Wnt5a mRNA Research Articles

The effect of eight weeks of moderate and high intensity aerobic training on the gene expression of Mir-145, Wnt3a and Dab2 in the heart tissue of type 2 diabetic rats.

Pathological hypertrophy of heart tissue has been attributed to changes in some microRNAs and their target genes in heart tissue. This study intended to study the effects of eight weeks of moderate and high intensity aerobic training (MIT&HIT) on the mRNA of Mir-145, Wnt3a, and Dab2 in heart tissue of type 2 diabetic rats. To implement this experimental research, 60 male Wistar rats were randomly divided into 6 groups, including Healthy-control (HC), Diabetic-control (DC), Moderate intensity training (MIT), Diabetes-MIT (DMIT), high intensity training (HIT) and Diabetes-HIT (DHIT). The aerobic training was conducted with moderate (50-60% VO2max) and high (85-90% VO2max) intensity, 5days a week, for 8weeks. The Mir-145, Wnt3a and Dab2 gene expression in the heart tissue samples was measured by Real Time PCR. Data were analyzed by one-way ANOVA and Tukey post hoc test at the P < 0.05. Moderate and high intensity aerobic training was associated with non-significant increase in Mir-145 mRNA of Heart tissue in type 2 diabetic rats than the diabetic control group(P < 0.05). Moderate and high intensity aerobic training was associated with significant increase in Wnt3a mRNA (P = 0.001) and significant decrease in Dab-2 mRNA (P = 0.001) of Heart tissue in type 2 diabetic rats than the diabetic control group. The Dab-2 mRNA was significantly lower of heart tissue in the diabetes- high intensity training group than the diabetes- moderate intensity training group (P = 0.001). It seems that moderate and high intensity aerobic exercise can help regulate the genes of the physiological hypertrophy pathway of the heart tissue in diabetes.

Protective effect of propofol via the regulation of ovarian granulosa cell proliferation and apoptosis.

Propofol is an anesthetic frequently used in surgery. Accumulating evidence suggests that propofol exhibits an effect on cell viability, apoptosis and invasion in several types of cancer cells. MicroRNAs (miRNAs) have been reported to play pivotal roles in the development of polycystic ovary syndrome (PCOS). However, the diagnostic applications of miR-451a in PCOS remain unknown. The present study aimed to elucidate the effects of propofol on ovarian granulosa cell proliferation and apoptosis and illustrate the specific mechanisms associated with this process. Human ovarian granulosa cell-like KGN cells, which were used as a representative of granulosa cells in the present study, were treated with different concentrations (0, 1, 5 and 10 µg/ml) of propofol for 48 h and cell proliferation and apoptosis were assessed using MTT and flow cytometry assays, respectively. Propofol treatment resulted in significant inhibition of cell viability and induction of apoptosis in KGN cells, which was accompanied with increased cleaved caspase 3 and suppressed pro-caspase 3 expression levels. Furthermore, propofol reduced Wnt3a and β-catenin protein and mRNA expression levels. miR-451a expression in KGN cells was evaluated by reverse transcription-quantitative PCR (RT-qPCR). miR-451a expression was upregulated in propofol-stimulated KGN cells. The data further demonstrated that miR-451a mimics suppressed cell proliferation and increased apoptosis of KGN cells compared with cells transfected with control mimics. Furthermore, the association between miR-451a and propofol was investigated. Rescue experiments were performed to investigate the anti-proliferative mechanism of propofol in ovarian granulosa cells. KGN cells were transfected with miR-451a inhibitor or inhibitor control sequences for 6 h and treated with 10 µg/ml propofol for an additional 48 h. The results from the MTT, RT-qPCR and western blot assays indicated that 10 µg/ml propofol inhibited cell viability, induced apoptosis, enhanced cleaved caspase 3 expression, reduced pro-caspase 3 levels and inhibited the protein and mRNA expression of Wnt3a and β-catenin. However, inhibition of miR-451a demonstrated the opposite effects. In conclusion, the results of the present study revealed that propofol exerted an anti-proliferative and apoptosis-inducing role in ovarian granulosa cells through mediation of miR-451a expression. In addition, the data indicated that miR-451a may be used as an effective therapeutic target for PCOS treatment.

LINGO-1 regulates Wnt5a signaling during neural stem and progenitor cell differentiation by modulating miR-15b-3p levels

BackgroundManipulation of neural stem and progenitor cells (NSPCs) is critical for the successful treatment of spinal cord injury (SCI) by NSPC transplantation, since their differentiation into neurons and oligodendrocytes can be inhibited by factors present in inflamed myelin. In this study, we examined the effects of LINGO-1 on spinal cord-derived NSPC (sp-NSPC) differentiation, the underlying mechanisms of action, and the functional recovery of mice after transplantation of manipulated cells.Methodssp-NSPCs were harvested from female adult C57/BL6 mice after SCI induced with an NYU impactor. These cells were infected with lentiviral vectors containing LINGO-1 shRNA sequence or a scrambled control and transplanted into SCI mice. Tuj-1- and GFAP-positive cells were assessed by immunofluorescence staining. Wnt5a, p-JNK, JNK, and β-catenin expression was determined by Western blot and RT-qPCR. miRNAs were sequenced to detect changes in miRNA expression. Motor function was evaluated 0–35 days post-surgery by means of the Basso Mouse Scale (BMS) and by the rotarod performance test.ResultsWe discovered that LINGO-1 shRNA increased neuronal differentiation of sp-NSPCs while decreasing astrocyte differentiation. These effects were accompanied by elevated Wnt5a protein expression, but unexpectedly, no changes in Wnt5a mRNA levels. miRNA-sequence analysis demonstrated that miR-15b-3p was a downstream mediator of LINGO-1 which suppressed Wnt5a expression. Transplantation of LINGO-1 shRNA-treated sp-NSPCs into SCI mice promoted neural differentiation, wound compaction, and motor function recovery.ConclusionsLINGO-1 shRNA promotes neural differentiation of sp-NSPCs and Wnt5a expression, probably by downregulating miR-15b-3p. Transplantation of LINGO-1 shRNA-treated NSPCs promotes recovery of motor function after SCI, highlighting its potential as a target for SCI treatment.

Dermal Fibroblasts from Chronic Wounds Exhibit Paradoxically Enhanced Proliferative and Migratory Activities that May be Related to the Non-Canonical Wnt Signaling Pathway

It is generally thought that dermal fibroblasts from chronic wounds are in a state of senescence, which contributes to the failure to heal. This assumption, based on limited experimental evidence, has led to the widespread use of therapeutic approaches focused on delivering new fibroblasts and/or increasing resident fibroblast activity to promote healing. In this study, we decided to re-visit the evidence for the relative inactivity of resident chronic wound fibroblasts. We therefore evaluated the proliferative and migratory activities of matching, patient-derived dermal fibroblasts from a chronic wound (wound dermal fibroblasts, or WDF), ipsilateral thigh newly created acute wound dermal fibroblasts (ADF, Day-3 after wounding the normal thigh skin), and ipsilateral thigh normal dermal skin fibroblasts (NDF). This approach was used in each of 10 consecutive non-selected individual patients with a venous leg ulcer, and allowed us to determine whether WDF are intrinsically less active than NDF and AWD. Cell migration and proliferation were quantified by a live-cell analysis system and MTT assay, respectively, in low (0.5%) or high (10%) levels of fetal bovine serum (FBS). In addition, the ability of patient-derived fibroblasts to modulate wound re-epithelialization in vivo was analyzed by transplantation in a mouse tail full-thickness wound model. Wnt5a mRNA, its ROR1 co-receptors, and ROR2 mRNA levels were determined by qRT-PCR. We report that WDF had increased -SMA and increased levels of Wnt5a. Moreover, using live-cell imaging in a scratch assay monolayer model, WDF showed baseline migratory activity similar to those of NDF and ADF, and such activity was not stimulated by FBS. WDF showed the same capacity to increase wound re-epithelialization as NDF and ADF. Together, these results suggest that WDF are not actually less "active" than NDF and ADF. This enhanced activity of chronic wound fibroblasts may lead to high energy requirements that contribute to a failure to heal. The findings may represent a new paradigm for wound chronicity, impaired healing, and high recurrence rates.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model.

ObjectiveThrough our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3a-secreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI. MethodsThirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×105 cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining. ResultsFourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups. ConclusionOur results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Insertion of gallic acid onto chitosan promotes the differentiation of osteoblasts from murine bone marrow-derived mesenchymal stem cells

Chitosan, a naturally occurring biodegradable and biocompatible polymer, has found use as a food additive, nutraceuticals, and functional foods in recent years. In this study, gallic acid-g-chitosan (GAC) was prepared by the insertion of GA onto plain chitosan (PC) via free radical-mediated grafting and its osteogenic effects were investigated in murine bone marrow-derived mesenchymal stem cells (mBMMSCs). Structural characterization of PC and GAC was performed using 1H NMR and FT-IR spectroscopy. The amount of GA successfully grafted onto PC was 111 mg GA/g GAC via the Folin-Ciocalteu's method. While PC and GAC promoted the increase in alkaline phosphatase activity and mineralization, GAC increased these factors significantly more than PC, indicating that the grafting of GA onto chitosan increased its osteogenic potential. Mechanistic study revealed that GAC activated Wnt1 and Wnt3a mRNA and protein expression as well as increased the translocation of β-catenin into the nucleus and upregulated the expression of β-catenin targeted genes including Runx2, osterix, type I collagen and cyclin D1. In addition, DKK-1, a Wnt antagonist, decreased GAC-mediated osteoblast differentiation in mBMMSCs through blocking the Wnt/β-catenin signaling pathway.

CircPVT1 promotes osteosarcoma glycolysis and metastasis by sponging miR-423-5p to activate Wnt5a/Ror2 signaling.

Osteosarcoma (OS) is the most prevalent form of bone cancer. It has a high metastatic potential and progresses rapidly. The molecular mechanisms of OS remain unclear and this study aims to examine the functional role of circPVT1 and miR‐423‐5p in OS. Quantitative RT‐PCR (qRT‐PCR) and western blotting were used to examine levels of miR‐423‐5p, circPVT1, Wnt5a, Ror2, and glycolysis‐related proteins, including HK2, PKM2, GLUT1, and LDHA. Colony formation and transwell assays were used to test the roles of miR‐423‐5p, circPVT1, and Wnt5a/Ror2 in OS cell proliferation, migration, and invasion. Dual luciferase assay and Ago2‐RIP were used to validate the interactions of miR‐423‐5p/Wnt5a, miR‐423‐5p/Ror2, and circPVT1/miR‐423‐5p. Glucose uptake assay and measurement of lactate production were performed to assess the glycolysis process. A nude mouse xenograft model was used to evaluate the effects of sh‐circPVT1 and miR‐423‐5p mimics on tumor growth and metastasis in vivo. miR‐423‐5p was reduced in both OS tissues and OS cell lines, while Wnt5a/Ror2 and circPVT1 were elevated. miR‐423‐5p bound to 3′‐UTR of Wnt5a and Ror2 mRNA, and inhibited glycolysis and OS cell proliferation, migration, and invasion by targeting Wnt5a and Ror2. circPVT1 interacted with miR‐423‐5p and activated Wnt5a/Ror2 signaling by sponging miR‐423‐5p. Knockdown of circPVT1 or overexpression of miR‐423‐5p suppressed OS tumor growth and metastasis in vivo. miR‐423‐5p inhibited OS glycolysis, proliferation, migration, and metastasis by targeting and suppressing Wnt5a/Ror2 signaling pathway, while circPVT1 promoted those processes by acting as a sponge of miR‐423‐5p.

MiR-503 Contributes to Glucocorticoid Sensitivity in Acute Lymphoblastic Leukaemia via Targeting WNT3A

Abnormal accumulation of lymphoblasts in the blood and bone marrow is the main characteristic of acute lymphoblastic leukaemia (ALL). Glucocorticoids are effective drugs for ALL, while glucocorticoid resistance is an obstacle to ALL therapy. MicroRNAs (miRNAs) are implicated in the drug resistance and modulate the response of ALL to glucocorticoids. The role of miR-503 in glucocorticoid sensitivity of ALL was investigated in this study. Firstly, T-leukaemic cells were isolated from patients with ALL. The human ALL cell line (CCRF/CEM) was incubated with dexamethasone to establish a glucocorticoid- resistant ALL cell line (CCRF/CEM-R). Data from MTT showed that IC50 (50% inhibitory concentration) of dexamethasone in T-leukaemic cells isolated from glucocorticoid-resistant ALL patients or CCRF/CEM-R was increased compared with IC50 in T-leukaemic cells isolated from glucocorticoid- sensitive ALL patients or CCRF/CEM. MiR- 503 was down-regulated in glucocorticoid-resistant leukaemic cells and CCRF/CEM-R. Secondly, overexpression of miR-503 sensitized CCRF/CEM-R to dexamethasone. Moreover, over-expression of miR- 503 also promoted the sensitivity of ALL cells to dexamethasone. Thirdly, miR-503 bound to WNT3A mRNA and negatively regulated the expression of WNT3A. Over-expression of miR-503 reduced protein expression of nuclear β-catenin, and over-expression of WNT3A attenuated the miR-503 overexpression- induced decrease in nuclear β-catenin. Lastly, the over-expression of miR-503-induced increased sensitivity of ALL-resistant cells and CCRF/ CEM-R to dexamethasone was attenuated by overexpression of WNT3A. In conclusion, miR-503 targeted WNT3A mRNA to sensitize ALL cells to glucocorticoids through inactivation of the Wnt/β-catenin pathway.

Activation of endothelial Wnt/β-catenin signaling by protective astrocytes repairs BBB damage in ischemic stroke.

The role of astrocytes in dysregulation of blood-brain barrier (BBB) function following ischemic stroke is not well understood. Here, we investigate the effects of restoring the repair properties of astrocytes on the BBB after ischemic stroke. Mice deficient for NHE1, a pH-sensitive Na+/H+ exchanger 1, in astrocytes have reduced BBB permeability after ischemic stroke, increased angiogenesis and cerebral blood flow perfusion, in contrast to wild-type mice. Bulk RNA-sequencing transcriptome analysis of purified astrocytes revealed that ∼177 genes were differentially upregulated in mutant astrocytes, with Wnt7a mRNA among the top genes. Using a Wnt reporter line, we confirmed that the pathway was upregulated in cerebral vessels of mutant mice after ischemic stroke. However, administration of the Wnt/β-catenin inhibitor, XAV-939, blocked the reparative effects of Nhe1-deficient astrocytes. Thus, astrocytes lacking pH-sensitive NHE1 protein are transformed from injurious to "protective" by inducing Wnt production to promote BBB repair after ischemic stroke.

The Expression of Wnt5a Gene Throughout Definitive Endoderm Induction Process in Induced Pluripotent Stem Cells

Background: Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely and differentiate into three germ layers of ectoderm, mesoderm, and endoderm. Definitive induction is the first and the most delicate stage of differentiation of various iPSC-derived organs. It has been found that the Wnt signaling pathway implicates in embryogenesis, organogenesis, and cell communication. Objectives: In the present study, we aimed to investigate the expression pattern of the Wnt5a gene as an indicator of non-canonical Wnt signaling activity during definitive endoderm induction of iPSCs. Methods: Human iPSCs (RSCB0042) were acquired from Royan stem cell bank of Royan Institute (Tehran, Iran). The iPSCs were cultured on a feeder layer of mitomycin-inactivated mouse embryonic fibroblasts (MEF), and iPSC colonies were collected for embryoid body (EB) generation by suspension culture method. Then endoderm induction step was performed using a series of small molecules. The quantitative real-time PCR was used to assess the mRNA expression of wnt5a, Nanog, OCT4, SOX17, and FOXA2 genes. Results: The production of efficient EBs confirmed by a decrease in Nanog and Oct4 gene expression and the success of DE (definite endoderm) induction step was confirmed by a high expression level of DE specific genes, Sox17, and FoxA2. A significant upregulation of Wnt5a in EB samples and a minor decrease at day 4 was observed. However, the differentiation process followed by an incremental fashion in Wnt5a mRNA expression starting from day 4 of differentiation among the samples of days 6 and 8 (DE stage). Conclusions: Our results suggest that Wnt5a is more activated at the later steps of endoderm induction rather than the early steps, which may be due to the stimulation of canonical Wnt signaling. Finding the expression level of Wnt5a could rise insights for developing more efficient differentiation induction protocols.

Smooth-muscle-derived WNT5A augments allergen-induced airway remodelling and Th2 type inflammation

Asthma is a heterogeneous disease characterized by chronic inflammation and structural changes in the airways. The airway smooth muscle (ASM) is responsible for airway narrowing and an important source of inflammatory mediators. We and others have previously shown that WNT5A mRNA and protein expression is higher in the ASM of asthmatics compared to healthy controls. Here, we aimed to characterize the functional role of (smooth muscle-derived) WNT5A in asthma. We generated a tet-ON smooth-muscle-specific WNT5A transgenic mouse model, enabling in vivo characterization of smooth-muscle-derived WNT5A in response to ovalbumin. Smooth muscle specific WNT5A overexpression showed a clear trend towards enhanced actin (α-SMA) expression in the ASM in ovalbumin challenged animals, but had no effect on collagen content. WNT5A overexpression in ASM also significantly enhanced the production of the Th2-cytokines IL4 and IL5 in lung tissue after ovalbumin exposure. In line with this, WNT5A increased mucus production, and enhanced eosinophilic infiltration and serum IgE production in ovalbumin-treated animals. In addition, CD4+ T cells of asthma patients and healthy controls were stimulated with WNT5A and changes in gene transcription assessed by RNA-seq. WNT5A promoted expression of 234 genes in human CD4+ T cells, among which the Th2 cytokine IL31 was among the top 5 upregulated genes. IL31 was also upregulated in response to smooth muscle-specific WNT5A overexpression in the mouse. In conclusion, smooth-muscle derived WNT5A augments Th2 type inflammation and remodelling. Our findings imply a pro-inflammatory role for smooth muscle-derived WNT5A in asthma, resulting in increased airway wall inflammation and remodelling.

Expression of Notch and Wnt/β-catenin signaling pathway in acute phase severe brain injury rats and the effect of exogenous thyroxine on those pathways.

With the rapid development of economy, transportation and industry, the incidence of severe traumatic brain injury (sTBI) is rising rapidly, which is one of the main traumatic diseases threatening human life. It is very difficult for sTBI patients to regenerate and repair the central nervous and recover the brain function. Moreover, no effective neuroprotective drug has been found in the treatment of sTBI patients. Seeking drugs to promote nerve repair has become a hot and difficult problem. It is widely accepted that thyroxine is one of the essential hormones in the human body, which not only promotes the growth and development of the nervous system, but also plays an important role in maintaining adult brain function. There are many reports of modern research on thyroxine, mainly focusing on the changes of thyroid hormone levels and their effects on the prognosis after injury. Besides, most of them are observed in clinical cases. Currently, there are few dynamic experimental studies about observing whether thyroxine can promote the repair of central nervous system at different stages after sTBI. In our previous experiment, we found that Wnt/β-catenin signaling pathway, whose functions are opposite to Notch signaling pathway, can be further activated by exogenous thyroxine in rats with sTBI. As a result, we are interested in the expression of Notch and Wnt/β-catenin signaling pathway in acute phase sTBI rats and the effect of thyroxine on those pathways. To investigate expression of Notch and Wnt/β-catenin signaling pathway in acute phase severe brain injury rats and the effect of thyroxine on those pathways by observing dynamically Notch and Wnt/β-catenin signaling pathway, NSS, GFAP, S100B, Bcl-2, Bax, etc. METHODS: 108 rats were randomly divided into Group A (normal control group), Group B (normal-thyroxine group), Group C (TBI group), Group D (TBI+ low-dose thyroxine group), Group E (TBI + moderate-dose thyroxine) and Group F (TBI + high-dose thyroxine) with 18 rats in each group. The animal model was established according to Feeney's free-fall method, and administered with thyroxine or physiological saline at 6 h after sTBI. Six rats in each group were randomly killed on the 1st, 3rd and 7th days after intragastric administration. The changes of brain pathology and NSS were observed. The level of Wnt3a, β-catenin, Notch1 and Hes1 mRNA was detected by RT-PCR method, and the level of GFAP and S100B protein in serum was detected by ELISA. The expression of Bcl-2 and Bax was detected by immunohistochemistry. (1) There was no significant change in brain pathology and NSS in groups A and B, but the changes of brain pathology and NSS in group D, E and F were significantly less than those in group C, especially in groups E and F. (2) RT-PCR showed that there was no change in the expression of Wnt3a mRNA, β-catenin mRNA, Notch1 and Hes1 mRNA in groups A and B. Compared with group C, the expression of Wnt3a mRNA and β-catenin mRNA in group D increased significantly on the 7th day after sTBI, especially in groups E and F; expression of Notch1 and Hes1 mRNA in groups D, E and F increased gradually with time, especially in group F. (3) ELISA showed that Compared with group C, GFAP and S100B in group D did not change significantly at 3 time points, GFAP in groups E and F decreased gradually with time and reached the lowest value on the 7th day, and S100B in groups E and F decreased gradually with time, especially in group F. (4) Compared with group C, the expression of BCL-2 in brain tissue of groups D, E and F increased gradually with time, and peaked on the 7th day, and the increase of E and F was more obvious. The expression of Bax in brain tissue of group D, E and F decreased gradually with time. Exogenous thyroxine has no effect on Notch and Wnt/β-catenin signaling pathway in normal rats. After TBI, exogenous thyroxine can activate Notch and Wnt/β-catenin, and have a synergistic effect on the repair of central nervous system, which may be related to the up-regulation of Notch and Wnt/β-catenin signaling pathway mRNA expression and the increase of BDNF and NGF, and resist apoptosis in the brain of sTBI rats.

Repairing effects of glucosamine sulfate in combination with etoricoxib on articular cartilages of patients with knee osteoarthritis

PurposeTo evaluate the repairing effects of glucosamine sulfate combined with etoricoxib on articular cartilages of patients with knee osteoarthritis (KOA).MethodsA total of 106 KOA patients were randomly divided into control (n = 40) and experimental groups (n = 66) and treated with etoricoxib alone and glucosamine sulfate plus etoricoxib, respectively. Changes in WOMAC score and clinical efficacy were observed. The synovial fluid was extracted. Bone metabolism indices, growth factors, inflammatory factors, matrix metalloproteinases (MMPs), and NO-induced apoptosis-related factors were measured by ELISA. JNK and Wnt5a mRNA levels were determined using RT-PCR.ResultsAfter treatment, the total WOMAC scores of both groups significantly declined (P < 0.05), being lower in experimental group. The total effective rate of experimental group was higher (P < 0.05). BGP and OPG levels rose, especially in experimental group (P < 0.05). CTX-II, COMP, and RANKL levels decreased, particularly in experimental group (P < 0.05). TGF-β, IGF-1, and FGF-2 levels increased, especially in experimental group (P < 0.05). Both groups, particularly experimental group, had decreased levels of IL-1β, IL-17, IL-18, TNF-α, MMP-3, MMP-9, and MMP-13 (P < 0.05). JNK and Wnt5a mRNA levels of both groups dropped, which were lower in experimental group (P < 0.05). NO and LPO levels reduced, being lower in experimental group. SOD level rose, especially in experimental group (P < 0.05).ConclusionGlucosamine sulfate plus etoricoxib can repair the articular cartilages of KOA patients. Probably, JNK and Wnt5a are downregulated to inhibit the secretion of MMPs through lowering the levels of inflammatory factors, thereby delaying cartilage matrix degradation. NO-induced chondrocyte apoptosis may be suppressed via the SOD pathway.

Effect of electroacupuncture on neurological function and Wnt signaling pathway in ischemic brain tissue of cerebral infarction rats

To explore the mechanism of electroacupuncture (EA) underlying improvement of cerebral infarction (CI) by investigating its influence on expression of cerebral Wnt7a, lymphoid enhancer factor-1 (LEF1), glycogen synthase kinase 3β(GSK-3β) and Dickkopf-1(DKK1) mRNA and proteins in CI rats. A total of 280 male Wistar rats were randomly divided into blank control (n＝10), sham-operation, model and EA groups，and 90 rats of the last 3 groups were further divided into 1, 3, 6, 9, 12 and 24 h, and 3, 7 and 12 d subgroups with 10 rats in each subgroup. The CI model was established by occlusion of the middle cerebral artery (MCAO). The sham-operation group received the same surgical operation but without thread embolus insertion. EA (2 Hz/15 Hz, 2 mA) was applied to "Shuigou" (GV26) for 20 min, once a day for 1, 3, 7 and 12 d, respectively. The neurological deficit was evaluated by using Neurological Severity Scores (NSS). The expression levels of Wnt7a，LEF1, GSK-3β and DKK1 mRNAs and proteins in the right ischemic brain tissues were detected by Quantative real-time PCR and Western blot, respectively. After MCAO, the NSS score was significantly increased in the model and EA groups relevant to the blank control and sham-operation groups (P<0.01) and gradually decreased with the prolongation of ischemia time. After EA, the NSS scores were notably decreased on day 3, 7 and 12 in the EA group compared with the model group (P<0.05, P<0.01). After modeling, the expression levels of Wnt7a and LEF1 mRNAs from 3 h to 12 d, Wnt7a and LEF1 proteins from 6 h to 12 d were considerably increased (P<0.01, P<0.05), while those of GSK-3β mRNA at 9, 12 and 24 h, GSK-3β protein at 24 h and 3 d, and DKK1 mRNA at 24 h and 3 d and DKK1 protein at 3 d were obviously decreased in the model group relevant to the sham-operation group (P<0.05, P<0.01). After the intervention, the expression levels of Wnt7a mRNA at 12 h to 3 d, Wnt7α protein from 24 h to 12 d, LEF1 mRNA from 24 h to 12 d, and LEF1 protein from 3 d to 12 d were further apparently up-regulated (P<0.01, P<0.05), while those of GSK-3β mRNA at 9 h, 3，7 and 12 d, and GSK-3β protein at 12 h, 7 d and 12 d, and DKK1 mRNA at 12 h, 24 h and 3 d, and DKK1 protein at 24 h to 12 d were obviously down-regulated in the EA group relevant to the model group (P<0.01, P<0.05). No significant difference was found between the blank and sham-operation groups in the NSS scores and expression levels of Wnt7a, LEF1, GSK-3β and DKK1 mRNAs and proteins at all the time points (P>0.05). EA of GV26 can significantly improve the neurological deficit symptoms in MCAO rats, which may be associated with its effects in up-regulating the expression of Wnt7a and LEF1 mRNAs and proteins, and in down-regulating the expression of GSK-3β and DKK1 mRNAs and proteins.

WNT7A Promotes EGF-Induced Migration of Oral Squamous Cell Carcinoma Cells by Activating β-Catenin/MMP9-Mediated Signaling.

Aims and hypothesisEpidermal growth factor (EGF) has been shown to induce the migration of various cancer cells. However, the underlying signaling mechanisms for EGF-induced migration of oral squamous cell carcinoma (OSCC) remain to be elucidated. WNT7A, a member of the family of 19 Wnt secreted glycoproteins, is commonly associated with tumor development. It is mostly unknown whether and, if so, how EGF modulates WNT7A in OSCC cells. The role of WNT7A in OSCC was thus investigated to explore the underlying signaling mechanisms for EGF-induced migration of OSCC.MethodsCell migration was measured by Wound healing assay and Transwell assay. Western blotting was carried out to detect the expression of WNT7A, MMP9, β-catenin, p-AKT, and p-ERK. The cells were transfected with plasmids or siRNA to upregulate or downregulate the expression of WNT7A. The location of β-catenin was displayed by immunofluorescence microscopy. Immunohistochemistry was carried out to confirm the relation between WNT7A expression and OSCC progression.ResultsThe present study showed that the levels of WNT7A mRNA and protein were increased by EGF stimulation in OSCC cells. Besides, it was proved that p-AKT, but not p-ERK, mediated the expression of WNT7A protein induced by EGF. Furthermore, the inhibition of AKT activation prevented the EGF-induced increase of WNT7A and matrix metallopeptidase 9 (MMP9) expression and translocation of β-catenin from the cytoplasm to the nucleus. Moreover, histological analysis of OSCC specimens revealed an association between WNT7A expression and poor clinical prognosis of the disease.ConclusionsThe data in this paper indicated that WNT7A could be a potential oncogene in OSCC and identified a novel PI3K/AKT/WNT7A/β-catenin/MMP9 signaling for EGF-induced migration of OSCC cells.

BRD7-Mediated miR-3148 Inhibits Progression of Cervical Cancer by Targeting Wnt3a/β-Catenin Pathway.

Abnormal expression of miRNAs is closely related to the occurrence and development of tumors, and thus has become the most concerned biomolecule in the field of tumors. But so far, miRNAs that are truly recognized and studied for their function are only a small part, and their mechanism in tumors needs to be further studied. In this study, we identify that miR-3148 is downregulated in the development of cervical cancer. In cervical cancer cells, upregulated miR-3148 inhibits cell proliferation and promotes apoptosis, suggesting that miR-3148 acts as a tumor suppressor gene. Furthermore, we explored the mechanism of miR-3148 in cervical cancer cells from both transcriptional and post-transcriptional levels. Our research reveals that on the one hand, bromodomain containing 7 (BRD7) acts as a transcription factor to up-regulate the expression of miR-3148 at the transcriptional level; on the other hand, miR-3148 targets the 3'UTR of Wnt3a mRNA to inhibit Wnt3a expression at the post-transcriptional level, thereby suppressing Wnt3a/β-catenin signaling pathway and exerting its tumor suppressor role in cervical cancer. In conclusion, our study elucidates the mechanism of BRD7/miR-3148/Wnt3a/β-catenin pathway in cervical cancer and provides a new research direction for targeted therapy of cervical cancer.

STAT3-Induced Wnt5a Provides Chronic Lymphocytic Leukemia Cells with Survival Advantage.

The wingless and integration site growth factor-5a (Wnt5a) is a ligand of the receptor tyrosine kinase-like orphan receptor-1 (ROR1). Because both Wnt5a and ROR1 are expressed in circulating chronic lymphocytic leukemia (CLL) cells, and because in other cell types, STAT3, which is constitutively activated in CLL, induces Wnt5a signaling, we wondered whether STAT3 induces the expression of Wnt5a in CLL cells. Sequence analysis detected four putative STAT3 binding sites in close proximity to the Wnt5a gene promoter's start codon. Chromatin immunoprecipitation and EMSA revealed that STAT3 binds to the Wnt5a gene promoter, and a luciferase assay showed that STAT3 activates the Wnt5a gene. Additionally, transfection of peripheral blood CLL cells with STAT3 short hairpin RNA downregulated Wnt5a mRNA and protein levels, suggesting that STAT3 binds to the Wnt5a gene promoter and induces the expression of Wnt5a in CLL cells. Flow cytometry and confocal microscopy determined that both Wnt5a and its receptor ROR1 are coexpressed on the surface of CLL cells, and Western immunoblotting showed an inverse correlation between Wnt5a and ROR1 protein levels, implying that, regardless of CLL cells' ROR1 levels, blocking the interaction between Wnt5a and ROR1 might be beneficial to patients with CLL. Indeed, transfection of CLL cells with Wnt5a small interfering RNA reduced Wnt5a mRNA and protein levels and significantly increased the spontaneous apoptotic rate of CLL cells. Taken together, our data unravel an autonomous STAT3-driven prosurvival circuit that provides circulating CLL cells with a microenvironment-independent survival advantage.

Wnt5a is a transcriptional target of Gli3 and Trps1 at the onset of chondrocyte hypertrophy

During endochondral ossification, the differentiation of proliferating into hypertrophic chondrocytes is a key step determining the pace of bone formation and the future length of the skeletal elements. A variety of transcription factors are expressed at the onset of hypertrophy coordinating the expression of different signaling molecules like Bmps, Ihh and Wnt proteins. In this study, we characterized the murine Wnt5a promoter and provide evidence that two alternative Wnt5a transcripts, Ts1 and Ts2, are differentially expressed in the developing skeletal elements. Ts2 expression decreases while Ts1 expression increases during chondrocyte differentiation. The transcription factor Trps1 and the activator form of Gli3 (Gli3A), which is a mediator of Hedgehog signaling, activate Wnt5a expression. In Chromatin Immunoprecipitation and reporter gene assays, we identified two upstream regulatory sequences (URS) in the Wnt5a promoter mediating either activating or repressive functions. The activating URS1 is bound by Trps1 and Gli3A in vitro and in vivo to upregulate Wnt5a expression. Loss of both transcription factors decreases endogenous Wnt5a mRNA and protein levels during chondrocyte differentiation, thereby identifying Wnt5a as a target gene of Trps1 and Gli3A in chondrocytes.

PF363 UNDETECTABLE EXPRESSION OF WNT5A, A GENE ENCODING A ROR1 LIGAND, DEFINES MEMORY B-CELL LIKE CHRONIC LYMPHOCYTIC LEUKEMIA

Background: Chronic lymphocytic leukemia (CLL) is a malignancy with highly variable disease course among patients. Although many approaches distinguishing patients with worse and better prognosis have been published, IGHV somatic hypermutation status remains one of the strongest prognostic predictors. However, even within prognostically favorable IGHV-mutated (M-CLL) group, there is a subset of “grey-zone” patients with worse prognosis. We have shown recently that expression of WNT5A, a gene encoding a ligand activating WNT/PCP signaling pathway via ROR1 receptor, distinguishes the cases with worse prognosis within M-CLL (Janovska, Clin Cancer Res 2016). Still, over 80% of M-CLL patients had undetectable WNT5A expression, suggesting possible silencing of the gene expression through its methylation. Aims: To analyze methylation status of WNT5A promoter in the context of WNT5A mRNA expression, IGHV somatic hypermutations and methylation-based CLL classification (Querios, Leukemia 2015). Methods: Using bisulphite sequencing we analyzed methylation status of 3 CpG islands (CGI) located in WNT5A promoter and 5 marker CpG sites distinguishing methylation-based prognostic subgroups of CLL (located in B3GNTL1, CTBP2, TNF, SCARF1 and intergenic region on chromosome 14). mRNA expression was analyzed with quantitative real-time PCR using TaqMan probes (ThermoFisher Scientific): WNT5A, and HPRT1, TBP were used as controls. Results: Thirty-nine previously untreated CLL patients were chosen for the analysis. They were equally distributed among 4 groups based on IGHV status and WNT5A expression [WNT5A- M-CLL (10), WNT5A+ M-CLL (9), WNT5A- IGHV-unmutated CLL (U-CLL; 10), and WNT5A+ U-CLL (10), respectively]. Within the first of the three CpG islands of the WNT5A promoter (CGI1), we identified three CpG sites whose methylation status significantly correlated with the WNT5A expression in M-CLL (P = 0,004; P = 0,004; P = 0,0268; Mann-Whitney test). Furthermore, complete demethylation of the three CpG sites was associated with high WNT5A expression in M-CLL. In fact, complete demethylation of the whole tested CGI1 region was noted in these patients. Methylation analysis of 5 marker CpGs (Querios, Leukemia 2015) enabled us to classify patients into three methylation-based prognostic groups: 9/10 WNT5A- M-CLL patients belonged to the memory B-cell like CLL (m-CLL) group, and 7/9 WNT5A+ M-CLL patients to the intermediate CLL (i-CLL). U-CLL patients irrespective of WNT5A expression were assigned as naive B-cell like CLL (n-CLL) in 19/20 cases.Summary/Conclusion: Within M-CLL, methylation of the 3 CpGs located in the CGI1 of WNT5A promoter correlated significantly with WNT5A expression. Higher WNT5A expression was associated with complete demethylation of CGI1 locus. Our classification based on WNT5A expression and IGHV status overlapped with the methylation-profile-based CLL classification (m-CLL, i-CLL, n-CLL). Furthermore, memory B-cell like CLL group was associated with undetectable WNT5A level in our cohort. Supported by grants: MUNI/A/1105/2018, CEITEC2020 LQ1601, MZCR-RVO 65269705. Spanish Ministry of Health, Plan Estatal de I + D + I, ISCIII, FEDER, FIS PI15/01763ISIII PI15/01763 and PI18/01484.

Effect of exogenous thyroxine on regeneration and repair of central nervous system in rats with severe traumatic brain injury

Objective To investigate the effect of exogenous thyroxine on the regeneration and repair of central nervous system in rats with severe traumatic brain injury. Methods A total of 108 male SD rats were randomly divided into Group A (normal group), Group B (thyroxine group), Group C (TBI group), Group D [TBI+ low dose of thyroxine (6.5 μg/kg) group], Group E [TBI+ medium dose of thyroxine (10 μg/kg) group)] and Group F [TBI+ high dose of thyroxine (15.4 μg/kg)] by random number table method, with 18 rats in each group. The animal model was established according to the Feeney method. Groups A and C were given saline and thyroxine 6 hours after TBI. At 1 day, 3 days and 7 days after injury, six rats were selected from each group according to random number table method for brain tissue harvest. The pathological changes of brain tissues were observed, and the expressions of Wnt3a and β-catenin, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) were detected by RT-PCR and immunohistochemistry respectively. Results (1) There were no pathological changes in brain tissue in Groups A and B. The pathological changes in brain tissue of TBI rats in Groups D, E and F were significantly mitigated than those in Group C, and the effect of Groups E and F were more obvious. (2) RT-PCR showed that the expressions of Wnt3a and β-catenin mRNA in hippocampus of Group D at 7 days after injury were significantly higher [(6.46±2.02) vs. (4.08±1.06); (13.53±1.64) vs. (2.11±0.63); P<0.05); Wnt3a mRNA expression of Groups E and F were increased significantly at 3 days and 7 days after injury [(5.45±1.54) vs. (2.78±1.04), (8.59±1.88) vs. (4.08±1.06), (5.40±1.38) vs. (2.78±1.04), (8.63±1.74) vs. (4.08±1.06); P<0.05)]. The expressions of β-catenin mRNA were increased significantly at 1 day, 3 days, and 7 days after injury [(3.10±0.59) vs. (1.45±0.28), (5.28±0.62) vs. (1.45±0.28), (4.12±0.43) vs. (1.64±0.31), (8.78±0.92) vs. (1.64±0.31), (23.80±2.38)vs. (2.11±0.63), (25.94±3.79) vs. (2.11±0.63); P<0.05)]. The expressions of Wnt3a and β-catenin in hippocampus of Group B did not change significantly. (3) Immunohistochemical staining showed that the expression of BDNF and NGF in hippocampus of B did not change significantly after thyroxine treatment, but that of BDNF and NGF in Group D did not change significantly after treatment. The expressions of BDNF and NGF of Groups E and F were significantly higher than that in Group C and reached the peak at 3 days after injury [BDNF: (77.3±5.4) vs. (55.0±5.6), (78.0±7.8) vs. (55.0±5.6); NGF: (83.7±5.8) vs. (62.2±10.3), (86.8±4.8) vs. (62.2±10.3); P<0.05]. Conclusions Exogenous thyroxine has no effect on the Wnt/β-catenin pathway in normal rats. But exogenous thyroxine can significantly promote the centralis regeneration and repair in TBI rats, which might be associated with its role in upregulating the expression of mRNA and increasing the secretion of BDNF and NGF. Key words: Thyroxine; Brain injuries; Nerve regeneration; Wnt/β-catenin signaling pathway