Wnt Inhibitory Factor-1 Expression Research Articles

WNT-inhibitory factor 1-mediated glycolysis protects photoreceptor cells in diabetic retinopathy

BackgroundIn diabetic retinopathy (DR), hypoxia-inducible factor (HIF-1α) induces oxidative stress by upregulating glycolysis. This process leads to neurodegeneration, particularly photoreceptor cell damage, which further contributes to retinal microvascular deterioration. Further, the regulation of Wnt-inhibitory factor 1 (WIF1), a secreted Wnt signaling antagonist, has not been fully characterized in neurodegenerative eye diseases. We aimed to explore the impact of WIF1 on photoreceptor function within the context of DR.MethodTwelve-week-old C57BL/KsJ-db/db mice were intravitreally injected with WIF1 overexpression lentivirus. After 4 weeks, optical coherence tomography (OCT), transmission electron microscopy (TEM), H&E staining, and electroretinography (ERG) were used to assess the retinal tissue and function. The potential mechanism of action of WIF1 in photoreceptor cells was explored using single-cell RNA sequencing. Under high-glucose conditions, 661 W cells were used as an in vitro DR model. WIF1-mediated signaling pathway components were assessed using quantitative real-time PCR, immunostaining, and western blotting.ResultTypical diabetic manifestations were observed in db/db mice. Notably, the expression of WIF1 was decreased at the mRNA and protein levels. These pathological manifestations and visual function improved after WIF1 overexpression in db/db mice. TEM demonstrated that WIF1 restored damaged mitochondria, the Golgi apparatus, and photoreceptor outer segments. Moreover, ERG indicated the recovery of a-wave potential amplitude. Single-cell RNA sequencing and in vitro experiments suggested that WIF1 overexpression prevented the expression of glycolytic enzymes and lactate production by inhibiting the canonical Wnt signaling pathway, HIF-1α, and Glut1, thereby reducing retinal and cellular reactive oxygen species levels and maintaining 661 W cell viability.ConclusionsWIF1 exerts an inhibitory effect on the Wnt/β-catenin-HIF-1α-Glut1 glycolytic pathway, thereby alleviating oxidative stress levels and mitigating pathological structural characteristics in retinal photoreceptor cells. This mechanism helps preserve the function of photoreceptor cells in DR and indicates that WIF1 holds promise as a potential therapeutic candidate for DR and other neurodegenerative ocular disorders.Graphical

WIF1 was downregulated in cervical cancer due to promoter methylation.

Wnt inhibitory factor 1 (WIF1) is frequently downregulated in a variety of cancer due to promoter methylation. However, the methylation status of the WIF1 promoter in cervical cancer remains unclear. This study aimed to elucidate the mechanism by which WIF1 promoter methylation contributes to cervical cancer development. The expression of WIF1 in cervical cancer tissues was examined by immunohistochemistry. The methylation status of the WIF1 promoter in cervical cancer cells was detected by methylation specific PCR. WIF1 mRNA levels and protein levels were detected by PCR and Western blot analysis. We found that WIF1 expression was low in cervical cancer tissues compared to adjacent normal cervical tissues. The WIF1 promoter was methylated in the cervical cancer SiHa cell line but not in the normal cervical epithelial cell line Ect1. Correspondingly, WIF1 mRNA levels and protein levels were significantly lower in SiHa cells than in Ect1 cells. Treatment with 5-aza-2-deoxycytidine (AZA) led to the upregulation of WIF1 mRNA and protein levels in SiHa cells, but the effects were abrogated by treatment with WIF1 siRNA. In addition, AZA treatment induced apoptosis and inhibited the invasion of SiHa cells, and the effects were abrogated by WIF1 siRNA. The protein levels of survivin, c-myc and cyclinD1 were significantly lower in SiHa cells treated with AZA, but their levels were upregulated after treatment with WIF1 siRNA. In conclusion, the methylation of the WIF1 promoter leads to the downregulation of WIF1 and the activation of Wnt/β-catenin signaling in cervical cancer cells. WIF1 is a tumor suppressor that is inactivated in cervical cancer.

Effects of phthalates on human chorionic trophoblast cells and mouse embryonic development

Phthalate exposure is associated with reproductive health, but the mechanism is unclear. This study used human chorionic trophoblast epithelial cells (HTR8/Svneo cells) and mouse embryos as objects aims to explore the effects of phthalate plasticizers on germ cells and fertility and the possible signalling pathways. In the present study, high concentrations of MEHP for 24 h significantly inhibited the proliferation and viability of HTR8/SVneo cells. Compared with the negative control (NC) group, the MEHP medium and high concentration groups promoted the apoptosis of HTR8/SVneo cells and inhibited the cell cycle, HTR8/SVneo cells were blocked in G1/G0 phase and could not enter S phase, and cell meiosis was inhibited. Western blot experiments showed that there was no difference in the protein expression of wnt inhibitory factor 1 (WIF1) and β-catenin in HTR8/SVneo cells between the MEHP exposure groups and the NC groups. In vitro embryo culture experiments found that there was no difference in blastocyst formation rate among groups after exposure to DEHP for 2 h. Immunofluorescence showed that the expression of WIF1 decreased in the low concentration group, and there was no difference in the medium and high concentration groups, while the expression of β-catenin was increased in both the low concentration group and the high concentration group. Our data suggest that exposure to phthalate plasticizers can affect the viability, cell cycle and apoptosis of trophoblast cells, resulting in abnormal expression of the embryonic WIF1/β-catenin signalling pathway and impaired fertility.

METTL3 is aberrantly expressed in endometriosis and suppresses proliferation, invasion, and migration of endometrial stromal cells.

Endometriosis (EM) is one of the leading gynecological disorders, and associated with excessive functioning of endometrial stromal cells (ESCs). The current study was conducted to determine the expression and role of methyltransferase-like 3 (METTL3) in the proliferation, invasion, and migration of ESCs in EM. The documented expression levels of METTL3, microRNA (miR)-21-5p, and WNT inhibitory factor 1 (WIF1) in eutopic (Eut) and ectopic (Ect) endometrial tissues and ESCs were determined by a combination of real-time quantitative polymerase chain reaction and Western blot assay. After transfection with pcDNA3.1-METTL3, miR-21-5p mimic, and WIF1 small interfering RNA, cell counting kit-8, colony formation, and Transwell assays were performed in the Ect ESCs (Ect-ESCs). Subsequently, the binding of miR-21-5p to METTL3 was analyzed, along with quantification of the N6-methyladenosine (m6A) level, the enrichments of METTL3 and m6A on WIF1, and the mRNA stability of WIF1. In our findings, METTL3 was downregulated in the EM tissues and cells. METTL3 overexpression intrinsically reduced the proliferation, invasion, and migration of Ect-ESCs. miR-21-5p inhibited the METTL3 expression while METTL3 enhanced the mRNA stability and expression of WIF1 via m6A modification. Additionally, a negative correlation of METTL3 was identified with miR-21-5p along with a positive correlation with the WIF1 mRNA in EM tissues. The miR-21-5p overexpression or WIF1 downregulation enhanced the proliferation, invasion, and migration of Ect-ESCs. Collectively, miR-21-5p inhibited the METTL3-mediated m6A modification and mRNA stability of WIF1, thereby facilitating the proliferation, invasion, and migration of Ect-ESCs.

20(S)- Protopanaxadiol suppresses hepatic stellate cell activation via WIF1 demethylation-mediated inactivation of the Wnt/β-catenin pathway

Background20(S)-protopanaxadiol (PPD), one of the main components of ginseng, has anti-inflammatory, anti-estrogenic, and anti-tumor activities. It is known that activated hepatic stellate cells (HSCs) are the primary producers of extracellular matrix (ECM) in the liver, and the Wnt/β-catenin pathway participates in the activation of HSCs. We aimed to explore whether PPD inhibits liver fibrosis is associated with the Wnt/β-catenin pathway inactivation. MethodsThe anti-fibrotic roles of PPD were examined both in vitro and in vivo. We also examined the levels of Wnt inhibitory factor 1 (WIF1), DNA methyltransferase 1 (DNMT1) and WIF1 methylation. ResultsPPD obviously ameliorated liver fibrosis in carbon tetrachloride (CCl4)-treated mice and reduced collagen deposition. PPD also suppressed the activation and proliferation of primary HSCs. Notably, PPD inhibited the Wnt/β-catenin pathway, reduced TCF activity, and increased P-β-catenin and GSK-3β levels. Interestingly, WIF1 was found to mediate the inactivation of the Wnt/β-catenin pathway in PPD-treated HSCs. WIF1 silencing suppressed the inhibitory effects of PPD on HSC activation and also restored α-SMA and type I collagen levels. The downregulation of WIF1 expression was associated with the methylation of its promoter. PPD induced WIF1 demethylation and restored WIF1 expression. Further experiments confirmed that DNMT1 overexpression blocked the effects of PPD on WIF1 expression and demethylation and enhanced HSC activation. ConclusionPPD up-regulates WIF1 levels and impairs Wnt/β-catenin pathway activation via the down-regulation of DNMT1-mediated WIF1 methylation, leading to HSC inactivation. Therefore, PPD may be a promising therapeutic drug for patients with liver fibrosis.

Gene Methylation and Silencing of WIF1 Is a Frequent Genetic Abnormality in Mantle Cell Lymphoma.

We have previously shown that the Wnt canonical pathway (WCP) is constitutively active in most cases of mantle cell lymphoma (MCL). Here, we aimed to elucidate the mechanisms underlying this biochemical deregulation. We hypothesized that gene methylation/silencing of WIF1 (Wnt inhibitory factor-1), a physiologic inhibitor of WCP, contributes to the deregulation of WCP and promotes cell growth in MCL. In support of this hypothesis, we found that the expression of WIF1 was detectable in none of the 4 MCL cell lines, and in only 2 of 5 tumors (40%) examined. Using methylation-specific PCR, we found evidence of gene methylation of WIF1 in 4 of 5 cell lines (80%) and in 24 of 29 (82%) tumors. The addition of the demethylation agent 5-aza-2′-deoxycytidine to Mino and JeKo-1, two WIF1-negative cell lines, restored the expression of WIF1 mRNA in these cells. Gene transfection of WIF1 into JeKo-1 and Mino cells significantly reduced cell growth, and this finding correlated with substantial downregulations of various proteins in WCP, such as β-catenin and pGSK-3β. In conclusion, our results support the concept that gene methylation/silencing of WIF1 is a frequent event in MCL, and this abnormality contributes to the aberrant activation of WCP. These results have provided further evidence that aberrant Wnt signaling is pathogenetically important in MCL and it may represent a potential therapeutic target.

Pinostilbene hydrate suppresses hepatic stellate cell activation via inhibition of miR-17–5p-mediated Wnt/β-catenin pathway

BackgroundIn the development of liver fibrosis, activated hepatic stellate cells (HSCs) contribute to the synthesis and deposition of extracellular matrix (ECM) proteins. HSC activation is considered as a central driver of liver fibrosis. Recently, microRNAs (miRNAs) have been reported to act as key regulators in HSC activation. PurposePinostilbene hydrate (PSH), a methylated derivative of resveratrol, has demonstrated anti-inflammatory, antioxidant and anti-tumour activities. However, the effects of PSH on HSC activation remain unclear. MethodsThe effects of PSH on HSC activation were examined. Moreover, the roles of WNT inhibitory factor 1 (WIF1) and miR-17–5p in the effects of PSH on HSC activation were examined. ResultsPSH induced a significant reduction in HSC proliferation. PSH also effectively inhibited HSC activation, with reduced α-SMA and collagen expression. Notably, it was found that Wnt/β-catenin signalling was involved in the effects of PSH on HSC activation. PSH resulted in Wnt/β-catenin signalling inactivation, with a reduction in TCF activity as well as β-catenin nuclear translocation. Further studies showed that PSH inhibited Wnt/β-catenin signalling via regulation of WIF1 and miR-17–5p. Reduced HSC activation caused by PSH could be restored by loss of WIF1 or miR-17–5p mimics. Luciferase reporter assays further confirmed that WIF1 was a target of miR-17–5p. ConclusionPSH has a significant protective effect against HSC activation. In addition, we demonstrate that PSH enhances WIF1 expression and inhibits Wnt/β-catenin signalling via miR-17–5p, contributing to the suppression of HSC activation.

Changes of WIF-1 and WT-1 genes expression following the anti-cancer effects of omega-3 and omega-6 on gastric cancer cells

Gastric cancer is one of the most common cancers. Environmental factors, especially diet, play an important role in causing gastric cancer. The omega-3 (ω−3) and omega-6 (ω−6) from the polyunsaturated fatty acids (PUFAs) group as the natural compounds indicated significant effects on inhibiting various types of tumors. In this study, we investigated the anti-cancer properties of ω−3 and ω−6 on gastric cancer cells with focusing on WNT signaling pathway via WNT inhibitory Factor-1 (WIF-1) and The Wilms Tumor-1 (WT-1) genes.AGS cells were used to investigate the effects of ω−3 and ω−6 on cell viability, apoptosis and the expression of WIF-1 and WT-1 genes in gastric cancer. AGS cells were treated with different concentrations of ω−3 and ω−6 for 24, 48, and 72 h, and then cell viability was assessed by MTT assay. The apoptosis rate was assessed using the flow cytometery test in every three times. The mRNA expression of WIF-1 and WT-1 were examined by quantitative real-time PCR (qRT-PCR).Our study revealed that ω−3 and ω−6 provide various antitumor activities such as inhibition of cell viability in a dose and time-dependent manner. The results of flow cytometery showed that cytotoxicity effects are due to the ability of ω−3 and ω−6 to induce apoptosis. In addition, ω−3 and ω−6 significantly increased the expression of WIF-1, whereas WT-1 level decreased in AGS cells compared to the control group.These results suggested that ω−3 and ω−6 can serve as therapeutics in against gastric cancer by targeting of the WNT signaling pathway.

A long non-coding RNA, HOTAIR, promotes cartilage degradation in osteoarthritis by inhibiting WIF-1 expression and activating Wnt pathway

BackgroundLong noncoding RNAs (lncRNAs) are recently found to be critical regulators of the epigenome. However, our knowledge of their role in osteoarthritis (OA) development is limited. This study investigates the mechanism by which HOTAIR, a key lncRNA with elevated expression in OA, affects OA disease progression.ResultsHOTAIR expression was greatly elevated in osteoarthritic compared to normal chondrocytes. Silencing and over-expression of HOTAIR in SW1353 cells respectively reduced and increased the expression of genes associated with cartilage degradation in OA. Investigation of molecular pathways revealed that HOTAIR acted directly on Wnt inhibitory factor 1 (WIF-1) by increasing histone H3K27 trimethylation in the WIF-1 promoter, leading to WIF-1 repression that favours activation of the Wnt/β-catenin pathway.ConclusionsActivation of Wnt/β-catenin signalling by HOTAIR through WIF-1 repression in osteoarthritic chondrocytes increases catabolic gene expression and promotes cartilage degradation. This is the first study to demonstrate a direct link between HOTAIR, WIF-1 and OA progression, which may be useful for future investigations into disease biomarkers or therapeutic targets.

A study on the mechanism of Wnt inhibitory factor 1 in osteoarthritis.

IntroductionIn our study we aimed to investigate the mechanism of Wnt inhibitory factor 1 (WIF1) on regulating chondrocyte proliferation and apoptosis via reactive oxygen species (ROS) and the Wnt/βcatenin signaling pathway in osteoarthritis (OA).Material and methodsOsteoarthritis chondrocytes were treated with interleukin 1β (IL-1β) to simulate an inflammatory condition. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were applied for detecting WIF1 expression in OA chondrocytes. MTT assay and flow cytometry were carried out to analyze the cell proliferation and apoptosis. Content of ROS was detected using flow cytometry, and activity of the Wnt/βcatenin signaling pathway was detected using immunofluorescence, western blot and luciferase reporter assay. Western blot and enzyme-linked immunosorbent assay (ELISA) were performed to detect the expression of apoptosis-related proteins and secretion of matrix metalloproteinases (MMPs).ResultsWIF1 expression in OA chondrocytes was significantly lower than in normal chondrocytes. After WIF1 cDNA transfection, the aberrantly high ROS level in OA chondrocytes was down-regulated, which led to the increase of proliferation and reduction of apoptosis. The Wnt/βcatenin signaling pathway was suppressed by WIF1 overexpression and the secretion of MMPs was therefore reduced.ConclusionsUp-regulation of WIF1 would promote proliferation and suppress apoptosis of OA chondrocytes through eliminating ROS production and reduce secretion of MMPs via blocking the Wnt/βcatenin signaling pathway.

MiR-552-5p facilitates osteosarcoma cell proliferation and metastasis by targeting WIF1.

miR-552 promotes tumor growth and metastasis in colorectal cancer. However, the function of miR-552 in osteosarcoma remains unclear. The current study investigated the role and mechanism of miR-552-5p in the proliferation, migration and invasion of osteosarcoma cells. miR-552-5p was significantly upregulated in osteosarcoma tissues and cell lines compared with adjacent normal tissues and normal osteoblast cells. Knockdown of miR-552-5p significantly reduced the proliferation of MG63 and U2OS cells, and inhibited cell migration and invasion. Wnt inhibitory factor 1 (WIF1) was the direct target gene of miR-552-5p in osteosarcoma cells. Overexpression of miR-552-5p markedly decreased the expression of WIF1 in MG63 and U2OS cells. A negative association was identified between the expression levels of miR-552-5p and WIF1 in osteosarcoma tissues. Furthermore, the expression of WIF1 was downregulated in osteosarcoma tissues and cell lines. Finally, knockdown of WIF1 in MG63 and U2OS cells treated with miR-552-5p inhibitors rescued their ability to proliferate, migrate and invade. Overall, the results indicated that miR-552-5p promoted osteosarcoma development and progression by inhibiting WIF1. Therefore, miR-552-5p may serve as a new therapeutic target for treatment of patients with osteosarcoma.

Molecular cloning of WIF1 and HMGA2 reveals ear-preferential expression while uncovering a missense mutation associated with porcine ear size in WIF1.

Considerable diversity exists in porcine ear size, which is an important morphological feature of pig breeds. Previously, we localized four crucial candidate genes-high mobility group AT-hook 2 (HMGA2), LEM domain-containing 3 (LEMD3), methionine sulfoxide reductase B3 (MSRB3) and Wnt inhibitory factor 1 (WIF1)-on Sus Scrofa chromosome 5 affecting porcine ear size, then cloned LEMD3 and MSBR3. In this study, we performed rapid amplification of cDNA ends to obtain full-length cDNA sequences of 2338-bp WIF1 and 2998-bp HMGA2. Using quantitative real-time PCR, we revealed that WIF1 expression was highest in ear cartilage of 60-day-old pigs and that this is therefore a better candidate gene for ear size than HMGA2. We further screened coding sequence variants in both genes and identified only one missense mutation (WIF1:c.1167C>G) in a conserved epidermal growth factor-like domain from the mammalian WIF1 protein. The protein-altering mutation was significantly associated with ear size across the Large White×Minzhu hybrid and Beijing Black pig populations. When WIF1:c.1167C>G was included as fixed effect in the model to re-run a genome-wide association study in the Large White×Minzhu intercross population the P-value of the peak SNP on SSC5 from re-running the genome-wide association study dropped from 2.45E-12 to 7.33E-05. Taken together, the WIF1:c.1167C>G could be an important mutation associated with ear size. Our findings provide helpful information for further studies of the molecular mechanisms controlling porcine ear size.

UPA affects the CRSsNP nasal mucosa epithelium apoptosis by regulating WIF1

Chronic rhinosinusitis without nasal polyps (CRSsNP) is the main type of Chronic rhinosinusitis (CRS) and is a common otorhinolaryngologic disease worldwide. However, the mechanisms of CRSsNP remain poorly understood. In this study, C57BL/6J wild-type and urokinase-type plasminogen activator (uPA) gene knockout (uPA-/-) mice were used to construct the CRSsNP model. Primary human nasal epithelial cells (HNEC) were isolated from CRSsNP patient and treated with uPA knockdown/overexpression lentivirus. CCK-8 and Annexin-V/PI staining were used to detected cell proliferation and apoptosis. In vivo, we found that uPA depletion alleviated mucosal inflammation in the CRSsNP mice model. Wnt inhibitory factor 1 (WIF1) was upregulated in the uPA-/- CRSsNP mice model. In vitro, inhibition of uPA increased cell proliferation and decreased cell apoptosis. Mechanistically, uPA depletion upregulated WIF1 and BCL2 expression, and reduced the expression level of BAX in CRSsNP HNEC. In contrast, decreased cell proliferation and increased cell apoptosis were observed after uPA overexpression. Consistently, a reduction in WIF1 and BCL2 expression levels and an increase in the BAX expression level were observed upon uPA ectopic expression. Furthermore, WIF1 overexpression rescued the effects caused by uPA overexpression in vitro. In conclusion, uPA affects the CRSsNP nasal mucosal epithelium cell apoptosis by upregulating WIF1. To our knowledge, this is the first study to explore the role of uPA in CRSsNP to date.

Knockdown of HOXC6 inhibits glioma cell proliferation and induces cell cycle arrest by targeting WIF-1 in vitro and vivo

BackgroundHomeobox C6 (HOXC6) is one of several HOXC genes and is frequently overexpressed in multiple cancers. However, the function and mechanism of HOXC6 in glioma remain unclear. MethodsThe expression level of HOXC6 and its relationship with prognosis in glioma were determined through the TCGA database. The expressions of HOXC6 mRNA in glioblastoma tissues and normal brain tissues were detected by qRT-PCR and Western blot. To explore the role of HOXC6 in glioma, a lentiviral vector that expressed HOXC6-shRNA was constructed and transfected into glioma U87 cells. The expression levels of HOXC6 and WNT inhibitory factor 1 (WIF-1) in the glioma U87 cells after transfection with HOXC6-shRNA were measured by real-time PCR and Western blot． CCK-8, colony formation and EdU assays were used to measure the effects of HOXC6 on U87 cell proliferation, and flow cytometry was used to monitor the changes in the cell cycle and cell apoptosis after transfection with HOXC6-shRNA. Xenograft tumors were examined in vivo for the carcinogenic effects and prognostic value of HOXC6 in glioma tissues. ResultsIn this study, HOXC6 was highly expressed in human glioma tissues, and a high expression of HOXC6 was associated with poor prognosis in GBM patients. We demonstrated that HOXC6 was highly expressed in human GBM tissues and three glioma cell lines. The knockdown of HOXC6 expression significantly inhibited the proliferation and colony formation ability of U87 cells by blocking cell cycle progression in the G0/G1 phase and induced apoptosis. In addition, we found that the mRNA and protein levels of WIF-1 were substantially increased after transfection with HOXC6-shRNA compared with Ctrl-shRNA in vitro. Consistent with the results of the in vitro assays, the xenograft assay and immunohistochemistry also demonstrated that in response to HOXC6 inhibition, the tumor growth and Ki-67 expression level were inhibited and the WIF-1 expression was increased in vivo. ConclusionsIn conclusion, the results of the current study indicate that HOXC6 promotes glioma U87 cell growth through the WIF-1/Wnt signaling pathway and HOXC6 might be a novel target in clinical treatment for gliomas.

Association of TGF-β1 and WIF1 Expression with 36 Paired Primary/Recurrent Nonfunctioning Pituitary Adenomas: A High-Throughput Tissue Microarrays Immunohistochemical Study

This study was undertaken primarily to research transforming growth factor β1 (TGF-β1) and Wnt inhibitory factor 1 (WIF1) for the prediction of nonfunctioning pituitary adenoma (NFPAs) invasion and recurrence of tumor samples and the relations between quantitatively determined markers and clinical characters. We studied 104 patients, including 59 patients without recurrence and 45 patients with recurrence (9 patients with one surgery and 36 patients operated twice, both tumors being studied). All tissues were immunostained for TGF-β1 and WIF1 using tissue microarrays and confirmed with real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot. We found that invasion, TGF-β1, and WIF1 were significantly associated with recurrence and that age was associated with low expression of TGF-β1 and WIF1 (P < 0.001). There were no statistically significant differences in the expression of the 2 proteins between the noninvasive and the invasive groups. The expression of TGF-β1 and WIF1 in primary tumors in the recurrence group was lower than in the nonrecurrence group (P < 0.001). In the 36 paired primary or recurrent tumors, the expression of TGF-β1 and WIF1 in recurrent tumors was higher than the expression of primary tumors, which was confirmed with qRT-PCR and Western blot. Therefore, TGF-β1 and WIF1 seem to be related to recurrence or progression of pituitary adenomas. The expression of TGF-β1 and WIF1 in NFPAs correlated with cell proliferation and recurrence potential. They may be good markers of progressive behavior in NFPAs; however, the biologic mechanism needs further study.

Genistein inhibits invasion and migration of colon cancer cells by recovering WIF1 expression

Colon cancer is characterized by invasion and migration. DNA methylation of CpG islands in tumor suppressor genes is considered to be an epigenetic mechanism underlying cancer development. Epigenetic silencing of a gene may be reversed by drugs, including genistein. The present study aimed to determine the effect of genistein on Wnt inhibitory factor 1 (WIF1) and invasion, and migration of colon cancer cells. The viability of HT29 colon cancer cells was suppressed by genistein in a dose dependent manner. Following 72h of treatment with 10, 20 and 60µmol/l genistein, increased demethylation of WIF1 was induced in a dose‑dependent manner. Additionally, the invasive/migratory abilities of cells treated with genistein decreased in a dose‑dependent manner. Reverse transcription‑quantitative polymerase chain reaction and western blot analyses were performed to identify the mRNA and protein expression levels of invasion/migration‑associated factors. Following treatment with genistein, matrix metalloproteinase (MMP) 2 and MMP9 expression levels decreased, whereas the expression of metalloproteinase inhibitor 1 and E‑cadherin increased significantly. In addition, the expression levels of proto‑oncogene Wnt‑1 (Wnt‑1)/β‑catenin pathway‑associated factors, β‑catenin, c‑Myc proto‑oncogene protein and cyclinD1 decreased in a dose‑dependent manner following treatment with genistein. The invasive/migratory abilities of cells transfected with WIF1‑small interfering (si) RNA, and those transfected with WIF1‑siRNA and treated with genistein, increased notably compared with the control group. The present study demonstrated that genistein was able to inhibit the cell invasion and migration of colon cancer cells by inducing demethylation, and recovering the activity of WIF1 by altering the expression of invasion‑associated factors, and components of the Wnt signaling pathway.

LEF1, TFE3, and AR are putative diagnostic markers of solid pseudopapillary neoplasms.

The diagnosis of solid pseudopapillary neoplasms (SPNs) is challenging because some SPNs share many similar morphological and immunohistochemical features with other pancreatic neoplasms. In this study, we investigated potential diagnostic markers of SPN.Based on the SPN-specific upregulated genes from a previous DNA microarray and proteome study, we selected six immunohistochemical markers [beta-catenin, androgen receptor (AR), lymphoid enhancer-binding factor 1 (LEF1), transcription factor for immunoglobulin heavy-chain enhancer 3 (TFE3), fused in sarcoma (FUS), and WNT inhibitory factor 1 (WIF-1)]. We also evaluated the Ki-67 proliferative index to investigate its associations with prognosis. To validate these markers, we studied 91 SPNs as well as 51 pancreatic ductal carcinomas (PDC) and 48 neuroendocrine tumors (NET) as controls.We found frequent and diffuse nuclear expressions of β-catenin (98.9%), AR (81.3%), LEF1 (93.4%), TFE3 (74.7%), FUS (84.6%), and cytoplasmic expression of WIF-1 (96.7%) in SPNs. In contrast, PDCs and NETs showed no expression. (P < 0.001). When beta-catenin, LEF1, and TFE3 staining were combined, the sensitivity and specificity were 100% and 91.9%, respectively. Four (4.4%) SPNs showed distant metastasis and these tumors were associated with a relatively high Ki-67 proliferative index (≥ 5%; P = 0.013).We identified LEF1, TFE3, and AR as putative diagnostic markers of SPN, auxiliary to β-catenin. Incorporated into an immunohistochemical panel, these markers could be beneficial to distinguish SPN from PDC and NET. In addition, we suggest that the Ki-67 proliferative index can be a predictive marker of metastasis in SPNs.

The effect of EBV on WIF1, NLK, and APC gene methylation and expression in gastric carcinoma and nasopharyngeal cancer.

Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p.

Wnt inhibitory factor 1 (WIF1) methylation and its association with clinical prognosis in patients with chondrosarcoma

Chondrosarcoma (CS) is a rare cancer, but it is the second most common primary malignant bone tumor and highly resistant to conventional chemotherapy and radiotherapy. Aberrant DNA methylation in the promoter CpG island of Wnt inhibitory factor 1 (WIF1) has been observed in different cancers. However, no studies have shown the relationship between WIF1 methylation and CS. In this study, we found promoter methylated WIF1 in both CS cell lines (CS-1 and SW1353) and tumor tissues. Western blot analysis confirmed loss WIF1 expression and activation of Wnt pathway proteins (Wnt5a/b, LRP6, and Dvl2). We subsequently examined the correlation between levels of WIF1 methylation and overall survival (OS) and progression-free survival (PFS) in CS patient samples with a follow-up spanning 234 months (mean: 57.6 months). Kaplan-Meier survival curves and log-rank tests revealed that high levels of WIF1 methylation were associated with lower OS and PFS rates (p < 0.05). Multivariate Cox hazard analysis suggested that detection of high level methylation of WIF1 could be an independent prognostic factor in OS and PFS. In conclusion, we found that WIF1 is epigenetically silenced via promoter DNA methylation in CS and propose that WIF1 methylation may serve as a potential prognostic marker for patients with CS.

Hypermethylation of WIF1 and its inhibitory role in the tumor growth of endometrial adenocarcinoma.

Endometrial carcinoma is the most common malignancy of the female genital tract and is the fourth most common malignancy among women worldwide. Endometrial adenocarcinoma (EAC) accounts for ~80% of endometrial carcinoma cases. Numerous critical genetic events have been determined to serve an essential role in EAC progression; however, the precise molecular mechanisms underlying EAC progression remain unclear. Pyrosequencing and methylation-specific PCR were used to detect the methylation status of Wnt inhibitory factor 1 (WIF1). Immunohistochemistry and western blot were used to detect the expression of WIF1, Wnt family member 1 and other related pathways. The anticancer role of WIF1 in EAC was investigated in vitro and in vivo. Two of the three EAC cases exhibited significantly high methylation in five CpG sites, and the WIF1 methylation rate in EAC and endometrial tissues was 43.4 and 8%, respectively (P<0.05). The kappa consistency coefficient was −0.369 between methylation and mRNA expression (P<0.05) and WIF1 expression levels were significantly downregulated in EAC tissues compared with non-tumorous tissues (P<0.01). The 5-year overall survival rates were significantly lower for patients with tumors that negatively expressed WIF1 when compared with the 77.9% exhibited by those with positive WIF1 expression. Furthermore, the proliferation rate of KLE cells was significantly reduced following 5-aza-20-deoxycytidine treatment or WIF1 overexpression in vitro and in vivo, which may be associated with downregulated c-Myc and phosphorylated-extracellular signal-regulated kinase expression. These results demonstrated the important role of WIF1 in EAC tumorigenesis, and suggested that WIF1 may be a potential drug target in EAC treatment.