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Background: Evidence suggests that cortical anatomy may be aytpical in autism spectrum disorder. The wingless-type MMTV integration site family, member 2 (WNT2), a candidate gene for autism spectrum disorder, may regulate cortical development. However, it is unclear whether WNT2 variants are associated with altered cortical thickness in autism spectrum disorder. Methods: In a sample of 118 people with autism spectrum disorder and 122 typically developing controls, we investigated cortical thickness using FreeSurfer software. We then examined the main effects of the WNT2 variants and the interactions of group × SNP and age × SNP for each hemisphere and brain region that was altered in people with autism spectrum disorder. Results: Compared to neurotypical controls, people with autism spectrum disorder showed reduced mean cortical thickness in both hemispheres and 9 cortical regions after false discovery rate correction, including the right cingulate gyrus, the orbital gyrus, the insula, the inferior frontal gyrus (orbital part and triangular part), the lateral occipitotemporal gyrus, the posterior transverse collateral sulcus, the lateral sulcus and the superior temporal sulcus. In the full sample, 2 SNPs of WNT2 (rs6950765 and rs2896218) showed age × SNP interactions for the mean cortical thickness of both hemispheres, the middle-posterior cingulate cortex and the superior temporal cortex. Limitations: We examined the genetic effect for each hemisphere and the 9 regions that were altered in autism spectrum disorder. The age effect we found in this cross-sectional study needs to be examined in longitudinal studies. Conclusion: Based on neuroimaging and genetic data, our findings suggest that WNT2 variants might be associated with altered cortical thickness in autism spectrum disorder. Whether and how these WNT2 variants might involve cortical thinning requires further investigation. Trial registration: ClinicalTrials.gov no. NCT01582256. Protocol registration: National Institutes of Health no. NCT00494754.

The aim of this paper was to explore the role and mechanism of circHIPK3 in unexplained recurrent spontaneous abortion (URSA). The expression of circHIPK3 and miR-30a-3p mRNA in URSA villous tissues was detected by quantitative polymerase chain reaction. The effects of circHIPK3 on the proliferation and migration of villous trophoblasts were analysed by MTTassay and scratch assay. Hoechst/PI staining was used to detect the effect of circHIPK3 on villous trophoblast apoptosis. The binding of circHIPK3 to miR-30a-3p and miR-30a-3p to Wnt2 was analysed by dual-luciferase assay. When URSA occurred, the expression level of circHIPK3 was downregulated, while the expression level of miR-30a-3p was upregulated in villous trophoblasts. Inhibition of circHIPK3 in villous trophoblasts can reduce the proliferation and migration of villous trophoblasts while promoting their apoptosis. The dual-luciferase assay showed that circHIPK3 was able to interact with miR-30a-3p and increased the miR-30a-3p expression after inhibition of circHIPK3, and if miR-30a-3p was also inhibited it was able to reverse the effect of circHIPK3 on villous trophoblast proliferation and migration. It was demonstrated by prediction and dual-luciferase assay that miR-30a-3p binds to Wnt2, and when miR-30a-3p and Wnt2 are inhibited simultaneously, it has an inhibitory effect on the proliferation and migration process of villous trophoblasts. Downregulation of circHIPK3 expression in URSA leads to increased expression of miR-30a-3p, which in turn inhibits the expression of target gene Wnt2 and exerts a weakening effect on the proliferation and migration process of trophoblasts, thereby decreasing trophoblast invasiveness and shallow placental implantation, which in turn leads to recurrent spontaneous abortion.

Zika Virus (ZIKV) belongs to the family of flaviviruses, and is neurotrophic. It has been known to cause severe congenital disabilities including microcephaly in neonates. The virus has a specific preference towards neural stem cells (NSCs). ZIKV impairs proliferation and differentiation of NSCs during in-utero brain development of the fetus. However, molecular pathways involved in ZIKV induced alteration in NSCs are yet to be explored. In our previous study, we have described that ZIKV E protein dysregulates microRNA circuitry in NSCs and also impairs their proliferative and differentiation abilities. WNT signalling was found to be the target of differentially expressed miRNAs as suggested by PANTHER PATHWAY analysis of differentially expressed miRNA targets. In our current follow-up study, we investigate that WNT2 is downregulated in response to ZIKV E protein in human fetal NSCs and WNT2 is the molecular target of microRNA miR-204-5p. We provide pieces of evidences that miR-204-5p/WNT2 axis is involved in ZIKV induced impairment in the proliferation and immature differentiation of neural stem cells.

COL10A1 is a secreted, short-chain collagen found in several types of cancer. Studies have shown that COL10A1 aberrant expression is considered an oncogenic factor. However, its underlying mechanisms and regulation of gastric cancer remain undefined.The data on the expression of COL10A1, clinicopathological characteristics, and outcome of patients with GC were obtained from The Cancer Genome Atlas. The ALGGEN-PROMO database defined the related transcription factors. Quantitative real-time reverse transcription-polymerase chain reaction and western blotting analysis were used to identify the differential expression levels of COL10A1 and related transcription factors.We found that high COL10A1 expression is an independent risk factor for gastric cancer. Upregulation of LEF1 and Wnt2 was also observed in gastric cancer, suggesting a potential correlation between LEF1/COL10A1 regulation in the Wnt2 signaling pathway.High COL10A1 expression may contribute to poor outcomes via upregulation of LEF1 and Wnt2 in gastric cancer.

This study aimed to explore the role and underlying mechanism of action of Endoplasmic reticulum oxidoreductin-1 L (ERO1L) in lung adenocarcinoma (LUAD).The Gene expression profiling interactive analysis database was used to analyze the expression of ERO1L in LUAD cases. The expression of ERO1L and Wnt2 in LUAD tissue was evaluated using immunohistochemistry. We also used western blotting to assess the expression of ERO1L or Wnt2 and the phosphorylation of β-catenin in LUAD cell lines. Plasmid transfection and small interfering RNA were used for overexpression and knockdown of ERO1L in LUAD cells, respectively. The proliferation, invasion and migration of LUAD cells were analyzed using cell viability, Transwell invasion and wound healing assays. The growth of LUAD tumors in animal models was assessed using tumor xenograft experiments.This study revealed that elevated ERO1L expression was associated with a poor prognosis in LUAD patients. In addition, ERO1L expression was significantly associated with lymph-node metastasis, TNM stage and tumor size. The expression of Wnt2 was positively associated with ERO1L expression in LUAD tissue samples and cell lines. ERO1L overexpression upregulated the expression of Wnt2 and β-catenin phosphorylation in vitro. Additionally, ERO1L was essential for the ubiquitination of Wnt2. Last, ERO1L promoted the proliferation and metastasis of LUAD via the Wnt2 signaling pathway in vitro and in vivo.These findings suggest that ERO1L was highly expressed in LUAD tissue, and it promoted the proliferation and metastasis of LUAD by activating the Wnt2/β-catenin signaling pathway.

ObjectiveSolid tumours respond poorly to immune checkpoint inhibitor (ICI) therapies. One major therapeutic obstacle is the immunosuppressive tumour microenvironment (TME). Cancer-associated fibroblasts (CAFs) are a key component of the TME and negatively regulate antitumour T-cell response. Here, we aimed to uncover the mechanism underlying CAFs-mediated tumour immune evasion and to develop novel therapeutic strategies targeting CAFs for enhancing ICI efficacy in oesophageal squamous cell carcinoma (OSCC) and colorectal cancer (CRC).DesignAnti-WNT2 monoclonal antibody (mAb) was used to treat immunocompetent C57BL/6 mice bearing subcutaneously grafted mEC25 or CMT93 alone or combined with anti-programmed cell death protein 1 (PD-1), and the antitumour efficiency and immune response were assessed. CAFs-induced suppression of dendritic cell (DC)-differentiation and DC-mediated antitumour immunity were analysed by interfering with CAFs-derived WNT2, either by anti-WNT2 mAb or with short hairpin RNA-mediated knockdown. The molecular mechanism underlying CAFs-induced DC suppression was further explored by RNA-sequencing and western blot analyses.ResultsA negative correlation between WNT2+ CAFs and active CD8+ T cells was detected in primary OSCC tumours. Anti-WNT2 mAb significantly restored antitumour T-cell responses within tumours and enhanced the efficacy of anti-PD-1 by increasing active DC in both mouse OSCC and CRC syngeneic tumour models. Directly interfering with CAFs-derived WNT2 restored DC differentiation and DC-mediated antitumour T-cell responses. Mechanistic analyses further demonstrated that CAFs-secreted WNT2 suppresses the DC-mediated antitumour T-cell response via the SOCS3/p-JAK2/p-STAT3 signalling cascades.ConclusionsCAFs could suppress antitumour immunity through WNT2 secretion. Targeting WNT2 might enhance the ICI efficacy and represent a new anticancer immunotherapy.

The conclusive identity of Wnts regulating liver zonation (LZ) and regeneration (LR) remains unclear despite an undisputed role of β-catenin. Using single-cell analysis, we identified a conserved Wnt2 and Wnt9b expression in endothelial cells (ECs) in zone 3. EC-elimination of Wnt2 and Wnt9b led to both loss of β-catenin targets in zone 3, and re-appearance of zone 1 genes in zone 3, unraveling dynamicity in the LZ process. Impaired LR observed in the knockouts phenocopied models of defective hepatic Wnt signaling. Administration of a tetravalent antibody to activate Wnt signaling rescued LZ and LR in the knockouts and induced zone 3 gene expression and LR in controls. Administration of the agonist also promoted LR in acetaminophen overdose acute liver failure (ALF) fulfilling an unmet clinical need. Overall, we report an unequivocal role of EC-Wnt2 and Wnt9b in LZ and LR and show the role of Wnt activators as regenerative therapy for ALF.

WNT family genes have participated in the progression and development of many cancers, however, the association between colon adenocarcinoma (COAD) and WNTs have been rarely reported. This study investigated the significance of WNT genes expression in COAD from the standpoint of diagnosis and prognosis. The RNA-sequencing dataset of COAD was downloaded from The Cancer Genome Atlas and University of California, Santa Cruz Xena browser. The biology functions of WNT genes were investigated by biological analysis. Biological analysis of WNT family genes indicated that WNT genes were noticeably enriched in the complex process of WNT signaling pathway. The Pearson correlation analysis suggested WNT1 and WNT9B had a strong correlation. And receiver operating characteristic curves suggested that most of the genes could serve as a significant diagnostic makers in COAD (P < .05), especially WNT2 and WNT7B had high diagnostic values that the area under curve were 0.997 (95% confidence interval [0.994-1.000]) and 0.961 (95%CI [0.939-0.983]), respectively. And our multivariate survival analysis suggested the downregulated of WNT10B (P < .05) showed a favor prognosis in COAD overall survival. And the risk score model predicted that the upregulated expression of WNT10B might increase the risk of death. The very study we had conducted suggested that WNT genes had a certain connection with the diagnosis and prognosis of COAD. The messenger RNA expression of WNT2 and WNT7B might become potentially diagnostic biomarkers, and WNT10B might serve as an independent prognosis indicator for COAD.

Esophageal squamous cell carcinoma (ESCC) is an aggressive form of human squamous cell carcinomas with extremely aggressive pathological features. This study explores the functions of microRNA-149 (miR-149) and its interacted molecules in ESCC. The ESCC-related miRNA and messenger RNA (mRNA) datasets were applied to identify aberrantly expressed genes in ESCC. Forty-two patients with ESCC were included and their tissue samples were collected. miR-149 was poorly expressed whereas DNA methyltransferase 3 beta (DNMT3B) and ring finger protein 2 (RNF2) were abundantly expressed in ESCC tumor samples. Overexpression of miR-149 suppressed growth and invasiveness of ESCC cells in vitro and in vivo. DNMT3B bound to the promoter region of miR-149 to trigger its promoter methylation and downregulation. RNF2 mRNA was a target of miR-149. RNF2 overexpression blocked the inhibitory effect of miR-149 on ESCC cell growth. RNF2 activated the Wnt/β-catenin pathway to promote ESCC development. In conclusion, this study found that DNMT3B downregulates miR-149 level through methylation modification of the miR-149 promoter, while miR-149 suppresses RNF2 expression and inactivates the Wnt/β-catenin pathway to suppress growth of ESCC cells.

The hair follicle (HF) growth cycle is a complex, multistep biological process, for which dysfunction affects hair-related diseases in humans and wool production in animals. In this study, a treatment combination of 10 ng/mL insulin-like growth factor-1 (IGF-1) and 20 ng/mL epidermal growth factor (EGF) significantly increased the elongation length of hair shafts for cultured HFs. The combined treatment of IGF-1 and EGF enhanced the proliferation of HFs and promoted HF growth and development in vitro. In vivo, the combined treatment of IGF-1 and EGF was subcutaneously injected into the dorsal skin in HF synchronized rabbits. The IGF-1 and EGF combination promoted the transition of the hair cycle from telogen to anagen and stimulated the growth of hair shafts. This IGF-1 and EGF combination maintained the structure of the HF and enhanced the cell proliferation of outer root sheaths and the dermal papilla within rabbit skin. The combined treatment of IGF-1 and EGF regulated HF-related genes, including LEF1, CCND1 and WNT2, suggesting that IGF-1 and EGF play a positive role in HF growth and development. Utilization of the combined IGF-1 and EGF treatment may assist with hair and wool production and HF related diseases in mammals.

Atherosclerosis, is a chronic inflammatory disease, characterized by the narrowing of the arteries resulting from the formation of intimal plaques in the wall of arteries. Yet the molecular mechanisms responsible for maintaining the development and progression of atherosclerotic lesions have not been fully defined. In this study, we show that TGF-β activates the endothelial-to-mesenchymal transition (EndMT) in cultured human aortic endothelial cells (HAECs) and this transition is dependent on the key executor of the Wnt signaling pathway in vitro. This study presents the first evidence describing the mechanistic details of the TGF-β-induced EndMT signaling pathway in HAECs by documenting the cellular transition to the mesenchymal phenotype including the expression of mesenchymal markers α-SMA and PDGFRα, and the loss of endothelial markers including VE-cadherin and CD31. Furthermore, a short hairpin RNA (shRNA) screening revealed that Wnt2 signaling is required for TGF-β-mediated EndMT of HAECs. Also, we found that LDLR−/− mice fed on a high-fat western-type diet (21% fat, 0.2% cholesterol) expressed high levels of Wnt2 protein in atherosclerotic lesions, confirming that this signaling pathway is involved in atherosclerosis in vivo. These findings suggest that Wnt2 may contribute to atherosclerotic plaque development and this study will render Wnt2 as a potential target for therapeutic intervention aiming at controlling atherosclerosis.

The role of Wnt family proteins, E-cadherin, and β-catenin in non-small cell lung cancer (NSCLC) is unclear. In this study, we assessed the expression of these proteins as well as their reciprocal interaction and clinical relevance in NSCLC. Immunohistochemical expression of Wnt1, Wnt2, E-cadherin, and β-catenin was assessed in 208 patients with NSCLC who underwent curative pulmonary resection. Expression of Wnt1, Wnt2, and E-cadherin was found in 49.5%, 22.3%, and 37.4% of the patients, respectively, whereas expression of membranous and cytoplasmic β-catenin was found in 23.7% and 34.8% of the patients, respectively. The expression of Wnt1 and E-cadherin was lower in squamous cell carcinoma than in adenocarcinoma and large cell carcinoma, and the expression of both Wnt proteins, E-cadherin, and membranous β-catenin was lower in poorly differentiated compared with well-differentiated tumors. None of the analyzed proteins was associated with relapse-free or overall survival. Expression of Wnt1, Wnt2, E-cadherin, and β-catenin is a common occurrence in NSCLC and is related to tumor histology and grade. However, these proteins have no prognostic role in operable NSCLC

Background Colorectal adenoma (CRA) is a classical premalignant lesion, with high incidence and mainly coexisting with hyperplastic polyp (HPP). Hence, this study aimed to distinguish CRA from HPP by molecular expression profiling and advance the prevention of CRA and its malignance. Methods CRA and paired HPP biopsies were collected by endoscopy. Through RNA‐sequencing (RNA‐seq), the differentially expressed genes (DEGs) were obtained. Functional enrichment analysis was performed based on the DEGs. The STRING database and Cytoscape were used to construct the protein‐protein interaction (PPI) network and perform module analysis. Hub genes were validated by real‐time quantitative PCR (RT‐qPCR) and immunohistochemistry. The ROC curve was drawn to establish the specificity of the hub genes. Results 485 significant DEGs were identified including 133 up‐regulated and 352 down‐regulated. The top 10 up‐regulated genes were DLX5, MMP10, TAC1, ACAN, TAS2R38, WNT2, PHYHIPL, DKK4, DUSP27, and ABCA12. The top 10 down‐regulated genes were SFRP2, CHRDL1, KBTBD12, RERGL, DPP10, CLCA4, GREM2, TMIGD1, FEV, and OTOP3. Wnt signaling pathway and extracellular matrix (ECM) were up‐regulated in CRA. Three hub genes including WNT2, WNT5A, and SFRP1 were filtered out via Cytoscape. Further RT‐qPCR and immunohistochemistry confirmed that WNT2 was highly expressed in CRA. The area under the ROC curve (AUC) at 0.98 indicated the expression level of WNT2 as a candidate to differ CRA from HPP. Conclusion Our study suggests Wnt signaling pathway and ECM are enriched in CRA, and WNT2 may be used as a novel biomarker for distinguishing CRA from HPP and preventing the malignance of CRA., Collect colorectal hyperplastic polyp (HPP) and adenoma, and identify differentially expressed genes through comprehensive bioinformatic analysis. Then perform functional enrichment analysis and protein‐protein interaction, filter out significantly changed genes, and verify their expression at transcription and protein levels. WNT2 may be used as novel biomarker in distinguishing adenoma from HPP, and preventing the malignance of adenoma.

This study is to detect the expression of inflammatory factor or neutrophil-activating factor IL-8 and Wnt2 in gastric cancer (GC) and investigate the involvement of IL-8 and Wnt2 expressions in the clinicopathological indexes and prognosis.We detected the expression of IL-8 and Wnt2 in 100 GC tissues and 40 normal gastric mucosae using immunohistochemistry. The relationships between the IL-8 and Wnt2 expression and the clinicopathological characteristics were explored. The relationship between IL-8 expression, Wnt2 expression, and prognosis of GC was analyzed by survival curve and survival regression.The expression of IL-8 and Wnt2 in GC tissue was 64% and 75% respectively, which was significantly higher than that in adjacent normal gastric mucosa tissues, moreover, expressions of IL-8 and Wnt2 were positively correlated. The positive rate of IL-8 and Wnt2 expressions were correlated with lymph node metastasis and TNM staging （P0.01, and Wnt2 was also correlated with infiltration depth (P = 0.021), but there was no difference with age, sex, and differentiation (P0.05). The 3-year survival analysis showed that the survival rates of IL-8- and Wnt2-positive patients were 20% and 24%, respectively, which were significantly lower than those of negative patients. Cox regression analysis showed that IL-8 and Wnt2 may be independent factors affecting the prognosis of GC.Our data demonstrated that the overexpression of IL-8 and Wnt2 could be isolated prognostic factors in patients with GC and, possibly, may present new targets for the treatment of GC.

Introduction This study mainly investigated the role of miR‐199a‐5p in depression. Methods qRT‐PCR and western blotting were employed to detect the expressions of miR‐199a‐5p, CREB and BDNF. Sucrose preference test, forced swimming test, and tail suspension test were performed to evaluate depression‐related symptoms. MTT assays and flow cytometry were used to examine the cell reproduction and apoptotic cells of hippocampal neuron. Results The data demonstrated that the expression levels of miR‐199a‐5p in the cerebrospinal fluids and serums of depression patient and the hippocampus of chronic unpredictable mild stress (CUMS) mouse were significantly increased. However, the expressions of WNT2, p‐CREB, and BDNF were inhibited. In addition, miR‐199a‐5p‐inhibitor enhanced sucrose preferences of CUMS mouse and decreased immobile time in sucrose preference test and forced swimming test. Knockdown of WNT2 attenuated the effects of miR‐199a‐5p‐inhibitor on cell reproduction and apoptotic cells of hippocampal neuron and the expression of WNT2, p‐CREB, and BDNF. Conclusion MiR‐199a‐5p can target WNT2 to enhance the development of depression through regulation of the CREB/BDNF signaling. Trial registration: JNU‐Hos‐49284., miR‐199a‐5p can target WNT2 to enhance the developments of depressions through the regulations of CREB/BDNF signaling.

N-myc downstream-regulated gene 2 (NDRG2) is down-regulated in grade-III meningioma [anaplastic meningioma (AM)] and associated with clinically aggressive behavior. Current therapies in the treatment of high-grade meningioma are lacking with limited success. This study aims to validate the effect of NDRG2-targeted therapy using structurally related bioactive triterpene compounds derived from the edible mushroom Ganoderma lucidum (ganoderic acid A:GA-A/ganoderic acid DM:GA-DM) in human AM in relevant pre-clinical models. Tissue samples from the AM tumor regions of three human patients and control non-tumor samples were used to analyze the expression pattern of NDRG2. In vitro cell culture and in vivo cell-line-derived orthotopic xenograft animal models of AM were utilized to assess efficacy of treatment with GA-A/DM. Downregulation of NDRG2 expression was observed in surgically resected high-grade meningiomas compared to normal brain. These results prompt us to use NDRG2-targeting agents GA-A/DM. In vitro results showed that 72-h treatments of 25 µM GA-A/DM induced AM cell death, upregulate NDRG2 protein expression, downregulate NDRG2 promoter methylation in meningioma cells as compared to azacitidine and decitabine, the most commonly used demethylating agents. Our results also demonstrated that GA-A/DM does not have any detrimental effect on normal human neurons and arachnoid cells. GA-A/DM promoted apoptotic factors (Bax) while suppressing MMP-9, p-P13K, p-AKT, p-mTOR, and Wnt-2 protein expression. RNAi-mediated knockdown of NDRG2 protein expression increased tumor proliferation, while forced expression of wt-NDRG2 decreased proliferation in an in vitro model. Magnetic resonance (MR) imaging and Hematoxylin (H&E) staining demonstrated gross reduction of tumor volume in GA-A/DM treated mice at 5 weeks when compared with saline-treated orthotopic AM xenografted controls. There was an overall decrease in tumor cell proliferation with increased survival in GA-A/DM-treated animals. Enzyme assays showed that GA-A/DM did not negatively impact hepatic function. GA-A/DM may be a promising natural therapeutic reagent in the treatment of AM by suppressing growth via NDRG2 modulation and altering of intracellular signal pathways. We have shown it could potentially be an effective treatment for AM with decreased cellular proliferation in vitro, decreased tumor volume and increased survival in vivo.

AimsSinus venous valve (SVV) and sinoatrial node (SAN) develop together at the sinoatrial junction during embryogenesis. SVV ensures unidirectional cardiac input and SAN generates sinus rhythmic contraction, respectively; both functions are essential for embryonic survival. We aim to reveal the potential role of endocardial NOTCH signalling in SVV and SAN formation.Methods and resultsWe specifically deleted Notch1 in the endocardium using an Nfatc1Cre line. This deletion resulted in underdeveloped SVV and SAN, associated with reduced expression of T-box transcription factors, Tbx5 andTbx18, which are essential for the formation of SVV and SAN. The deletion also led to decreased expression of Wnt2 in myocardium of SVV and SAN. WNT2 treatment was able to rescue the growth defect of SVV and SAN resulted from the Notch1 deletion in whole embryo cultures. Furthermore, the Notch1 deletion reduced the expression of Nrg1 in the SVV myocardium and supplement of NRG1 restored the growth of SVV in cultured Notch1 knockout embryos.ConclusionOur findings support that endocardial NOTCH1 controls the development of SVV and SAN by coordinating myocardial WNT and NRG1 signalling functions.

WNT2 acts as a pro-angiogenic factor in placental vascularization and increases angiogenesis in liver sinusoidal endothelial cells (ECs) and other ECs. Increased WNT2 expression is detectable in many carcinomas and participates in tumor progression. In human colorectal cancer (CRC), WNT2 is selectively elevated in cancer-associated fibroblasts (CAFs), leading to increased invasion and metastasis. However, if there is a role for WNT2 in colon cancer, angiogenesis was not addressed so far. We demonstrate that WNT2 enhances EC migration/invasion, while it induces canonical WNT signaling in a small subset of cells. Knockdown of WNT2 in CAFs significantly reduced angiogenesis in a physiologically relevant assay, which allows precise assessment of key angiogenic properties. In line with these results, expression of WNT2 in otherwise WNT2-devoid skin fibroblasts led to increased angiogenesis. In CRC xenografts, WNT2 overexpression resulted in enhanced vessel density and tumor volume. Moreover, WNT2 expression correlates with vessel markers in human CRC. Secretome profiling of CAFs by mass spectrometry and cytokine arrays revealed that proteins associated with pro-angiogenic functions are elevated by WNT2. These included extracellular matrix molecules, ANG-2, IL-6, G-CSF, and PGF. The latter three increased angiogenesis. Thus, stromal-derived WNT2 elevates angiogenesis in CRC by shifting the balance towards pro-angiogenic signals. Electronic supplementary material The online version of this article (10.1007/s10456-019-09688-8) contains supplementary material, which is available to authorized users.

Recent studies have shown that the tumor microenvironment plays a significant role in the progression of solid tumors. As an abundant component of the tumor microenvironment, cancer‐associated fibroblasts (CAFs) have been shown to promote tumorigenesis and cancer aggressiveness, but their molecular characteristics remain poorly understood. In the present study, paired CAFs and normal fibroblasts (NFs) were isolated from five colorectal cancer (CRC) tissues from patients who underwent surgical resection. The gene expression profiles of CAFs and NFs identified by RNA sequencing were compared to understand the complex role of CAFs in cancer progression. Gene Set Enrichment Analysis revealed that the gene sets related to the Wnt signaling pathway were highly enriched in CAFs, as well as TGFβ signaling, which is considered to be a regulator of CAFs. Among the components of this pathway, Wnt2 was specifically expressed. The observations led us to speculate that Wnt2 is extremely involved in regulating CRC progression by CAFs. Thus, we performed immunohistochemical analysis on Wnt2 in 171 patients who underwent surgery for colorectal adenocarcinoma. Positive staining for Wnt2 was mainly observed in cancer stroma, although the immunoreactivity was weak in cancer cells. Wnt2 expression in CAFs was significantly correlated with depth of tumor (P, The analysis here involved comparing gene expression between cancer associated and normal fibroblasts using RNA sequencing, which identified that components of the Wnt signaling pathway were highly expressed in cancer associated fibroblasts, and Wnt2 in particular. We then conducted immunohistochemical analysis on Wnt2 in 171 patients who had undergone colorectal adenocarcinoma surgery. We found strong correlations between Wnt2 expression and progression‐related variables such as tumor depth and lymph node metastasis.

Aerobic glycolysis is a characteristic in cancers that is important for cancer cell proliferation. Emerging evidence shows that long non-coding RNA (LncRNA) participates in glucose metabolism and cell proliferation in cancer. This study explored the effect of LncRNA: SNHG9 in glioblastoma. The mRNA expression of SNHG9 in human glioma tissues and glioblastoma cell lines was measured by qRT-PCR. Glioblastoma cell lines (U87 and U251) were transfected with miR-199a-5p or SNHG9-expressing plasmid and cell viability as well as concentrations of glucose and lactate were measured. The extracellular acidification was evaluated by glycolysis stress test. The Wnt2 levels were determined by qRT-PCR and Western blot. Results showed that the mRNA expression of SNHG9 was elevated in glioblastoma tissues. The elevated SNHG9 expression was related to lower survival rate in patients with glioma. SNHG9 could downregulate miR-199a-5p and upregulate Wnt2 in glioblastoma cells. Overexpression of SNHG9 in glioblastoma cells promoted aerobic glycolysis and cell proliferation, which could be attenuated by miR-199a-5p. Results of this study indicated an effect of SNHG9/miR-199a-5p/Wnt2 axis in regulating cell growth and aerobic glycolysis in glioblastoma.

Lower lobe predominant pulmonary cysts occur in up to 90% of patients with Birt–Hogg–Dubé (BHD) syndrome, but the key pathologic cell type and signaling events driving this distinct phenotype remain elusive. Through examination of the LungMAP database, we found that folliculin (FLCN) is highly expressed in neonatal lung mesenchymal cells. Using RNA-Seq, we found that inactivation of Flcn in mouse embryonic fibroblasts leads to changes in multiple Wnt ligands, including a 2.8-fold decrease in Wnt2. This was associated with decreased TCF/LEF activity, a readout of canonical WNT activity, after treatment with a GSK3-α/β inhibitor. Similarly, FLCN deficiency in HEK293T cells decreased WNT pathway activity by 76% post-GSK3-α/β inhibition. Inactivation of FLCN in human fetal lung fibroblasts (MRC-5) led to ~ 100-fold decrease in Wnt2 expression and a 33-fold decrease in Wnt7b expression—two ligands known to be necessary for lung development. Furthermore, canonical WNT activity was decreased by 60%. Classic WNT targets such as AXIN2 and BMP4, and WNT enhanceosome members including TCF4, LEF1 and BCL9 were also decreased after GSK3-α/β inhibition. FLCN-deficient MRC-5 cells failed to upregulate LEF1 in response to GSK3-α/β inhibition. Finally, we found that a constitutively active β-catenin could only partially rescue the decreased WNT activity phenotype seen in FLCN-deficient cells, whereas silencing the transcription factor TFE3 completely reversed this phenotype. In summary, our data establish FLCN as a critical regulator of the WNT pathway via TFE3 and suggest that FLCN-dependent defects in WNT pathway developmental cues may contribute to lung cyst pathogenesis in BHD.

Background/objectives Wnt/β-catenin signalling plays vital roles in tissue homeostasis. Dysregulation of the pathway has been implicated in the pathogenesis of cancer and fibroses in numerous tissues, including the pancreas. We studied the effect of microenvironmental changes pertaining to fibrotic tissue remodelling on the expression of selected Wnt/β-catenin pathway proteins in the human exocrine pancreas. The role of acinar/stellate cross-talk on the expression of the proteins was elucidated in a long-term mouse co-culture system. Methods Expression of β-catenin, Wnt2, Wnt5a and SFRP4 was analysed immunohistochemically in normal and moderately or highly fibrotic human pancreata (n = 8). The effect of humoral interactions on the expression of the proteins was studied by immunocytochemical means in parallel mono- and co-cultures of mouse acinar and stellate cells (PSCs). Results In human pancreatic tissue, fibrotic microenvironment was associated with redistribution of the proteins in and between epithelial and stromal compartments, compared to acinar-rich tissue. In non-fibrotic and moderately fibrotic tissue the proteins appeared only in acinar cells whereas in highly fibrotic tissue stromal fibroblastoid/stellate cells and macrophages were their predominant locations. Subcellular changes in the expression of β-catenin and Wnt5a were detected. Our in vitro data suggest potential involvement of acinar cell/PSC cross-talk in mediating the changes observed in tissue specimens. Conclusions Wnt/β-catenin pathway-associated proteins are abundantly expressed in the exocrine pancreas with prominent changes in their cellular and subcellular expression patterns along with increasing levels of fibrosis. Diverse functions for Wnt/β-catenin signalling during the course of fibrotic remodelling in the exocrine pancreas are suggested.

Aberrant Wnt signaling is implicated in carcinogenesis triggering efforts for the development of new therapeutic agents, many of which have entered clinical trials. We extend our previous analysis of WNT3, FZD7, LEF1 expression levels in breast and colorectal cancer including WNT2, FZD4 and β-catenin expression, in an effort to delineate their relative expression levels along with concurrent expression patterns and possible prognostic value. We analyzed 82 breast and 102 colorectal carcinomas for relative mRNA expression levels of the investigated genes by RT-PCR relative quantification with the ΔΔCt method. Statistical analysis was performed in order to determine associations of relative mRNA expression and linear correlations. β-catenin expression was determined by immunochemistry. Regarding breast carcinomas, decreased relative mRNA expression levels of WNT2, FZD4 were found frequently and WNT2 expression was correlated with ER/ PR status (p = 0.045/p = 0.028), whereas β-catenin with grade (p = 0.026). In colorectal carcinomas, increased relative mRNA expression levels of WNT2 and FZD4 were found in 59% and 32% of cases respectively, whereas β-catenin showed decreased mRNA expression levels in 57% of cases and a correlation with pN-category (p = 0.037). Linear correlations were observed between WNT2/FZD4 (R=0.542, p 0.001), WNT2/β-catenin (R=0.254, p = 0.010), FZD4/β-catenin (R=0.406, p 0.001) expression and a correlation between mRNA expression and membranous/cytoplasmic β-catenin emerged (p = 0.039/0.046). Our results suggest a possible clinical significance for Wnt pathway gene expression levels in both tumour types. The concurrent expression of the investigated genes as well as the different expression profiles, underlines the complexity of this pathway and the necessity of patient selection in order to maximize the efficacy of drugs targeting Wnt pathway.

This study aimed to evaluate the role of miR-383 in the regulation of Wnt-2 signaling in the rat model of chronic stress. The male SD rats with depressive-like behaviors were stimulated with chronic unpredictable mild stress (CUMS) including ice-water swimming for 5 min, food deprivation for 24 h, water deprivation for 24 h, stimulating tail for 1 min, turning night into day, shaking for 15 min (once/s), and wrap restraint (5 min/time) every day for 21 days. The expression levels of miRNAs were detected by qRT-PCR, and the expression levels of Wnt2, depression-impacted proteins (GFAP, BDNF, CREB), brain neurotransmitters (5-HT, NE, DA) and apoptosis-related proteins (Bax and Bcl-2) were evaluated by qRT-PCR and western blot. Bioinformatic analysis and luciferase reporter assay were performed to determine the relationship between miR-383 and Wnt2. Ethological analysis was evaluated by sugar preference test, refuge island test and open field tests. Rescue experiments including knockdown of miR-383, overexpression and silencing of Wnt2 were performed to determine the role of miR-383. High expression levels of miR-383 were observed in the hippocampus of rats submitted to CUMS model. Downregulation of miR-383 significantly inhibited the apoptosis and inflammatory response of hippocampal neurons, and increased the expression levels of GFAP, BDNF and CREB which were impacted in depression, as well as neurotransmitters, then attenuated neural injury in rats induced by CUMS. Furthermore, Wnt family member 2 (Wnt2) was identified as a target of miR-383, and silencing of Wnt2 obviously attenuated the protective effect of miR-383 inhibitor on the apoptosis and inflammatory response in hippocampal neurons, as well as neural injury in CUMS-induced rats. Downregulation of miR-383 ameliorated the behavioral and neurochemical changes induced by chronic stress in rats by directly targeting Wnt2, indicating that the miR-383/Wnt2 axis might be a potential therapeutic target for MDD.

Small nucleolar RNA host gene 12 (SNHG12) has been indicated in the tumorigenesis of various human cancers, including clear cell renal cell carcinoma (ccRCC). However, the underlying mechanisms of SNHG12 driving progression of ccRCC remain incompletely understood. In the present study, we discovered that SNHG12 is up‐regulated in ccRCC and that overexpression of SNHG12 predicted poor clinical outcome of ccRCC patients. SNHG12 knockdown notably inhibited proliferation and migration of RCC cells. Furthermore, we discovered that miR‐30a‐3p, a putative ccRCC inhibitor, was competitively sponged by SNHG12. Via the crosstalk network, SNHG12 was capable of up‐regulating multiple target genes of miR‐30a‐3p, namely, RUNX2, WNT2 and IGF‐1R, which have been identified to facilitate tumorigenesis of ccRCC. Taken together, our present study suggested a novel ceRNA network, in which SNHG12 could promote the malignancy of ccRCC although competitively binding with miR‐30a‐3p and consequently release the expression of its downstream cancer‐related genes.

Background Gastric cancer (GC) is one of the most common malignant tumors worldwide. Currently, the overall survival rate of GC is still unsatisfactory despite progress in diagnosis and treatment. Therefore, studying the molecular mechanisms involved in GC is vital for diagnosis and treatment. CircRNAs, a type of noncoding RNA, have been proven to act as miRNA sponges that can widely regulate various cancers. By this mechanism, circRNA can regulate tumors at the genetic level by releasing miRNA from inhibiting its target genes. The WNT2/β-Catenin regulatory pathway is one of the canonical signaling pathways in tumors. It can not only promote the development of tumors but also provide energy for tumor growth through cell metabolism (such as glutamine metabolism). Methods Through RNA sequencing, we found that hsa_circ_0008259 (circLMO7) was highly expressed in GC tissues. After verifying the circular characteristics of circLMO7, we determined the downstream miRNA (miR-30a-3p) of circLMO7 by RNA pull-down and luciferase reporter assays. We verified the effect of circLMO7 and miR-30a-3p on GC cells through a series of functional experiments, including colony formation, 5-ethynyl-2′-deoxyuridine and Transwell assays. Through Western blot and immunofluorescence analyses, we found that WNT2 was the downstream target gene of miR-30a-3p and further confirmed that the circLMO7-miR-30a-3p-WNT2 axis could promote the development of GC. In addition, measurement of related metabolites confirmed that this axis could also provide energy for the growth of GC cells through glutamine metabolism. We found that circLMO7 could promote the growth and metastasis of GC in vivo by the establishment of nude mouse models. Finally, we also demonstrated that HNRNPL could bind to the flanking introns of the circLMO7 exons to promote circLMO7 cyclization. Results CircLMO7 acted as a miR-30a-3p sponge affecting the WNT2/β-Catenin pathway to promote the proliferation, migration and invasion of GC cells. Moreover, animal results also showed that circLMO7 could promote GC growth and metastasis in vivo. CircLMO7 could also affect the glutamine metabolism of GC cells through the WNT2/β-Catenin pathway to promote its malignant biological function. In addition, we proved that HNRNPL could promote the self-cyclization of circLMO7. Conclusions CircLMO7 promotes the development of GC by releasing the inhibitory effect of miR-30a-3p on its target gene WNT2.

In animals, body axis patterning is based on the concentration-dependent interpretation of graded morphogen signals, which enables correct positioning of the anatomical structures. The most ancient axis patterning system acting across animal phyla relies on β-catenin signaling, which directs gastrulation, and patterns the main body axis. However, within Bilateria, the patterning logic varies significantly between protostomes and deuterostomes. To deduce the ancestral principles of β-catenin-dependent axial patterning, we investigate the oral–aboral axis patterning in the sea anemone Nematostella—a member of the bilaterian sister group Cnidaria. Here we elucidate the regulatory logic by which more orally expressed β-catenin targets repress more aborally expressed β-catenin targets, and progressively restrict the initially global, maternally provided aboral identity. Similar regulatory logic of β-catenin-dependent patterning in Nematostella and deuterostomes suggests a common evolutionary origin of these processes and the equivalence of the cnidarian oral–aboral and the bilaterian posterior–anterior body axes., The authors show in Nematostella that the more orally expressed β-catenin targets repress the more aborally expressed β-catenin targets, thus patterning the oral-aboral axis. This likely represents the common mechanism of β-catenin-dependent axial patterning shared by Cnidaria and Bilateria.

Many hereditary cancer syndromes are associated with an increased risk of small and large intestinal adenocarcinomas. However, conditions bearing a high risk to both adenocarcinomas and neuroendocrine tumors are yet to be described. We studied a family with 16 individuals in four generations affected by a wide spectrum of intestinal tumors, including hyperplastic polyps, adenomas, small intestinal neuroendocrine tumors, and colorectal and small intestinal adenocarcinomas. To assess the genetic susceptibility and understand the novel phenotype, we utilized multiple molecular methods, including whole genome sequencing, RNA sequencing, single cell sequencing, RNA in situ hybridization and organoid culture. We detected a heterozygous deletion at the cystic fibrosis locus (7q31.2) perfectly segregating with the intestinal tumor predisposition in the family. The deletion removes a topologically associating domain border between CFTR and WNT2, aberrantly activating WNT2 in the intestinal epithelium. These consequences suggest that the deletion predisposes to small intestinal neuroendocrine tumors and small and large intestinal adenocarcinomas, and reveals the broad tumorigenic effects of aberrant WNT activation in the human intestine.

Wnts are the major ligands responsible for activating Wnt signaling pathway through binding to Frizzled proteins (Fzd) as the receptors. Among these ligands, Wnt2 plays the main role in the tumorigenesis of several human cancers especially colorectal cancer (CRC). Therefore, it can be considered as a potential drug target. The aim of this study was to identify potential drug candidates against two binding sites of Wnt2. Structure-based virtual screening approaches were applied to identify compounds against binding sites of Wnt2 for inhibiting the interaction Wnt2 and Fzd receptors. The best hit compounds from molecular docking of National Cancer Institute diversity set II database were used for structural similarity search on ZINC database, obtaining large hit compounds query to perform a virtual screening and retrieving potential lead compounds. Eight lead compounds were selected while their binding affinity, binding modes interactions, and molecular dynamics simulations studies were assessed. Molecular docking studies showed that eight selected lead compounds can bind to the desired binding sites of Wnt2 in a high affinity manner. Bioavailability analysis of the selected lead compounds indicated that they possessed significant drug like properties. Thus, these lead compounds were considered as potential drug candidates for inhibiting Wnt signaling pathway through combining with the binding sites of Wnt2 and hindering the interaction of Wnt2 and Fzd receptors. Our findings suggest that Wnt2 binding sites may be a useful target for treatment for CRC fueling the future efforts for developing new compounds against Wnt signaling pathway.

This study aims to illustrate the role of microRNA-548 (miR-548) in regulating the development of colorectal cancer (CRC) and the involvement of WNT2.MiR-548b levels in CRC species and paracancerous ones were detected. The relationship between miR-548b level and clinical parameters of CRC patients was analyzed. After overexpression of miR-548b, the changes in the proliferative and apoptotic capacities of Sw620 and HT29 cells were assessed by Cell Counting Kit-8 (CCK-8), colony formation assay, and flow cytometry, respectively. At last, the involvement of WNT2, the downstream gene of miR-548b, was detected by Luciferase assay and rescue experiments.Results manifested that miR-548b was lowly expressed in CRC species than paracancerous ones, and in vitro level of miR-548b was downregulated in CRC cell lines as well. Compared with CRC patients in T1-T2, miR-548b level was lower in T3-T4 CRC. Moreover, CRC patients with lymphatic metastasis had lower level of miR-548b than those without. Overexpression of miR-548b suppressed proliferative capacity and induced apoptosis in CRC cells. Besides, it was found that WNT2 was the downstream gene of miR-548b, and its level was negatively regulated by miR-548b in CRC. Furthermore, rescue experiments showed that WNT2 was responsible for CRC development regulated by miR-548b.MiR-548b is closely linked to tumor stage and lymphatic metastasis of CRC, and it alleviates the malignant development of CRC by targeting WNT2.

This study aimed to determine the WNT2 expression in patients with severe preeclampsia and to explore the function of WNT2 dysregulation on the biological behaviors of trophoblast cells. The WNT2 and β-catenin expression in the patients with early-onset and late-onset severe preeclampsia and normal controls was determined. Subsequently, WNT2 was overexpressed and knocked down in HTR8 cells and WNT2 signaling pathway in regulating trophoblast cell proliferation, migration, invasion, and apoptosis were evaluated in vitro. The mRNA and protein expression levels of WNT2 and β-catenin were decreased in patients with preeclampsia, especially early-onset severe preeclampsia. Overexpression of WNT2 promoted trophoblast cell proliferation, migration, and invasion and inhibited apoptosis in vitro, whereas knockdown of WNT2 had opposite effects. The findings of this study reveal that WNT2 and β-catenin were decreased expressed in patients with preeclampsia. Decreased expression of WNT2 may inhibit trophoblast cell proliferation, migration, and invasion but induced apoptosis. WNT2 may serve as a promising biomarker for early detection of preeclampsia.

Background MicroRNAs (miRNAs) play a role in regulating osteogenic differentiation (OD) of mesenchymal stem cells by inhibiting mRNAs translation under cyclic strain. miR-503-3p was downregulated in OD of human adipose-derived stem cells (hASCs) in vivo under cyclic strain in our previous study, while it might target the Wnt/β-catenin (W-β) pathway. In this study, we explored miR-503-3p’s role in OD of hASCs under cyclic strain. Methods OD of hASCs was induced by cyclic strain. Bioinformatic and dual luciferase analyses were used to confirm the relationship between Wnt2/Wnt7b and miR-503-3p. Immunofluorescence was used to detect the effect of miR-503-3p on Wnt2/Wnt7b and β-catenin in hASCs transfected with miR-503-3p mimic and inhibitor. Mimic, inhibitor, and small interfering RNA (siRNA) transfected in hASCs to against Wnt2 and Wnt7b. Quantitative real-time PCR (RT-PCR) and western blot were used to examine the OD and W-β pathway at the mRNA and protein levels, respectively. Immunofluorescence was performed to locate β-catenin. ALP activity and calcium were detected by colorimetric assay. Results Results of immunophenotypes by flow cytometry and multi-lineage potential confirmed that the cultured cells were hASCs. Results of luciferase reporter assay indicated that miR-503-3p could regulate the expression levels of Wnt2 and Wnt7b by targeting their respective 3′-untranslated region (UTR). Under cyclic strain, gain- or loss-function of miR-503-3p studies by mimic and inhibitor revealed that decreasing expression of miR-503-3p could significantly bring about promotion of OD of hASCs, whereas increased expression of miR-503-3p inhibited OD. Furthermore, miR-503-3p high-expression reduced the activity of the W-β pathway, as indicated by lowering expression of Wnt2 and Wnt7b, inactive β-catenin in miR-503-3p-treated hASCs. By contrast, miR-503-3p inhibition activated the W-β pathway. Conclusions Collectively, our findings indicate that miR-503-3p is a negative factor in regulating W-β pathway by Wnt2 and Wnt7b, which inhibit the OD of hASCs under cyclic strain.

The PTEN induced putative kinase 1 (PINK1) mutation is the second most common cause of autosomal recessive adolescent Parkinson's disease (PD). Furthermore, mitochondrial disorders and oxidative stress are important mechanisms in the pathogenesis of PD. Numerous members of the Wnt family have been found to be associated with neurodegenerative diseases. Therefore, the present study investigated the role of the Wnt2 gene in PINK1B9 transgenic flies, which is a PD model, and its underlying mechanism. It was identified that overexpression of Wnt2 reduced the abnormality rate of PD transgenic Drosophila and improved their flight ability, while other intervention groups had no significant effect. Furthermore, an increase in ATP concentration normalized mitochondrial morphology, and increased the mRNA expression levels of NADH‑ubiquinone oxidoreductase chain 1 (ND1), ND42, ND75, succinate dehydrogenase complex subunits B, Cytochrome b and Cyclooxygenase 1, which are associated with Wnt2 overexpression. Moreover, overexpression of Wnt2 in PD transgenic Drosophila resulted in the downregulation of reactive oxygen species and malondialdehyde production, and increased manganese superoxide dismutase (MnSOD), while glutathione was not significantly affected. It was found that overexpression of Wnt2 did not alter the protein expression of β‑catenin in PINK1B9 transgenic Drosophila, but did increase the expression levels of PPARG coactivator 1α (PGC‑1α) and forkhead box sub‑group O (FOXO). Collectively, the present results indicated that the Wnt2 gene may have a protective effect on PD PINK1B9 transgenic Drosophila. Thus, it was speculated that the reduction of oxidative stress and the restoration of mitochondrial function via Wnt2 overexpression may be related to the PGC‑1α/FOXO/MnSOD signaling pathway in PINK1 mutant transgenic Drosophila.

Mounting evidences have indicated that terminal differentiation-induced lncRNA (TINCR) contributes to various cellular processes, such as proliferation, apoptosis, autophagy, migration, invasion, and metastasis. However, the function of TINCR in regulating migration of MSCs is largely unknown. In this study, the effects of TINCR on the migration of rat MSCs from the bone marrow were studied by Transwell assays and wound healing assays. Our results suggested that TINCR positively regulated migration of rMSCs. miR-761 mimics suppressed rMSC migration, whereas miR-761 inhibitor promoted migration. Target prediction analysis tools and dual-luciferase reporter gene assay identified Wnt2 as a direct target of miR-761. miR-761 could inhibit the expression of Wnt2. Further, the investigation about the function of TINCR in miR-761-induced migration of rMSCs was completed. These results demonstrated that TINCR took part in the regulation of miR-761-induced migration in rMSCs through the regulation of Wnt2 and its Wnt2 signaling pathway. Taken together, our results demonstrate that lncRNA-TINCR functions as a competitive endogenous RNA (ceRNA) to regulate the migration of rMSCs by sponging miR-761 which modulates the role of Wnt2. These findings provide evidence that lncRNA-TINCR has a chance to serve as a potential target for enhancing MSC homing through the miR-761/Wnt2 signaling pathway.

Imatinib (IM) is a first-line therapeutic drug for chronic myeloid leukemia (CML), a hematological disease. Mutations in the BCR-ABL domain increase formation of IM resistance in CML. However, not all patients are BCR-ABL domain-mutant dependent. Investigating non-mutant mechanisms in the development of acquired IM resistance is a critical issue. We explored the mechanisms which influence IM efficacy and resistance in CML. Higher protective autophagy was identified in IM-resistant K562 (K562R) cells. Inhibition of autophagy by the inhibitors, chloroquine and 3-methyladenine, enhanced IM's efficacy in K562R cells. In addition, microRNA (miR)-199a/b-5p were downregulated in K562R cells compared to parent cells. Overexpression of miR-199a/b-5p reduced autophagy and induced cell apoptosis, resulting in enhanced IM's efficacy in K562R cells. Moreover, expression levels of the Wingless-type MMTV integration site family member 2 (WNT2), a positive regulator of autophagy, were significantly higher in K562R cells, and it was validated as a direct target gene of miR-199a/b-5p. Overexpressions of miR-199a/b-5p inhibited WNT2 downstream signaling. Furthermore, overexpression and knockdown of WNT2 influenced autophagy formation and CML drug sensitivity to IM. Overexpression of WNT2 could also reverse miR-199a/b-5p-enhanced IM efficacy in K562R cells. These results emphasized that miR-199a/b-5p inhibited autophagy via repressing WNT2 signaling and might provide novel therapeutic strategies for future IM-resistant CML therapy and drug development.

Filial imprinting is the behavior observed in chicks during the sensitive or critical period of the first 2–3 days after hatching; however, after this period they cannot be imprinted when raised in darkness. Our previous study showed that temporal augmentation of the endogenous thyroid hormone 3,5,3′-triiodothyronine (T3) in the telencephalon, by imprinting training, starts the sensitive period just after hatching. Intravenous injection of T3 enables imprinting of chicks on days 4 or 6 post-hatching, even when the sensitive period has ended. However, the molecular mechanism of how T3 acts as a determinant of the sensitive period is unknown. Here, we show that Wnt-2b mRNA level is increased in the T3-injected telencephalon of 4-day old chicks. Pharmacological inhibition of Wnt signaling in the intermediate hyperpallium apicale (IMHA), which is the caudal area of the telencephalon, blocked the recovery of the sensitive period following T3 injection. In addition, injection of recombinant Wnt-2b protein into the IMHA helped chicks recover the sensitive period without the injection of T3. Lastly, we showed Wnt signaling to be involved in imprinting via the IMHA region on day 1 during the sensitive period. These results indicate that Wnt signaling plays a critical role in the opening of the sensitive period downstream of T3.

Background: Acute myocardial infarction (AMI)-induced excessive myocardial fibrosis exaggerates cardiac dysfunction. However, serum Wnt2 or Wnt4 level in AMI patients, and the roles in cardiac fibrosis are largely unkown. Methods: AMI and non-AMI patients were enrolled to examine serum Wnt2 and Wnt4 levels by ELISA analysis. The AMI patients were followed-up for one year. 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 elevated Wnt2 and Wnt4 were correlated to adverse outcome of these patients. Knockdown of Wnt2 and Wnt4 significantly attenuated myocardial remodeling and cardiac dysfunction following experimental MI. In vitro, hypoxia enhanced the secretion and expression of Wnt2 and Wnt4 in neonatal rat cardiac myocytes (NRCMs) or fibroblasts (NRCFs). Mechanistically, the elevated Wnt2 or Wnt4 activated β-catenin /NF-κB signaling to promote pro-fibrotic effects in cultured NRCFs. In addition, Wnt2 or Wnt4 upregulated the expression of these Wnt co-receptors, frizzled (Fzd) 2, Fzd4 and (low-density lipoprotein receptor-related protein 6 (LRP6). Further analysis revealed that Wnt2 or Wnt4 activated β-catenin /NF-κB by the co-operation of Fzd4 or Fzd2 and LRP6 signaling, respectively. Interpretation: Elevated Wnt2 and Wnt4 activate β-catenin/NF-κB signaling to promote cardiac fibrosis by cooperation of Fzd4/2 and LRP6 in fibroblasts, which contributes to adverse outcome of patients with AMI, suggesting that systemic inhibition of Wnt2 and Wnt4 may improve cardiac dysfunction after MI.

AIM To investigate the potential role of microRNA-30a (miR-30a) in esophageal squamous cell carcinoma (ESCC). METHODS Expression of miR-30a-3p/5p was analyzed using microarray data and fresh ESCC tissue samples. Both in vitro and in vivo assays were used to investigate the effects of miR-30a-3p/5p on ESCC cell proliferation. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis was performed to explore underlying mechanisms involved in ESCC, and then, assays were carried out to verify the potential molecular mechanism of miR-30a in ESCC. RESULTS Low expression of miR-30a-3p/5p was closely associated with advanced ESCC progression and poor prognosis of patients with ESCC. Knock-down of miR-30a-3p/5p promoted ESCC cell proliferation. Increased miR-30a-3p/5p expression inhibited the Wnt signaling pathway by targeting Wnt2 and Fzd2. CONCLUSION Down-regulation of miR-30a-3p/5p promotes ESCC cell proliferation by activating the Wnt signaling pathway through inhibition of Wnt2 and Fzd2.

Breast cancer is one the most common cancers, making it the second leading cause of cancer-related death among women. Long non-coding RNAs (lncRNAs), with tightly regulated expression patterns, also serve as tumor suppressor during tumorigenesis. The present study aimed to elucidate the role of LINC00968 in breast cancer via WNT2-mediated Wnt2/β-catenin signaling pathway. Breast cancer chip GSE26910 was utilized to identify differential expression in LINC00968 and WNT2. The possible relationship among LINC00968, transcriptional repressor HEY and WNT2 was analyzed and then verified. Effects of LINC00968 on activation of the Wnt2/β-catenin signaling pathway was also tested. Drug resistance, colony formation, cell migration, invasion ability and cell apoptosis after transfection were also determined. Furthermore, tumor xenograft in nude mice was performed to test tumor growth and weight in vivo. WNT2 expression exhibited at a high level, whereas LINC00968 at a low expression in breast cancer which was also associated with poor prognosis in patients. LINC00968 targeted and negatively regulated WNT2 potentially via HEY1. Either overexpressed LINC00968 or silenced inhibited activation of the Wnt2/β-catenin signaling pathway, thereby reducing drug resistance, decreasing colony formation ability, as well as suppressing migration and invasion abilities of breast cancer cells in addition to inducing apoptosis. Lastly, in vivo experiment suggested that LINC00968 overexpression also suppressed transplanted tumor growth in nude mice. Collectively, overexpressed LINC00968 contributes to reduced drug resistance in breast cancer cells by inhibiting the activation of the Wnt2/β-catenin signaling pathway through silencing WNT2. This study offers a new target for the development of breast cancer treatment.

Mechanistic insight into estrogen deficiency by polycystic ovary syndrome (PCOS) remains a longstanding challenge in reproductive medicine. Recent advance suggest that Wingless-type MMTV integration site family members (WNTs), in concert with its Frizzled (FZD) receptors, regulate normal folliculogenesis, luteogenesis and ovarian steroidogenesis. However, no studies have so far investigated any causality between WNT-FZDs interactions and disrupted estrogen synthesis under certain pathological conditions. Here, we show that (i) FZD3 expression was significantly up-regulated in the cumulus cells (CCs) from PCOS patients. This up-regulation, along with the activation of WNT2/β-Catenin pathway, was tightly associated with insulin resistance and estrogen deficiency, two hallmarks of PCOS. (ii) Overexpression of exogenous FZD3 in human granulosa cell COV434 impaired long-term FSH incubation-induced CYP19A1 transactivation and the recruitment of β-Catenin onto CYP19A1 promoter, and subsequently compromised FSH-stimulated estrogen production. (iii) Conversely, inhibition of FZD3 expression exhibited a therapeutic effect on estrogen synthesis in PCOS CCs. Thus, excessive FZD3 expression in CCs may act as a brake on steroidogenic activation that is normally overcome by FSH stimulation. Future endeavor in this field should help to elucidate the complicated crosstalk between energy metabolism and endocrine cells through WNT/FZD signaling molecules.

Background/Purpose Central nervous system (CNS) patterning genes are recognized as candidate genes for autism spectrum disorders (ASDs) based on neuroimaging and neuropathological evidence. Several genes that regulate CNS development are shown to be associated with ASD. Our previous family-based association study also revealed that a specific haplotype of WNT2 (wingless-type MMTV integration site family member 2) gene was overtransmitted to probands with ASD. Whether the CNS patterning genes moderate the clinical phenotype of ASD is unclear. This study investigated the genetic associations of WNT2 , engrailed 2 ( EN2 ), and forkhead box P2 ( FOXP2 ) with the clinical symptom severity. Methods The sample included 391 patients (males, 88.3%; mean age±standard deviation, 9.5±4.4 years) diagnosed with ASDs. Tag single nucleotide polymorphisms (SNPs) of EN2 , WNT2 , and FOXP2 were genotyped. The single-locus and multilocus markers were tested for association. Results We found that multilocus markers of WNT2 were associated with stereotyped behaviors whereas the markers of FOXP2 tended to be associated with social deficits. Moreover, an SNP of WNT2 showed a trend to be associated with less inattentive symptoms. Conclusions Our findings that WNT2 and FOXP2 may moderate the clinical phenotypes of ASD provide evidence to support the possible universal effect of WNT2 and FOXP2 on neurodevelopmental symptom dimensions. Such findings warrant further validation in other independent samples. Trial registration: Clinical trial registration identifier: NCT00494754

The canonical WNT signaling pathway is crucial for intestinal stem cell renewal and aberrant WNT signaling is an early event in colorectal cancer (CRC) development. Here, we show for the first time that WNT2 is one of the most significantly induced genes in CRC stroma as compared to normal stroma. The impact of stromal WNT2 on carcinoma formation or progression was not addressed so far. Canonical WNT/β-catenin signaling was assessed using a 7TGP-reporter construct. Furthermore, effects of WNT2 on fibroblast migration and invasion were determined using siRNA-mediated gene silencing. Tumor cell invasion was studied using organotypic raft cultures and in vivo significance was assessed via a xenograft mouse model. We identified cancer-associated fibroblasts (CAFs) as the main source of WNT2. CAF-derived WNT2 activated canonical signaling in adenomatous polyposis coli/β-catenin wild-type colon cancer cells in a paracrine fashion, whereas no hyperactivation was detectable in cell lines harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Furthermore, WNT2 activated autocrine canonical WNT signaling in primary fibroblasts, which was associated with a pro-migratory and pro-invasive phenotype. We identified FZD8 as the putative WNT2 receptor in CAFs. Three-dimensional organotypic co-culture assays revealed that WNT2-mediated fibroblast motility and extracellular matrix remodeling enhanced cancer cell invasion of cell lines even harboring mutations in the adenomatous polyposis coli/β-catenin pathway. Thus, suggesting a tumor-promoting influence on a broad range of CRC. In line, WNT2 also promotes tumor growth, invasion and metastasis in vivo. Moreover, high WNT2 expression is associated with poor prognosis in human CRC. The identification of the pro-malignant function of stromal derived WNT2 in CRC classifies WNT2 and its receptor as promising stromal targets to confine cancer progression in combination with conventional or targeted therapies.

Members of the wnt gene family encode secreted glycoproteins that mediate critical intercellular communications in metazoans. Large-scale genome and transcriptome analyses have shown that this family is composed of 13 distinct subfamilies. These analyses have further established that the number of wnt genes per subfamily varies significantly between metazoan phyla, highlighting that gene duplication and gene loss events have shaped the complements of wnt genes during evolution. In sea urchins, for example, previous work reported the absence of representatives of both the WNT2 and WNT11 subfamilies in two different species, Paracentrotus lividus and Strongylocentrotus purpuratus. Recently, however, we identified a gene encoding a WNT2 ortholog in P. lividus and, based on that finding, we also reanalyzed the genome of S. purpuratus. Yet, we found no evidence of a bona fide wnt2 gene in S. purpuratus. Furthermore, we established that the P. lividus wnt2 gene is selectively expressed in vegetal tissues during embryogenesis, in a pattern that is similar, although not identical, to that of other P. lividus wnt genes. Taken together, this study amends previous work on the P. lividus wnt complement and reveals an unexpected variation in the number of wnt genes between closely related sea urchin species.

Purpose: To investigate inhibitory effect of Astragalus polysaccharide (APS) on osteoporosis in ovariectomized rats by regulating FoxO3a/Wnt2 signaling pathway. Methods: Postmenopausal osteoporosis (PMOP) animal model was developed by excising the bilateral ovaries of rats. The model rats were administered with APS (200 mg/kg, 400 mg/kg, 800 mg/kg) by intragastric administration once daily for 12 weeks. Bone density, bone metabolism index and oxidative stress index were measured in all groups. Furthermore, the regulation of APS of FoxO3a / Wnt2 signaling pathway was observed. Results: APS has an estrogen-like effect, which can increase bone mass, lower serum ALP and BGP values, increase blood calcium content, and increase bone density of the femur and vertebrae in rats. At the same time, APS can increase the bone mineral content of the femur, increase the maximum stress, maximum load and elastic modulus of the ovariectomized rats, improve oxidative stress in rats by increasing the gene expression of β-catenin and Wnt2 mRNA and inhibiting the gene expression of FoxO3a mRNA. Conclusion: Astragalus polysaccharide can effectively alleviate oxidative stress-mediated osteoporosis in ovariectomized rats, which may be related to its regulation of FoxO3a/Wnt2/β-catenin pathway.

International audience; A short anogenital distance (AGD) in males is a marker for incomplete masculinization and a predictor of adverse effects on male reproductive health. For this reason, AGD is used to assess the endocrine disrupting potential of chemicals for risk assessment purposes. The molecular mechanisms underpinning this chemically induced shortening of the AGD, however, remains unclear. Although it is clear that AR-mediated signaling is essential, evidence also suggest the involvement of other signaling pathways. This study presents the first global transcriptional profile of the anogenital tissue in male rat fetuses with chemically induced short AGD, also including comparison to normal male and female control animals. The anti-androgenic drug finasteride (10mg/kg bw/day) was used to induce short AGD by exposing time-mated Sprague Dawley rats at gestation days (GD) 7-21. The AGD was 37% shorter in exposed male fetuses compared to control males at GD21. Transcriptomics analysis on anogenital tissues revealed a sexually dimorphic transcriptional profile. More than 350 genes were found to be differentially expressed between the three groups. The expression pattern of four genes of particular interest (Esr1, Padi2, Wnt2 and Sfrp4) was also tested by RT-qPCR analyses, indicating that estrogen and Wnt2 signaling play a role in the sexually dimorphic development of the anogenital region. Our transcriptomics profiles provide a stepping-stone for future studies aimed at characterizing the molecular events governing development of the anogenital tissues, as well as describing the detailed Adverse Outcome Pathways for short AGD; an accepted biomarker of endocrine effects for chemical risk assessment.