) HCP5 in the development of gastric cancer (GC).MethodsDetection of HCP5, miR-186-5p, and WNT5A expression in clinical GC tissues and adjacent healthy tissues was performed, followed by Pearson correlation analysis. BGC-823 and AGS cells, with interferences of HCP5, miR-186-5p, and WNT5A, were cultured under hypoxia. MTT, colony formation assay, Caspase-3 activity assay, and transwell assay were applied for the determination of cell proliferation, viability, apoptosis, and invasion, respectively. Expressions of WNT5A and protein markers of epithelial-mesenchymal transition (EMT) in cells were detected by western blotting. And the binding of HCP5 and WNT5A to miR-186-5p was validated using dual-luciferase reporter assay.ResultsIn GC tissues, an increase in HCP5 and WNT5A expressions and a reduction in miR-186-5p expression were found, and the negative correlation between miR-186-5p and HCP5/WNT5A was proven. Subsequently, under hypoxia, an increase in HCP5 and WNT5A expressions and a decrease in miR-186-5p expression in GC cells were confirmed. In addition, in GC cells under hypoxia, the inhibition of HCP5 suppressed cell biological activity and EMT, while the inhibition of miR-186-5p or the overexpression of WNT5A led to the opposite changes.ConclusionAn upregulation of WNT5A expression by HCP5 competitively binding to miR-186-5p promotes GC cell development.

Long intergenic noncoding RNA 00473 promoting migration and invasion of trophoblastic cell line HTR-8/SVneo via regulating miR-424-5p-mediated wnt3a/β-catenin signaling pathway.

Preeclampsia (PE) is a serious obstetric complication. Recent studies point out that the functions of long intergenic noncoding RNA 00473 (linc00473), miR-424-5p, and Wnt/β-catenin signaling pathway were involved in the invasion and migration of extravillous trophoblast. Here, we investigated the role and mechanism of linc00473 in HTR-8/SVneo trophoblastic cell line and its role in PE. The expression levels of linc00473 and miR-424-5p in placental tissues and the transfection efficiency of miR-424-5p were detected by quantitative real-time polymerase chain reaction (qRT-PCR). HTR-8/SVneo cell invasion and proliferation were determined by transwell and Cell Counting Kit-8 (CCK-8) assays. The protein expressions of wnt3a, p-GSK3β, GSK3β, active β-catenin, and total β-catenin were detected by Western blot. The apoptosis and migration of HTR-8/SVneo cells were detected by flow cytometry and wound healing assays. The targeting relationships between linc00473, miR-424-5p, and wnt3a were predicted by ENCORI database and TargetScan V7.2 and were determined using dual-luciferase reporter assay. The expression level of linc00473 was low and miR-424-5p was high in placenta of PE patients. Linc00473 can target miR-424-5p, while miR-424-5p target wnt3a. High expression of linc00473 and wnt3a promoted cell proliferation, migration, invasion, and inhibited cell apoptosis. However, miR-424-5p mimic inhibited HTR-8/SVneo cells proliferation, migration, invasion, while promoted cell apoptosis, partially reversed the effect of linc00473, while wnt3a overexpression partially counteracted the effect of miR-424-5p mimic. Linc00473 mediates the regulation of Wnt/β-catenin signaling pathway by miR-424-5p to affect the invasion and migration ability of trophoblastic cell line HTR-8/SVneo. It indicated that linc00473 is involved in PE and could be a therapeutic target.

CircVAPA exerts oncogenic property in non-small cell lung cancer by the miR-876-5p/WNT5A axis.

Non-small cell lung cancer (NSCLC) is one of the most fatal malignant tumors. Emerging studies have clarified the crucial roles of circular RNAs (circRNAs) in the tumorigenesis of cancers. CircVAPA was demonstrated to function in some human cancers. The present study aimed to investigate the role of circVAPA in NSCLC. A quantitative real-time polymerase chain reaction was used to measure the expression of genes. Actinomycin D and RNase R were employed to examine the stability of circVAPA. Cell-counting kit-8, 5-ethynyl-2'-deoxyuridine, Transwell and sphere formation assays, and well as western blot analysis, were conducted to examine the changes of NSCLC cells in response to circVAPA knockdown. A luciferase reporter assay was conducted for the molecular mechanism. Our findings demonstrated high expression of circVAPA in tissues and cell lines of NSCLC. Knockdown of circVAPA had a suppressive effect on cell proliferation, migration, invasion and stemness, and also inhibited tumor growth in vivo. Mechanistically, circVAPA acted as a competing endogenous RNA to up-regulate WNT5A by sponging miR-876-5p. Moreover, circVAPA activated Wnt/β-catenin signaling by up-regulation of WNT5A. Rescue assays showed that silencing of miR-876-5p or overexpression of WNT5A reversed the circVAPA knockdown-mediated inhibition on cellular processes in NSCLC. CircVAPA promotes aggressive phenotypes of NSCLC cells by the miR-876-5p/WNT5A axis activating Wnt/β-catenin signaling.

Overexpression of aberrant Wnt5a and its effect on acquisition of malignant phenotypes in adult T-cell leukemia/lymphoma (ATL) cells

Wnt5a is a ligand of the non-canonical Wnt signaling pathway involved in cell differentiation, motility, and inflammatory response. Adult T-cell leukemia/lymphoma (ATL) is one of the most aggressive T-cell malignancies caused by infection of human T-cell leukemia virus type1 (HTLV-1). Among subtypes of ATL, acute-type ATL cells are particularly resistant to current multidrug chemotherapies and show remarkably high cell-proliferative and invasive phenotypes. Here we show a dramatic increase of WNT5A gene expression in acute-type ATL cells compared with those of indolent-type ATL cells. Treatment with IWP-2 or Wnt5a-specific knockdown significantly suppressed cell growth of ATL-derived T-cell lines. We demonstrated that the overexpression of c-Myb and FoxM1 was responsible for the synergistic activation of the WNT5A promoter. Also, a WNT5A transcript variant without the exon4 (the ΔE4-WNT5A mRNA), encoding ΔC-Wnt5 (1-136aa of 380aa), is overexpressed in acute-type ATL cells. The ΔC-Wnt5a is secreted extracellularly and enhances cellular migration/invasion to a greater extent compared with wildtype (WT)-Wnt5a. Moreover, the ΔC-Wnt5a secretion was not suppressed by IWP-2, indicating that this mutant Wnt5a is secreted via a different pathway from the WT-Wnt5a. Taken together, synergistic overexpression of the ΔC-Wnt5a by c-Myb and FoxM1 may be responsible for the malignant phenotype of acute-type ATL cells.

Secreted frizzled-related protein 5 (SFRP5) protects ATDC5 cells against LPS-induced inflammation and apoptosis via inhibiting Wnt5a/JNK pathway

BackgroundSecreted frizzled-related protein 5 (SFRP5) is an endogenous inhibitor of Wnt5a (wingless-type family member 5a), which has been implicated in anti-inflammatory response. In this study, we aimed to investigate whether SFRP5 could protect chondrocytes against LPS-induced inflammation and apoptosis.MethodsATDC5 cells that overexpressed with SFRP5 or not were challenged with LPS to observe the effects of SFRP5 overexpression on LPS-triggered inflammation and apoptosis as well as Wnt5a/JNK activation. Wnt5a was elevated in ATDC5 cells in the presence of SFRP5 overexpression, to determine whether Wnt5a/JNK signaling was involved in the actions of SFRP5.ResultsThe mRNA and protein levels of SFRP5 was significantly reduced by LPS in a concentration-dependent manner. Overexpression of SFRP5 in ATDC5 cells inhibited LPS-induced inflammation and apoptosis, as evidenced by decreased production of TNF-α, IL-1β, IL-6, and ROS, together with a reduced ratio of TUNEL-positive cells, a lower expression of Bax and cleaved caspase 3, but a higher expression of Bcl-2. Meanwhile, SFRP5 overexpression also repress Wnt5a and phosphorylated JNK expression. However, the overexpression of Wnt5a considerably weakened the inhibitory effect of SFRP5 on LPS-triggered inflammation and apoptosis. Besides, the level of Wnt5a and JNK phosphorylation, which was inhibited by SFRP5 overexpression, was also partially recovered by Wnt5a overexpression.ConclusionSFRP5 could alleviate LPS-induced ATDC5 cell inflammation and apoptosis; these actions may rely on repressing Wnt5a/JNK activation.

DJ-1 modulates Nrf2-mediated MRP1 expression by activating Wnt3a/β-catenin signalling in A549 cells exposed to cigarette smoke extract and LPS

BackgroundChronic obstructive pulmonary disease (COPD) is an inflammatory disease characterized by airway obstruction and abnormal inflammatory responses. Multidrug resistance-related protein 1 (MRP1) can reduce lung inflammation and damage by excreting various toxic exogenous substances and certain pro-inflammatory molecules. AimsWe studied whether DJ-1 modulates nuclear factor erythroid 2-related factor 2 (Nrf2) by activating the Wnt3a/β-catenin signalling pathway to further regulate MRP1 expression and pulmonary antioxidant defences in alveolar epithelial (A549) cells treated with smoke extract (CSE) and lipopolysaccharide (LPS). Main methodsMarker expression was studied by western blot analysis, quantitative real-time PCR and immunofluorescence staining of A549 cells. Key findingsA549 cells exposed to CSE and LPS showed downregulation of DJ-1, Wnt3a, MRP1 and haem oxygenase-1 (HO-1) and upregulation of inflammatory factors. Additionally, Nrf2 protein levels were significantly decreased, while there was no change in Nrf2 mRNA levels. Overexpression of DJ-1 and Wnt3a activated Nrf2 signalling, increased MRP1 and HO-1 levels and decreased IL-6 protein expression, while knockdown of DJ-1 and Wnt3a had the opposite effects. Furthermore, DJ-1 overexpression and DJ-1 knockdown increased and decreased, respectively, the levels of Wnt3a and β-catenin. Interestingly, Nrf2 and Wnt3a deficiency reduced the protective effects of Wnt3a and DJ-1, respectively, in A549 cells. However, the levels of DJ-1 and Wnt3a were not altered by Wnt3a and Nrf2 deletion, respectively. SignificanceIn A549 cells treated with CSE and LPS, DJ-1 regulates Nrf2-mediated MRP1 expression and antioxidant defences by activating the Wnt3a/β-catenin signalling pathway. These findings may provide potential therapeutic targets for COPD intervention.