Expression Of Wif1 Research Articles

Clinical study of Wnt inhibitory factor-1 expression and its association with disease severity in non-segmental vitiligo

BackgroundVitiligo is classified as an acquired chronic depigmentation disorder that includes the destruction of epidermal melanocytes. It affects 0.5–1% of the population all over the world. Wnt signaling pathway is vital in melanocytes differentiation and development. WIF-1 is an antagonist of the Wnt signaling pathway; it hinders Wnt from binding its receptors. The present study aims to detect WIF-1 expression in vitiligo skin and if it relates to the disease's severity.ResultsThis case–control study included 70 subjects: 35 vitiligo patients and 35 healthy controls. Skin WIF-1 expression was estimated using quantitative real-time PCR. Assessment of the vitiligo disease activity score and vitiligo area severity index score was determined. WIF-1 expression showed significant elevation in the skin of vitiligo patients compared to the healthy control group.ConclusionOverexpression of WIF-1 may participate in the pathogenesis of vitiligo; hence, it should be a future therapeutic target.

FGF Signaling Regulates Development of the Anterior Fontanelle.

The calvarial bones of the infant skull are connected by transient fibrous joints known as sutures and fontanelles, which are essential for reshaping during birth and postnatal growth. Genetic disorders such as Apert, Pfeiffer, Crouzon, and Bent bone dysplasia linked to FGFR2 variants often exhibit multi-suture craniosynostosis and a persistently open anterior fontanelle (AF). This study leverages mouse genetics and single-cell transcriptomics to determine how Fgfr2 regulates closure of the AF closure and its transformation into the frontal suture during postnatal development. We find that cells of the AF, marked by the tendon/ligament factor SCX, are spatially restricted to ecto- or endocranial domains and undergo regionally selective differentiation into ligament, bone, and cartilage. Differentiation of SCX+ AF cells is dependent on FGFR2 signaling in cells of the osteogenic fronts which, when fueled by FGF18 from the ectocranial mesenchyme, express the secreted WNT inhibitor WIF1 to regulate WNT signaling in neighboring AF cells. Upon loss of Fgfr2 , Wif1 expression is lost, and cells of the AF retain a connective tissue-like fate failing to form the posterior frontal suture. This study provides new insights into regional differences in suture development by identifying an FGF-WNT signaling circuit within the AF that links frontal bone advancement with suture joint formation.

Cyanidin Ameliorates Bisphenol A-Induced Alzheimer's Disease Pathology by Restoring Wnt/β-Catenin Signaling Cascade: an In Vitro Study.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder causing memory loss and cognitive decline, linked to amyloid-beta (Aβ) plaques and hyperphosphorylated tau protein accumulation in the brain. Environmental pollutant bisphenol A (BPA) has been implicated in AD pathology due to its neurotoxic effects. This study aims to evaluate cyanidin from flower bracts of Musa acuminata Colla (red variety; AAA group) for its neuroprotective properties against BPA-induced AD pathology. The extraction of cyanidin was optimized using 70% ethanol in acidified water, showing promising anti-acetylcholinesterase activity. Cyanidin was effectively purified from the resultant extract and characterized using spectroscopic techniques. Two gradient doses of cyanidin (90 and 10µg/ml) were determined based on cell viability assay. The role of cyanidin in promoting nerve growth and differentiation was assessed in PC12 cells for up to 72h. A discernible and statistically significant difference was assessed in neurite extension at both doses at 72h, followed by pre-treatment with cyanidin. BPA stimulation significantly increased the p-tau expression compared to the control (p < 0.0001). Pre-treatment with cyanidin reduced the tau expression; however, a significant difference was observed compared to control cells (p = 0.0003). Cyanidin significantly enhanced the mRNA expression of Wnt3a (p < 0.0001), β-catenin (p = 0.0004), and NeuroD1 (p = 0.0289), and decreased the expression of WIF1(p = 0.0040) and DKK1 (p < 0.0001), which are Wnt antagonist when compared to cells stimulated with BPA. Conclusively, our finding suggests that cyanidin could agonize nerve growth factor and promote neuronal differentiation, reduce tau-hyperphosphorylation by restoring the Wnt/β-catenin signaling cascade, and thereby render its neuroprotective potential against BPA-induced AD pathology.

MiR-181b-1-3p affects the proliferation and differentiation of chondrocytes in TD broilers through the WIF1/Wnt/β-catenin pathway

Thiram is a plant fungicide, its excessive use has exceeded the required environmental standards. It causes tibial dyschondroplasia (TD) in broilers which is a common metabolic disease that affects the growth plate of tibia bone. It has been studied that many microRNAs (miRNAs) are involved in the differentiation of chondrocytes however, their specific roles and mechanisms have not been fully investigated. The selected features of tibial chondrocytes of broilers were studied in this experiment which included the expression of miR-181b-1-3p and the genes related to WIF1/Wnt/β-catenin pathway in chondrocytes through qRT-PCR, western blot and immunofluorescence. The correlation between miR-181b-1-3p and WIF1 was determined by dual luciferase reporter gene assay whereas, the role of miR-181b-1-3p and WIF1/Wnt/β-catenin in chondrocyte differentiation was determined by mimics and inhibitor transfection experiments. Results revealed that thiram exposure resulted in decreased expression of miR-181b-1-3p and increased expression of WIF1 in chondrocytes. A negative correlation was also observed between miR-181b-1-3p and WIF1. After overexpression of miR-181b-1-3p, the expression of ACAN, β-catenin and Col2a1 increased but the expression of GSK-3β decreased. It was observed that inhibition of WIF1 increased the expression of ALP, β-catenin, Col2a1 and ACAN but decreased the expression of GSK-3β. It is concluded that miR-181b-1-3p can reverse the inhibitory effect of thiram on cartilage proliferation and differentiation by inhibiting WIF1 expression and activating Wnt/β-catenin signaling pathway. This study provides a new molecular target for the early diagnosis and possible treatment of TD in broilers.

Potential of miR-181a-5p and miR-630 as clinical biomarkers in NSCLC

BackgroundThe development of drug resistance and high mortality rates are the major problems observed in non-small cell lung cancer (NSCLC). Biomarkers indicating and predicting disease development towards these unfavorable directions are therefore on high demand. Many studies have demonstrated that changes in miRNAs expression may be associated with a response to treatment and disease prognosis, thus suggesting its potential biomarker value for a broad spectrum of clinical applications. The aim of the present study was to investigate the expression level of miR-181a-5p, miR-630, and its targets in NSCLC tumor tissue and plasma samples; and to analyze its association with NSCLC patient’s response to treatment and disease prognosis.MethodsThe study was performed in 89 paired tissue specimens and plasma samples obtained from NSCLC patients who underwent surgical treatment at the Department of Thoracic Surgery and Oncology of the National Cancer Institute. Analysis of miR-181a-5p and miR-630 expression was performed by qRT-PCR using TaqMan miRNA specific primers. Whereas BCL2, LMO3, PTEN, SNAI2, WIF1 expression levels were identified with KAPA SYBR FAST qPCR Kit. Each sample was examined in triplicate and calculated following the 2-ΔΔCt method. When the p-value was less than 0.05, the differences were considered statistically significant.ResultsIt was found that miR-181a-5p and miR-630 expression levels in NSCLC tissue and plasma samples were significantly decreased compared with control samples. Moreover, patients with low miR-181a-5p expression in tumor tissue and plasma had longer PFS rates than those with high miRNA expression. Decreased miR-630 expression in tumor was statistically significantly associated with better NSCLC patients’ OS. In addition, the expression of miR-181a-5p, as well as miR-630 in tumor tissue, are the statistically significant variables for NSCLC patients’ OS. Moreover, in NSCLC patient plasma samples circulating miR-181a-5p can be evaluated as significant independent prognostic factors for OS and PFS.ConclusionsOur findings indicate the miR-181a-5p and miR-630 expression levels have the potential to prognose and predict and therefore improve the treatment individualization and the outcome of NSCLC patients. Circulating miR-181a-5p has the potential clinical value as a non-invasive biomarker for NSCLC.

WIF1 promoter hypermethylation induce endometrial carcinogenesis through the Wnt/β-catenin signaling pathway.

This study explores the mechanism underlying WIF1 promoter methylation and its relationship with the pathogenesis of endometrial carcinoma. WIF1 promoter methylation was detected using methylation-specific polymerase chain reaction (MSP). WIF1 expression was examined through qRT-PCR and western blotting. Furthermore, 5-aza-2'-deoxycytidine (5-Aza) was used to demethylate the WIF1 promoter. The roles of WIF1 were investigated using in vitro loss- and gain-of-function assays. Xenograft models were used to analyze WIF1 expression and downstream genes, and results were confirmed using immunofluorescence and western blotting. WIF1 promoter methylation in endometrial cancer cells was significantly higher than that in normal cells, but the WIF1 mRNA and protein levels were reduced. The expression of WIF1 increased significantly after 5-Aza treatment (p<.05). Thus, 5-Aza treatment can inhibit the proliferation of endometrial cancer cells and induce apoptosis, while knockdown of WIF1 significantly inhibits the effects of 5-Aza. 5-Aza treatment can also inhibit Wnt pathway genes, including phosphorylation of β-catenin protein, c-Myc, and CyclinD1, inhibit downstream functional genes, and activate the tumor suppressor gene APC, which can be blocked by WIF1 knockdown in endometrial carcinoma cells. Finally, 5-Aza inhibited the proliferation of subcutaneous tumor-bearing nude mice with endometrial cancer cells, but the effect was weaker than that of WIF1 overexpression. Our research shows that WIF1 promoter hypermethylation may promote the progression of endometrial cancer by downregulating WIF1 expression, activating the Wnt/β-catenin pathway, and promoting proliferation and inhibiting apoptosis. WIF1 may be a potential biological target for gene therapy and drug development for the treatment of endometrial cancer.

Wnt/β-Catenin Signaling Activation Induces Differentiation in Human Limbal Epithelial Stem Cells Cultured Ex Vivo.

Human limbal epithelial stem cells (hLESCs) continuously replenish lost or damaged human corneal epithelial cells. The percentage of stem/progenitor cells in autologous ex vivo expanded tissue is essential for the long-term success of transplantation in patients with limbal epithelial stem cell deficiency. However, the molecular processes governing the stemness and differentiation state of hLESCs remain uncertain. Therefore, we sought to explore the impact of canonical Wnt/β-catenin signaling activation on hLESCs by treating ex vivo expanded hLESC cultures with GSK-3 inhibitor LY2090314. Real-time qRT-PCR and microarray data reveal the downregulation of stemness (TP63), progenitor (SOX9), quiescence (CEBPD), and proliferation (MKI67, PCNA) genes and the upregulation of genes for differentiation (CX43, KRT3) in treated- compared to non-treated samples. The pathway activation was shown by AXIN2 upregulation and enhanced levels of accumulated β-catenin. Immunocytochemistry and Western blot confirmed the findings for most of the above-mentioned markers. The Wnt/β-catenin signaling profile demonstrated an upregulation of WNT1, WNT3, WNT5A, WNT6, and WNT11 gene expression and a downregulation for WNT7A and DKK1 in the treated samples. No significant differences were found for WNT2, WNT16B, WIF1, and DKK2 gene expression. Overall, our results demonstrate that activation of Wnt/β-catenin signaling in ex vivo expanded hLESCs governs the cells towards differentiation and reduces proliferation and stem cell maintenance capability.

Epigenetic reader MeCP2 repressed WIF1 boosts lung fibroblast proliferation, migration and pulmonary fibrosis

Epigenetic has been implicated in pulmonary fibrosis. However, there is limited information regarding the biological role of the epigenetic reader MeCP2 in pulmonary fibrosis. The aim of this study was to investigate the role of MeCP2 and its target WIF1 in pulmonary fibrosis. The pathological changes and collagen depositions was analyzed by H&E, Masson’s Trichrome Staining and Sirius Red staining. MeCP2, WIF1, α-SMA, Wnt1, β-catenin, and collagen I expression were analyzed by western blotting, RT-qPCR, immunohistochemistry, immunofluorescence, respectively. The effects of MeCP2 on pulmonary fibrosis involve epigenetic mechanisms, using cultured cells, animal models, and clinical samples. Herein, our results indicated that MeCP2 level was up-regulated, while WIF1 was decreased in Bleomycin (BLM)-induced mice pulmonary fibrosis tissues, patients pulmonary fibrosis tissues and TGF-β1-induced lung fibroblast. Knockdown of MeCP2 by siRNA can rescue WIF1 downregulation in TGF-β1-induced lung fibroblast, inhibited lung fibroblast activation. The DNA methylation inhibitor 5-azadC-treated lung fibroblasts have increased WIF1 expression with reduced MeCP2 association. In addition, we found that reduced expression of WIF1 caused by TGF-β1 is associated with the promoter methylation status of WIF1. Moreover, in vivo studies revealed that knockdown of MeCP2 mice exhibited significantly ameliorated pulmonary fibrosis, decreased interstitial collagen deposition, and increased WIF1 expression. Taken together, our study showed that epigenetic reader MeCP2 repressed WIF1 facilitates lung fibroblast proliferation, migration and pulmonary fibrosis.

Eugenol exposure inhibits embryonic development and swim bladder formation in zebrafish

Eugenol is a natural phenolic essential oil extracted from cloves, that has analgesic and anesthetic effects and is widely used in fishery anesthesia. However, the potential safety risks of aquaculture production associated with the massive use of eugenol and its developmental toxicity during early life stages of fish have been overlooked. In this study, zebrafish (Danio rerio) embryos at 24 hours post-fertilization (hpf) were exposed to eugenol at concentrations of 0, 10, 15, 20, 25, or 30 mg/L for 96 h. Eugenol exposure delayed the hatching of zebrafish embryos, and reduced the body length and the inflation rate of the swim bladder. The accumulated number of dead zebrafish larvae in the eugenol-exposed groups was higher than that of the control group, and it was dose-dependent. Real-time quantitative polymerase chain reaction (qPCR) analysis showed that the Wnt/β-catenin signaling pathway that regulates the development of the swim bladder during the hatching and mouth-opening stages was inhibited after eugenol exposure. Specifically, the expression of wif1, a Wnt signaling pathway inhibitor, was significantly up-regulated, whereas the expression of fzd3b, fzd6, ctnnb1, and lef1 involved in the Wnt/β-catenin pathway was significantly down-regulated. These results suggest that the failure of zebrafish larvae to inflate their swim bladders as a result of eugenol exposure may be caused by the inhibition of the Wnt/β-catenin signaling pathway inhibited. In addition, the inability to catch food due to the abnormal development of the swim bladder may be the key to the death of zebrafish larvae during the mouth-opening stage.

Single mRNA detection of Wnt signaling pathway in the human limbus

Limbal epithelial stem/progenitor cells (LSCs) are adult stem cells located at the limbus, tightly regulated by their close microenvironment. It has been shown that Wnt signaling pathway is crucial for LSCs regulation. Previous differential gene profiling studies confirmed the preferential expression of specific Wnt ligands (WNT2, WNT6, WNT11, WNT16) and Wnt inhibitors (DKK1, SFRP5, WIF1, FRZB) in the limbal region compared to the cornea. Among all frizzled receptors, frizzled7 (Fzd7) was found to be preferentially expressed in the basal limbal epithelium. However, the exact localization of Wnt signaling molecules-producing cells in the limbus remains unknown. The current study aims to evaluate the in situ spatial expression of these 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7. Wnt ligands, DKK1, and Fzd7 expression were scattered within the limbal epithelium, at a higher abundance in the basal layer than the superficial layer. SFRP5 expression was diffuse among the limbal epithelium, whereas WIF1 and FRZB expression was clustered at the basal limbal epithelial layer corresponding to the areas of high levels of Fzd7 expression. Quantitation of the fluorescence intensity showed that all 4 Wnt ligands, 3 Wnt inhibitors (WIF1, DKK1, FRZB), and Fzd7 were highly expressed at the basal layer of the limbus, then in a decreasing gradient toward the superficial layer (P < 0.05). The expression levels of all 4 Wnt ligands, FRZB, and Fzd7 in the basal epithelial layer were higher in the limbus than the central cornea (P < 0.05). All 4 Wnt ligands, 4 Wnt inhibitors, and Fzd7 were also highly expressed in the limbal stroma immediately below the epithelium but not in the corneal stroma (P < 0.05). In addition, Fzd7 had a preferential expression in the superior limbus compared to other limbal quadrants (P < 0.05). Taken together, the unique expression patterns of the Wnt molecules in the limbus suggests the involvement of both paracrine and autocrine effects in LSCs regulation, and a fine balance between Wnt activators and inhibitors to govern LSC fate.

Effect of Sox18 on the Induction Ability of Dermal Papilla Cells in Hu Sheep.

Sox18 is a developmental gene that encodes transcription factors. It has been indicated as be a key gene affecting the growth and development of hair follicles, in which dermal papilla cells (DPCs) have been demonstrated to play an important role through their ability to induce the formation of hair follicles. Pre-laboratory studies have found that Sox18 is differentially expressed in the dermal papilla cells of different pattern types of Hu sheep. We speculated that Sox18 plays an important role in the dermal papilla cells of Hu sheep. In our study, we analyzed the effect of Sox18 on the induction ability of DPCs in order to elucidate the function and molecular mechanism of Sox18 in the DPCs of Hu sheep. We first identified the expression of Sox18 in the DPCs of Hu sheep by immunofluorescence staining. We then used alkaline phosphatase staining, cell morphology observations and RT-PCR to detect the effect of Sox18 on the induction of DPCs after overexpression of or interference with Sox18. We also used RT-PCR, WB and immunofluorescence staining to detect the effect of Sox18 on the Wnt/β-catenin signal pathway in DPCs. We found that Sox18 was specifically expressed in the DPCs of Hu sheep, and that Sox18 could enhance the alkaline phosphatase activity in the DPCs of Hu sheep and accelerate cell agglutination. The results of RT-PCR revealed that Sox18 promoted the mRNA expression of Versican, HHIP and FGFRI, and inhibited the mRNA expression of BMP4 and WIF1. Further studies showed that Sox18 promoted the expression of β-catenin and activated the Wnt/β-catenin signal pathway in DPCs. When the Wnt/β-catenin signal pathway of DPCs was activated, the induction ability of DPCs was enhanced. Overall, we believe that Sox18 could enhance the induction ability of DPCs in Hu sheep and regulate the induction ability of DPCs through the Wnt/β-catenin signal pathway.

Creb5 coordinates synovial joint formation with the genesis of articular cartilage

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

Prostate Cancer Secretome and Membrane Proteome from Pten Conditional Knockout Mice Identify Potential Biomarkers for Disease Progression.

Prostate cancer (PCa) is the second most common cause of mortality among men. Tumor secretome is a promising strategy for understanding the biology of tumor cells and providing markers for disease progression and patient outcomes. Here, transcriptomic-based secretome analysis was performed on the PCa tumor transcriptome of Genetically Engineered Mouse Model (GEMM) Pb-Cre4/Ptenf/f mice to identify potentially secreted and membrane proteins—PSPs and PMPs. We combined a selection of transcripts from the GSE 94574 dataset and a list of protein-coding genes of the secretome and membrane proteome datasets using the Human Protein Atlas Secretome. Notably, nine deregulated PMPs and PSPs were identified in PCa (DMPK, PLN, KCNQ5, KCNQ4, MYOC, WIF1, BMP7, F3, and MUC1). We verified the gene expression patterns of Differentially Expressed Genes (DEGs) in normal and tumoral human samples using the GEPIA tool. DMPK, KCNQ4, and WIF1 targets were downregulated in PCa samples and in the GSE dataset. A significant association between shorter survival and KCNQ4, PLN, WIF1, and F3 expression was detected in the MSKCC dataset. We further identified six validated miRNAs (mmu-miR-6962-3p, mmu-miR- 6989-3p, mmu-miR-6998-3p, mmu-miR-5627-5p, mmu-miR-15a-3p, and mmu-miR-6922-3p) interactions that target MYOC, KCNQ5, MUC1, and F3. We have characterized the PCa secretome and membrane proteome and have spotted new dysregulated target candidates in PCa.

The effect of ω-6 fatty acid on WT1 and WIF-1 genes expression and inducing apoptosis in A375 melanoma cell line

BackgroundAn exciting question as to whether nutritional linoleic acid (LA, Omega-6, or ω-6) fatty acid interferes with tumorigenesis is remained to be entirely determined in well experimentally qualified investigations. To elucidate the particular roles of LA in tumorigenesis of melanoma skin cancer, we evaluated the effect of this fatty acid on a melanoma cell line, A375, in terms of tumor growth characteristics by MTT and Flow Cytometry. In particular, we have analyzed the expression levels of two genes (WIF-1 and WT1) involved during Wnt signaling pathway as a molecular base for the formation and progression of many cancer types. MethodsIn order to analyze the cell viability, MTT assay was performed following the treatment of cultured human melanoma cell line, A375, by various concentrations of ω-6 in 24 h, 48 h, and 72 h time intervals, and apoptosis was determined using Flow Cytometry. The expression of WIF-1 and WT1 genes were measured using quantitative real-time PCR, which were further analyzed by Rest 2.0.13 Software. ResultsOur findings revealed that during treatment of cells with all the concentrations of ω-6 in various time intervals, the expression of the WIF-1 gene was significantly (p < 0.05) increased in contrast to decreasing WT1 expression levels in a time- and concentration-dependent manner. The cell viability was also shown to be decreased with an IC50 value of 1000 μM. The differences were more substantial following 72 h of treatment. DiscussionOur results supported the contributory role of ω-6 on the tumor growth of melanoma cells, highlighting the potential of this fatty acid as an influencing element which at least in part exerts its inhibitory roles by interfering with Wnt signaling molecules WIF-1 and WT1.

Wif1 Mediates Coordination of Bone Morphogenetic Protein and Wnt Signaling in Neural and Glioma Stem Cells

Wnts, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) are paracrine signaling pathways implicated in the niche control of stem cell fate decisions. BMP-on and Wnt-off are the dominant quiescent niche signaling pathways in many cell types, including neural stem cells (NSCs). However, among the multiple inhibitory family members of the Wnt pathway, those with direct action after BMP4 stimulation in NSCs remain unclear. We examined 11 Wnt inhibitors in NSCs after BMP4 treatment. Wnt inhibitory factor 1 (Wif1) has been identified as the main factor reacting to BMP4 stimuli. RNA sequencing confirmed that Wif1 was markedly upregulated after BMP4 treatment in different gene expression analyses. Similar to the functional role of BMP4, Wif1 significantly decreased the cell cycle of NSCs and significantly inhibited cell proliferation (P < 0.05). Combined treatment with BMP4 and Wif1 significantly enhanced the inhibition of cell growth compared with the single treatment (P < 0.05). Wif1 expression was clearly lower in glioblastoma and low-grade glioma samples than in normal samples (P < 0.05). A functional analysis revealed that both BMP4 and Wif1 could decrease glioma cell growth. These effects were abrogated by the BMP inhibitor Noggin. The collective findings demonstrate that Wif1 plays a key role in quiescent NSC homeostasis and glioma cell growth downstream of BMP-on signaling. The functional roles of Wif1/BMP4 in glioma cells may provide a technical basis for regenerative medicine, drug discovery, and personal molecular therapy in future clinical treatments.

Cellular retinol binding protein-1 inhibits cancer stemness via upregulating WIF1 to suppress Wnt/\u03b2-catenin pathway in hepatocellular carcinoma

BackgroundCRBP-1, a cytosolic chaperone of vitamin A, is identified in a serious number of cancers; however, its biological role in hepatocellular carcinoma (HCC) needs to be further explored. The aim of our present study is to explore the roles and mechanisms of CRBP-1 in regulating liver cancer by using in vitro and in vivo biology approaches.MethodsThe expression level of CRBP-1 was detected using immunohistochemistry in HCC and matching adjacent non-tumorous liver tissues. Following established stable CRBP-1 overexpressed HCC cell lines, the cell growth and tumorigenicity were investigated both in vitro and in vivo. Intracellular retinoic acid was quantified by ELISA. The relationship between CRBP-1 and WIF1 was validated by using dual luciferase and ChIP analyses.ResultsThe low expression of CRBP-1 was observed in HCC tissues compared to the normal liver tissues, while high CRBP-1 expression correlated with clinicopathological characteristics and increased overall survival in HCC patients. Overexpression of CRBP-1 significantly inhibited cell growth and tumorigenicity both in vitro and in vivo. Moreover, overexpression of CRBP-1 suppressed tumorsphere formation and cancer stemness related genes expression in HCC. Mechanically, CRBP-1 inhibited Wnt/β-catenin signaling pathway to suppress cancer cell stemness of HCC. Furthermore, our results revealed that CRBP-1 could increase the intracellular levels of retinoic acid, which induced the activation of RARs/RXRs leading to the transcriptional expression of WIF1, a secreted antagonist of the Wnt/β-catenin signaling pathway, by physically interacting with the region on WIF1 promoter.ConclusionOur findings reveal that CRBP-1 is a crucial player in the initiation and progression of HCC, which provide a novel independent prognostic biomarker and therapeutic target for the diagnosis and treatment of HCC.

The status of WIF1 methylation in cell-free DNA is associated with the insusceptibility for gefitinib in the treatment of lung cancer.

Targeted cancer therapy has shed light on the treatment of tumor, especially for patients with non-small cell lung cancer. However, only a limited portion of NSCLC patients carrying specific mutations showed an ideal drug response. In addition, DNA methylation status showed a great potential for cancer detection and prognosis prediction. Bisulfite sequencing was performed to analyze the DNA methylation of WIF1 promoter in cfDNA and tumor tissue samples collected from NSCLC patients. PFS and OS analyses were carried out to evaluate the prognosis of gefitinib treatment in patients with differential levels of WIF1 DNA methylation. Quantitative real-time PCR was used to analyze the expression of WIF1 mRNA, while immunohistochemistry was performed to assess the expression of WIF1 protein. Furthermore, ELISA was carried out to evaluate the WIF1 activity in plasma. The DNA methylation level of WIF1 promoter was lower in the cfDNA of NSCLC patients with a complete or partial response to gefitinib, and NSCLC patients with hypomethylated WIF1 showed better PFS and OS. The DNA methylation of WIF1 promoter in the resected tumor tissues was consistent with WIF1 DNA methylation in cfDNA, indicating that cfDNA was mainly derived from lung cancer tissues. As a result, the expression of WIF1 in tissue samples and the WIF1 activity in plasma was inhibited in patients with hypermethylated WIF1. Moreover, the cell viability of gefitinib-resistant cells was decreased by the suppressed WIF1 methylation in vitro. And the expression level of WIF1 mRNA was higher in gefitinib-resistant cells overexpressing ALKBH5, a known suppressor of WIF1 methylation. In summary, the findings of this study demonstrated that the level of WIF1 methylation in cfDNA was associated with the insusceptibility of gefitinib in the treatment of lung cancer.

Exosomal circ_0030167 derived from BM-MSCs inhibits the invasion, migration, proliferation and stemness of pancreatic cancer cells by sponging miR-338-5p and targeting the Wif1/Wnt8/β-catenin axis.

Pancreatic cancer (PC) is one of the most lethal malignant tumors and has the lowest survival rate due to early metastasis and drug resistance. Exosomes derived from bone marrow mesenchymal stem cells (BM-MSCs) have emerged as crucial regulators of the progression of various tumors. These vesicles contain abundant circRNAs that have important biological functions. This study aimed to elucidate the role of exosomal circRNAs in PC progression. In this study, we successfully isolated BM-MSCs from human bone marrow based on their surface marker expression and osteogenic and adipogenic differentiation potential. We found that BM-MSC-derived exosomes significantly reduced the invasion, migration, and proliferation of PC cells, as well as tumor stemness. According to whole-transcriptome resequencing and clustering heat map analysis, we identified the key molecule circ_0030167 and miR-338-5p, its downstream target. We revealed that circ_0030167 mainly regulates miR-338-5p, enhances Wif1 expression, and inhibits the Wnt8/β-catenin pathway, thereby inhibiting the stemness of PC cells and tumor progression. Overall, BM-MSC exosomal circ_0030167 contributes to the progression and stemness of PC cells via the miR-338-5p/wif1/Wnt 8/β-catenin axis. Our study provides a new perspective for the treatment of PC.

DNA methylation and gene expression of sFRP2, sFRP4, Dkk 1, and Wif1 during osteoblastic differentiation of bone marrow derived mesenchymal stem cells

ObjectivesBone marrow derived mesenchymal stem cells (BMSCs) are an irresistible choice for use in stem cell therapy and regenerative medicine. BMSCs osteoblastic differentiation is also important in bone development, diseases, malignancies, and cancers studies. Wnt signaling pathway antagonists, Dickkopf-1 (Dkk 1), Secreted Frizzled-Related Proteins (sFRPs), and Wnt Inhibitory Factor 1 (Wif1) play important roles in inducing osteoblastic differentiation. This study is the first to investigate the association between DNA methylation and gene expression of Dkk1, sFRP2, sFRP4, and Wif1 during BMSCs osteoblastic differentiation. MethodsHuman BMSCs were isolated and characterized using flow cytometry. Then, cells were treated with osteo-differentiation medium for three weeks. Alizarin red S staining and polymerase chain reaction (PCR) (alkaline phosphatase/osteocalcin) were performed for confirmation. The expression of Dkk 1, sFRP2, sFRP4, and Wif1 genes was evaluated at days 7, 14, and 21 using real-time PCR. Methylation-specific PCR (MSP) was performed to detect the methylation status of the promoters of the genes. ResultsData showed significant decreases (P < 0.05) during various days of BMSCs differentiation, while the promoters of the genes remained mostly un-methylated. ConclusionsThe down-regulation of Dkk 1, sFRP2, sFRP4, and Wif1 regulates various stages of human BMSCs during osteoblastic differentiation. DNA methylation does not interfere in the down-regulation of these genes, except for Wif1. We propose that the Wnt antagonist gene promoters should remain un-methylated during osteoblastic differentiation of BMSCs and that the down-regulation of these genes may contribute to other epigenetic mechanisms, other than DNA methylation, which implicitly indicates the role of DNA methylation in osteogenic cancers.

Indirubin inhibits Wnt/\u03b2-catenin signal pathway via promoter demethylation of WIF-1

BackgroundPsoriasis is a common inflammatory skin disease. Abnormal proliferation of keratinocytes is one of the psoriatic histopathological features. Indirubin has an essential effect on the proliferation and activation of keratinocytes; however, in psoriasis, the specific mechanism of action of indirubin on keratinocytes is unclear. In the present study, we revealed the effects of indirubin on DNA methyltransferase 1 (DNMT1), wnt inhibitory factor 1 (wif-1), and wnt/β-catenin signal pathway, in the meantime, we explored the effects of indirubin on proliferation, cell cycle and the apoptosis of HaCaT cells.MethodsThe expression of DNMT1, wif-1, Frizzled2, Frizzled5, and β-catenin in HaCaT cells treated with different concentrations of indirubin were detected by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The expression levels of DNMT1 and wif-1 were observed after treated with different concentrations of indirubin by enzyme-linked immunosorbent assay (ELISA). The wif-1 promoter methylation status was detected by DNA methylation-specific PCR (MSP). The transcriptional activities of wif-1 and β-catenin were discovered by a luciferase reporter gene system. Cell viability was determined by Cell Counting Kit-8 (CCK8) method. The cell cycle was detected by flow cytometry. The apoptotic cells were surveyed by the apoptosis kit. The expression of Inolucrin, Loricrin, Filaggrin, Keratin 17, and transcriptional activation of transglutaminase 1(TGase1) were detected by Western blotting.ResultsIndirubin inhibited the expression of DNMT1 and the methylation of the wif-1 promoter. In the wnt signal pathway, indirubin restored the protein expression of wif-1 and inhibited expression of Frizzled2, Frizzled5, and β-catenin. Besides, indirubin inhibited the proliferation of HaCaT cells, induced apoptosis, and arrest cell cycle. We also reported that indirubin could down-regulate the expression of Involucrin, TGase 1, and keratin 17, but the expression of Filaggrin and Loricrin had no significant effect.ConclusionOur research showed that indirubin promoted the demethylation of wif-1 and suppressed the wnt/β-catenin signal pathway, thereby exerted an anti-proliferative effect. This study reveals the anti-proliferation mechanism of indirubin, which may provide an effective option for the treatment of proliferative diseases.