WIF1 Research Articles

Methylation profiles of genes in breast cancer luminal HER2-negative primary tumor during regional lymphnode metastasis

Aberrant methylation is strongly associated with development of cancer, but limited data exist on correlation between methylation and regional lymph node metastasis (RLNM). The aim of this research was to study using of methylation levels of WIF1, RASSF1A, CDO1 and MEST aberrant methylated genes in a primary breast cancer for prediction of regional lymph node metastases. We used MS-HRM (Methylation Sensitive High Resolution Melting) to assess methylation levels. The results were confirmed by pyrosequencing. The study included 66 women with LumA and 46 women with HER2- (LumB-), 22 and 26 of them had metastasis in at least one lymph node respectively. It was found that methylation levels between LumA and LumB subtypes differed significantly in genes: WIF1 (p<0.001), CDO1 (p=0.002) and MEST (p=0.033). In the Lum A subtype statistically significant differences in level of methylation of WIF1 gene between patients with metastases in RLNM and patients without metastases were found (p=0.03). Analysis of tumors longer than 2 cm in the LumA subtype, revealed an increase of statistical significance of WIF1 gene - p=0.009 (AUC (95%CI) = 0.76 (0.59-0.93)). In LumB- subtype RASSF1A, CDO1 and MEST had statistically significant differences in methylation level between groups (p=0.03, p=0.048 and p=0.045 respectively). ROC analysis showed that combining of three genes by logistic regression, AUC (95%CI) was 0.74 (0.6-0.88). Analysis of tumors longer than 2 cm, did not increase statistical significance for these genes (p=0.046; p=0.089 and p=0.076, respectively). Thus, the study of methylation in primary tumors may be useful for prediction of lymph node metastasis, as well as for better understanding of biological process inside breast cancer.

Prognostic Role of RASSF1A, SOX17 and Wif-1 Promoter Methylation Status in Cell-Free DNA of Advanced Gastric Cancer Patients.

Epigenetic modification of several genes is a key component in the development of gastric cancer. The methylation status of RASSF1A, SOX17 and Wif-1 genes was evaluated in the cell free circulating DNA of 70 patients with advanced gastric cancer, using methylation-specific PCR. Patients with higher cell-free DNA concentration seem to have lower PFS, than patients with lower cell-free DNA concentration (p = 0.001). RASSF1A was the tumor suppressor gene, most frequently methylated in metastatic gastric cancer patients, followed by SOX17 and Wif-1 (74.3%, 60.0% and 47.1%, respectively). Patients having the SOX17 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p < 0.001). Patients having the Wif-1 promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.001). Patients having the RASSF1A promoter methylated, had lower progression free survival and overall survival, than unmethylated ones (p = 0.004). Promoter methylation of the examined genes was significantly associated with a decrease in progression free survival and overall survival, comparing to that of patients without methylation. Simultaneous methylation of the above genes was associated with even worse progression free survival and overall survival. The methylation of RASSF1A, SOX-17 and Wif-1 and genes, is a frequent epigenetic event in patients with advanced gastric cancer.

Метилирование гена WIF1 при различных видах патологии эндометрия

Метилирование гена WIF1 при различных видах патологии эндометрия

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.

Molecular Biology of Osteosarcoma.

Osteosarcoma (OS) is the most frequent primary bone cancer in children and adolescents and the third most frequent in adults. Many inherited germline mutations are responsible for syndromes that predispose to osteosarcomas including Li Fraumeni syndrome, retinoblastoma syndrome, Werner syndrome, Bloom syndrome or Diamond–Blackfan anemia. TP53 is the most frequently altered gene in osteosarcoma. Among other genes mutated in more than 10% of OS cases, c-Myc plays a role in OS development and promotes cell invasion by activating MEK–ERK pathways. Several genomic studies showed frequent alterations in the RB gene in pediatric OS patients. Osteosarcoma driver mutations have been reported in NOTCH1, FOS, NF2, WIF1, BRCA2, APC, PTCH1 and PRKAR1A genes. Some miRNAs such as miR-21, -34a, -143, -148a, -195a, -199a-3p and -382 regulate the pathogenic activity of MAPK and PI3K/Akt-signaling pathways in osteosarcoma. CD133+ osteosarcoma cells have been shown to exhibit stem-like gene expression and can be tumor-initiating cells and play a role in metastasis and development of drug resistance. Although currently osteosarcoma treatment is based on adriamycin chemoregimens and surgery, there are several potential targeted therapies in development. First of all, activity and safety of cabozantinib in osteosarcoma were studied, as well as sorafenib and pazopanib. Finally, novel bifunctional molecules, of potential imaging and osteosarcoma targeting applications may be used in the future.

Promoter Hypermethylation of Wnt/β-catenin Signaling Pathway Inhibitor WIF-1 Gene and its Association with MTHFR C677T Polymorphism in Patients with Colorectal Cancer.

ObjectivesColorectal cancer (CRC) is a common malignancy with a high rate of mortality. The dysregulation of genes involved in the Wnt/β-catenin signaling pathway is a common finding in cancers. Wnt-inhibitory factor-1 (WIF-1) suppresses the Wnt/β-catenin signaling pathway and its inactivation by genetics and epigenetic changes may cause cancer. We investigated the DNA methylation status of the WIF-1 gene in patients with CRC and its interaction with MTHFR C677T polymorphism, a known modifier of methylation reaction.MethodsWe investigated 50 cancerous tissues and the adjacent non-cancerous tissue. Genomic DNA was extracted using a commercial kit and was treated by sodium bisulfite. Methylation-specific PCR was used for methylation analysis, and restriction fragment length polymorphism PCR to analyze the C677T polymorphism of the MTHFR gene.ResultsThe frequency of WIF1 promoter DNA methylation was significantly higher in cancerous tissue than adjacent non-cancerous tissue (52.0% vs. 8.0%; p < 0.001). WIF1 promoter DNA methylation status showed a significant association only with tumor location (p = 0.009). Carriers of TT genotype and T allele of MTHFR C677T polymorphism had a significantly higher frequency of unmethylated WIF1 gene than methylated WIF-1 gene in cancerous tissue (p = 0.025 and p = 0.001, respectively).ConclusionsPromoter DNA hypermethylation of the WIF-1 gene is a significant risk factor for CRC development, which was significantly associated with tumor location only. The significant association of TT genotype and T allele of MTHFR C677T polymorphism with unmethylated WIF-1 gene suggests a protective role for this common polymorphism against methylation-induced development of CRC.

P829 Methylation patterns in dysplasia in inflammatory bowel disease patients

Abstract Background Inflammatory Bowel Disease (IBD) with colonic involvement increases colorectal cancer risk. However, the distinction between IBD related and sporadic dysplasia in IBD patients is difficult. Some data favours the importance of abnormal DNA methylation in IBD-related carcinogenesis. Our study aimed to define methylation patterns in patients with a colonic cancer or dysplasia diagnosis following an IBD diagnosis. Methods Multicentric cross-sectional study- 91 samples from colonic mucosa with/without dysplasia from 9 patients with IBD-related dysplasia/cancer and 26 patients with IBD and sporadic dysplasia/cancer were included. Methylation patterns of CpG islands in the promoter regions of 67 genes were studied by Methylation-specific Multiplex Ligation-dependent Probe Amplification. Results Mean age at IBD diagnosis: 42±16 years; at dysplasia diagnosis: 56± ± 14 years. Twenty-nine patients had ulcerative colitis. Twenty-five patients had at least 1 lesion endoscopically described as adenoma-like, 4 at least 1 non-adenoma like, 3 had cancer and 3 had dysplasia in flat mucosa. No patient had both adenoma-like and non-adenoma-like lesions. Patients with an IBD-related lesion were significantly younger at IBD diagnosis (p = 0.003) and at dysplasia/cancer diagnosis (p = 0.039). Promoter methylation of IGF2, RARB, ESR1, CHFR, CDH13, WT1, GATA5, WIF1 genes was significantly associated to dysplasia/cancer; methylation of MSH6, TIMP3 was significantly associated to IBD-related dysplasia/cancer. Promoter methylation of MSH6, MSH3, RUNX3, CRABP1, TP73, RARB, CDH13, PAX5, WT1, THBS1, TP53, SFRP1, WIF1, APAF1, BCL2 genes was significantly associated to active IBD. Conclusion Methylation analysis, namely of MSH6, may contribute to the classification of dysplastic lesions in IBD– to be further tested in prospective studies.

Противоопухолевая активность индол-3-карбинола в клетках рака молочной железы: фенотип - генетический паттерн - обращение ДНК-метилирования

The effect of indole-3-carbinol on a number of functions and characteristics of MDA-MB-231 breast cancer cells and MCF-10A healthy breast tissue cells has been studied. It was shown that indole-3-carbinol significantly reduced the proliferation and migration of MDA-MB-231 cells and does not affect these functions in MCF-10A cells. Incubation of MDA-MB-231 tumor cells with indole-3-carbinol (100 uM) for 48 h resulted in a marked decrease in the expression of the Wnt cascade genes, CCND1 (by 28%), SP1 (by 44%), CDK6 (by 47%), as well as EGFR (by 64%) and FASN (by 22%) genes. Incubation of MCF-10A cell line under the same conditions induced a noticeable decrease in expression of only two genes, EGFR (by 16%) and CDK6 (by 9%). In addition, indole-3-carbinol was also shown to manifest a selective DNA demethylation activity in breast tumor cells and to reverse the process of abnormal methylation and functional blockage of the anti-tumor WIF1 gene. These data indicate that drags containing indole-3-carbinol as an active component can be potential regulators of epigenetic processes in the treatment of breast cancer and other tumors. indole-3-carbinol, breast cancer, epigenetics, Wnt

In silico approach for the prediction of functional nsSNPs in WIF1 gene of WNT pathway

In silico approach for the prediction of functional nsSNPs in WIF1 gene of WNT pathway

Plasma clearance of RAS mutation under therapeutic pressure is a rare event in metastatic colorectal cancer.

In metastatic colorectal cancer (mCRC), circulating tumor DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. Patients with a RAS mutated tumor included in the prospective PLACOL study were monitored for ctDNA. Analyses were based on optimized targeted next-generation sequencing and/or droplet-based digital polymerase chain reaction (ddPCR). For plasma samples without detectable mutations at progression disease, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. Among the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only two out of the eight patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only 2 samples among 36 were confirmed for clearance of RAS mutation in our series. In conclusion, this study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored.

623P - Plasma clearance of RAS mutation under therapeutic pressure is a rare event in metastatic colorectal cancer

BackgroundIn metastatic colorectal cancer (mCRC), circulating tumour DNA (ctDNA) monitoring can be used to genotype tumors and track clonal evolution. We investigated the clearance of RAS mutated clones under chemotherapy pressure by ctDNA analysis in patients with a RAS mutated mCRC. MethodsPatients with a RAS mutated tumour included in the prospective PLACOL study, were monitored for ctDNA. Analyzes were based on optimized targeted next generation sequencing and/or droplet-based digital PCR (ddPCR). For plasma samples without detectable mutations, we tested the methylation status of WIF1 and NPY genes using methylation-ddPCR (met-ddPCR) to validate the presence of ctDNA. ResultsAmong the 36 patients with positive plasma samples for RAS mutations at inclusion, 28 (77.8%) remained RAS positive at disease progression, and 8 (22.2%) became negative. Subsequent met-ddPCR for methylated markers showed that only 2 out of the 8 patients with RAS negative plasma had detectable ctDNA at progression. Therefore, only two samples among 36 were confirmed for clearance of RAS mutation in our series. ConclusionsThis study suggests that the clearance of RAS mutations in patients treated by chemotherapy for a RAS mutated mCRC is a rare event. Monitoring tumor mutations in plasma samples should be combined with a strict control of the presence of ctDNA. The therapeutic impacts of RAS clearance need to be further explored. Legal entity responsible for the studyThe authors. FundingThe Ministère de l’Enseignement Supérieur et de la Recherche, the Université Paris-Descartes, the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (INSERM), the Institut National du Cancer (INCA, n° 2009-1-RT-03-US-1 and 2009-RT-03-UP5-1), the Association pour la recherche contre le cancer (ARC, no. SL220100601375), the Agence Nationale de la Recherche (ANR Nanobiotechnologies; no. ANR-10-NANO-0002-09), the SIRIC CARPEM, the ligue nationale contre le cancer (LNCC, Program “Equipe labelisée LIGUE”; no. EL2016.LNCC/VaT) and Advanced Merieux Research Grant and canceropole funding (no. 2011-1-LABEL-UP5-2). DisclosureH. Blons: Advisory / Consultancy: AstraZeneca; Advisory / Consultancy: Merck; Advisory / Consultancy: Amgen; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Pfizer. V. Taly: Advisory / Consultancy: Servier; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Raindance Technologies. W. Shu-Fang: Research grant / Funding (institution): Servier. S. Pernot: Advisory / Consultancy: Amgen; Advisory / Consultancy: Sanofi. J. Taieb: Advisory / Consultancy, Speaker Bureau / Expert testimony: Merck; Advisory / Consultancy, Speaker Bureau / Expert testimony: Roche; Advisory / Consultancy, Speaker Bureau / Expert testimony: Amgen; Advisory / Consultancy, Speaker Bureau / Expert testimony: Eli Lilly; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sanofi; Advisory / Consultancy, Speaker Bureau / Expert testimony: Celgene; Advisory / Consultancy, Speaker Bureau / Expert testimony: Sirtex; Advisory / Consultancy, Speaker Bureau / Expert testimony: Shire; Advisory / Consultancy, Speaker Bureau / Expert testimony: MSD; Advisory / Consultancy, Speaker Bureau / Expert testimony: Servier; Advisory / Consultancy, Speaker Bureau / Expert testimony: Pierre Fabre. P. Laurent-Puig: Advisory / Consultancy: Amgen; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Boehringer Ingelheim; Advisory / Consultancy: Roche; Advisory / Consultancy: Lilly. A. Zaanan: Advisory / Consultancy: Amgen; Advisory / Consultancy: Baxter; Advisory / Consultancy: Lilly; Advisory / Consultancy: Merck-Serono; Advisory / Consultancy: MSD; Advisory / Consultancy: Sanofi; Advisory / Consultancy: Roche; Advisory / Consultancy: Servier. All other authors have declared no conflicts of interest.

Angiopoietin-like protein 8 (betatrophin) may inhibit hepatocellular carcinoma through suppressing of the Wnt signaling pathway.

Objective(s):Hepatocellular carcinoma (HCC) is one of the leading fatal neoplasms and the most common primary liver malignancy worldwide. Peptide hormone ANGPTL8 (betatrophin) may act as an important regulator in HCC development through the Wnt/β-catenin pathway. We aimed to evaluate the effects of recombinant ANGPTL8 on Wnt/β-catenin signaling in human liver carcinoma cells (HepG2) and their viability. Materials and Methods:The expression of ANGPTL8 was conducted in the pET-21b-E. coli Bl21 (DE3) system and the produced peptide was purified. HepG2 cells were treated with different concentrations of ANGPTL8 (25, 50, 100, 150, 200, and 250 ng/ml) for 24, 48, and 72 hr. MTT assay was performed to detect the viability of treated cells, and apoptotic induction by ANGPTL8 was measured by flow cytometry assay. Finally, using qRT-PCR the mRNA levels of Wnt signaling modulators WIF-1 and β-catenin were evaluated in treated cells. Results:MTT assay showed that ANGPTL8 inhibits proliferation of HepG2 cells moderately in a time-independent manner. The highest concentration of the ANGPTL8, 250 ng/ml, reduced cell proliferation after 24, 48, and 72 hr similarly about 30%. In the same concentration of ANGPTL8, after 24 hr of treatment flow cytometry assay revealed a mild increase in early and late apoptosis up to 7.7 and 14.3%, respectively. The qRT-PCR showed that in a concentration-dependent and time-independent fashion, the expression of WIF-1 and β-catenin genes respectively increased and decreased significantly (P<0.05). Conclusion:Our findings suggest that ANGPTL8 may act as a moderate suppressor against HCC cell proliferation possibly via affecting Wnt signaling modulators.

Gene expression and DNA methylation regulation of arsenic in mouse bladder tissues and in human urothelial cells.

According to a report of the International Agency for Research on Cancer, arsenic and inorganic arsenic compounds are classified into Group 1 carcinogens with regard to human health. Epidemiological studies indicate that arsenic is one of the main risk factors for the development of bladder cancer. In the present study, arsenic-altered gene expression in mouse bladder tissues and in human urothelial cells was compared. In the mouse model, sodium arsenite-induced mouse urothelial hyperplasia and intracellular inclusions were present. Following DNA array analysis, four genes with differential expression were selected for quantitative real-time PCR assay. The genes were the following: Cystathionine β-synthase (CBS), adenosine A1 receptor (ADORA1), metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) and Wnt inhibitory factor 1 (Wif1). The results indicated a significant increase in the levels of Cbs and Adora1. The analysis of the DNA CpG methylation levels of the mouse Cbs and Adora1 genes revealed no significant change. In contrast to these observations, the four genes were further analyzed in the human normal urothelial cell line SV-HUC1. The data indicated that WIF1 gene expression was decreased by sodium arsenite, whereas this was not noted for CBS, MALAT1 and ADORA1. Sodium arsenite decreased mRNA and protein expression levels of the WIF1 gene. In addition, the methylation levels of the WIF1 gene were increased. Sodium arsenite inhibited cell proliferation and promoted cell migration as demonstrated in cell functional assays. The gene status was compared in 8 human urothelial cell lines, and WIF1 mRNA expression levels were determined to be higher, whereas DNA CpG methylation levels were lower in SV-HUC1 cells compared with those noted in the other 7 bladder cancer cell lines. In summary, the data indicated that sodium arsenite decreased WIF1 gene expression and promoted cell migration. The increased methylation levels of WIF1 DNA CpG could be a potential biomarker for bladder cancer.

Genetic variant of WIF1 gene is functionally associated with developmental dysplasia of the hip in Han Chinese population

Developmental dysplasia of the hip (DDH) is a common skeletal disorder. Studies have demonstrated a significant role of WIF1 gene in skeletal development. The present study was conducted to reveal the association between DDH and gene WIF1. A two-stage case-control candidate gene association study was conducted in total 1573 samples (586 DDH patients and 987 healthy controls) in this study. Polymorphism rs3782499 was genotyped in all samples. Difference of WIF1 expression in hip joint tissue was compared between the patients and the controls. WIF1 expression was compared among different genotypes in DDH patients. The SNP rs3782499 was found significantly associated with DDH in the two-stage study with 585 patients and 987 controls. There was a significant difference in allele frequency (p = 4.37 * 10−5) and genotype distribution in a recessive model (AG + GG vs. AA). DDH patients were found to have significantly higher WIF1 expression than controls. Moreover, Patients with rs3782499 genotype AA have a significantly increased expression of WIF1 than those with GG. To conclude, polymorphism rs3782499 of WIF1 gene is a functional variant regulating the expression of WIF1 in DDH in Chinese Han population, which might be a potential biomarker for the early diagnosis of DDH.

RAS mutation analysis in circulating tumor DNA from patients with metastatic colorectal cancer: the AGEO RASANC prospective multicenter study

BackgroundRAS mutations are currently sought for in tumor samples, which takes a median of almost 3weeks in western European countries. This creates problems in clinical situations that require urgent treatment and for inclusion in therapeutic trials that need RAS status for randomization. Analysis of circulating tumor DNA might help to shorten the time required to determine RAS mutational status before anti-epidermal growth factor receptor antibody therapy for metastatic colorectal cancer. Here we compared plasma with tissue RAS analysis in a large prospective multicenter cohort. Patients and methodsPlasma samples were collected prospectively from chemotherapy-naive patients and analyzed centrally by next-generation sequencing (NGS) with the colon lung cancer V2 Ampliseq panel and by methylation digital PCR (WIF1 and NPY genes). Tumoral RAS status was determined locally, in parallel, according to routine practice. For a minimal κ coefficient of 0.7, reflecting acceptable concordance (precision±0.07), with an estimated 5% of non-exploitable data, 425 subjects were necessary. ResultsFrom July 2015 to December 2016, 425 patients were enrolled. For the 412 patients with available paired plasma and tumor samples, the κ coefficient was 0.71 [95% confidence interval (CI), 0.64–0.77] and accuracy was 85.2% (95% CI, 81.4% to 88.5%). In the 329 patients with detectable ctDNA (at least one mutation or one methylated biomarker), the κ coefficient was 0.89 (95% CI, 0.84–0.94) and accuracy was 94.8% (95% CI, 91.9% to 97.0%). The absence of liver metastases was the main clinical factor associated with inconclusive circulating tumor DNA results [odds ratio=0.11 (95% CI, 0.06–0.21)]. In patients with liver metastases, accuracy was 93.5% with NGS alone and 97% with NGS plus the methylated biomarkers. ConclusionThis prospective trial demonstrates excellent concordance between RAS status in plasma and tumor tissue from patients with colorectal cancer and liver metastases, thus validating plasma testing for routine RAS mutation analysis in these patients. Clinical Trial registrationClinicaltrials.gov, NCT02502656.

Aberrant expression of the sFRP and WIF1 genes in invasive non-functioning pituitary adenomas

Non-functioning pituitary adenomas (NFPAs) are the most common pituitary tumors and mainly invade the sphenoid, cavernous sinus or dura mate. Aberrant regulation of the Wnt signaling pathway plays an important role in tumorigenesis. This study was designed to investigate the relationships between secreted frizzled-related proteins (sFRPs), WIF1 genes and the invasion of NFPAs by tissue microassays (TMAs) of samples from 163 patients. Significantly weaker staining of WIF1 and sFRP4 were detected in the invasive group compared with the non-invasive group by TMAs (p = 0.002, p < 0.001). Univariate analysis showed a significant correlation between tumor invasion and low expression of WIF1 and sFRP4 (p = 0.002, p < 0.001). A similar trend was observed when analyzing the mRNA and protein levels through RT-PCR and western blot experiments. Methylation of the WIF1 promoter was significantly increased in invasive NFPAs compared with the noninvasive group (p = 0.004). The average progression free survival time in the high WIF1 group was longer than that in the low WIF1 group (p = 0.025). Furthermore, RT-PCR measured the levels of 11 miRNAs targeting WIF1 according to the Targetscan database and PubMed. The levels of miRNA-137, miRNA-374a-5p and miRNA-374b-5p in the invasive group were 0.037-fold, 0.577-fold and 0.44-fold that of the noninvasive group (p = 0.003, p = 0.049 and p = 0.047). Overexpression of miRNA-137 could inhibit the proliferation and invasion of GH3 cells through cell viability and Transwell experiments (p < 0.05). Furthermore, the WIF1 level was upregulated after overexpression of miRNA-137 compared with miRNA-137-NC (control miRNA) in GH3 cells. Our data suggest that WIF1 may be potential biomarker for the aggressiveness of NFPAs. miRNA-137 plays an important role in the Wnt signaling pathway by affecting promoter methylation of WIF1.

Guanosine monophosphate reductase 1 is a potential therapeutic target for Alzheimer\u2019s disease

Alzheimer’s disease (AD) is a severe neurodegenerative disorder for which identification of differentially expressed genes is one way to find new therapeutic targets. Here, we conducted analysis to identify age-independent, AD-specific genes. We found that the MET, WIF1, and NPTX2 genes are downregulated in AD. WIF1 and MET are implicated in Wnt and MET signaling and regulate GSK3β activity and are thus linked with AD. Importantly, we found that the GMPR gene exhibited a gradual increase in AD progression. A logistic model based on GMPR has good ability to classify AD cases. GMPR’s product GMPR1 is in the AMPK and adenosine receptor pathways and is thus associated with Tau phosphorylation in AD. This allows GMPR1 to be a therapeutic target. Therefore, we screened five possible inhibitors to GMPR1 by docking GMPR1 with 1,174 approved drugs. Among them, lumacaftor is ideal. We then tested the effects of lumacaftor on AD model mice. After 20 days of oral administration, we observed that β-Amyloid accumulation was slowed down, and phosphorylation of Tau was almost eliminated in the treated mice. We highlight the elevated expression level of GMPR in AD and propose a therapeutic strategy of inhibiting GMPR1 with lumacaftor.

Methylation Status and mRNA Expression of DKK-3 and WIF-1 in Acute Myeloid Leukemia Patients

To analyze the promoter methylation status, mRNA expression and clinical significance of DKK-3 and WIF-1 genes in patients with acute myeloid leukemia(AML). Methylation specific polymerase chain reaction (MS-PCR) mothod was carried out to detect DKK-3 and WIF-1 gene promoter methylation status in bone marrow specimen from 56 patients with AML and 20 patients with iron deficiency anaemia(IDA) as control; then the real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of DKK-3, WIF-1 gene and β -catenin in the above-mentioned specinens, and their relationship with the clinical features and survival time was analyzed. The promoter methylation rate of DKK-3 and WIF-1 gene in AML patients were significantly higher than that in control group(χ2=15.330,P<0.001; χ2=17.371,P<0.001). There was no relationship between DKK-3 and WIF-1 gene promoter methylation rate and AML patient's sex, age, clinical typing. The relative expression of DKK-3 and WIF-1 gene mRNA in AML group were 0.840±0.320 and 0.792±0.313, which were lower than those in control group (1.134±0.392 and 1.047±0.334) respectively, the difference was statistically significant (t=3.415,P=0.000; t=3.070, P=0.003). The relative expression of β-catenin mRNA in AML bone marrow specimens in AML group was 0.756±0.304, which was higher than that in control group(0.342±0.105), the difference was statistically significant (t=5.943, P=0.001). The expression of DKK-3 and WIF-1 gene mRNA negatively correlated with β-catenin mRNA(r=-0.543; r=-0.562). Kaplan Meier survival curve analysis showed that overall survival time in AML patients with DKK-3 gene methylation was shorter than that in the AML patients with DKK-3 gene unmethylation(χ2=3.957, P=0.042). Futhermore, the orerall survival time in AML patients with WIF-1 gene methylation was also shorter than that in AML patients with WIF-1 gene unmethylation (χ2=4.520, P=0.029). Wnt/β-catenin signaling pathway is abnormally activated in AML patients, the DKK-3 and WIF-1 gene promoter methylation may be involved in Wnt pathways activation and the pathogenesis of AML.

Value of the level of methylation of RASSF1A and WIF-1 in tissue and serum in neoadjuvant chemotherapeutic assessment for advanced breast cancer.

This study assessed the clinical efficacy of the neoadjuvant chemotherapy TAC scheme in treatment of patients with locally advanced breast cancer, and the value of the level of Ras association domain family 1A (RASSF1A) gene methylation and the Wnt inhibitory factor (WIF)-1 gene in tissue and serum of patients in clinical outcome prediction. In total, 126 patients were consecutively selected to receive TAC scheme (docetaxel, pirarubicin/epirubicin and cyclophosphamide) for at least four cycles with the total effective rate. The incidence of complications, progression-free survival and survival rate were recorded. Tumor tissues and peripheral blood samples collected in this study was used to detect methylation positive rate of RASSF1A and WIF-1 by methylation-specific PCR method and the relative level of expression of RASSF1A and WIF-1 mRNA by reverse transcription PCR method. Of the 126 patients, there were 18 cases with complete response (CR), 32 cases with partial response (PR), 50 cases with stable disease (SD), and 26 cases with disease progression (PD) with a total effective rate of 79.37%. Comparison on baseline data of effective group and ineffective group showed no difference (P>0.05), and comparison on adverse reactions occurrence showed no difference (P>0.05). Progression-free survival of the effective group was prolonged with a significant increase in survival rate (P<0.05). Positive rates of RASSF1A methylation and WIF-1 in tissue and serum of the patients in the effective group were significantly lower than those in the ineffective group, but the mRNA of RASSF1A and WIF-mRNA was significantly higher than the ineffective group (P<0.05). The sensitivity of clinical outcome prediction using tissue RASSF1A methylation was 67.0%, the specificity 15.4%, positive predictive value 69.0% and negative predictive value 31.0%. The above-mentioned indexes of tissue WIF-1 were 76.0, 31.4, 72.2 and 27.8, respectively. The indexes of serum RASSF1A were 85.0, 50.0, 76.2 and 23.8%, respectively, and the indexes of serum WIF-1 were 94.0, 75.0, 81.0 and 19.0%, respectively. The receiver operating characteristic curve analysis suggested that the accuracy of clinical outcome prediction using tissue RASSF1A mRNA level was 0.812. The sensitivity 85.2%, the specificity 76.3% and the critical value 0.4256. These indexes of tissue WIF-1 were 0.833, 86.7%, 75.4% and 0.3562 for CR, PR, SD and PD, respectively. These indexes of serum RASSF1A were 0.864, 88.3%, 77.4% and 0.2564, respectively, and for serum WIF-1 were 0.882, 89.4%, 73.5% and 0.1562, respectively. In conclusion, the detection of RASSF1A and WIF-1 gene methylation and level of mRNA expression in tissue and serum of patients with locally advanced breast cancer has an important application value in predicting clinical efficacy of neoadjuvant chemotherapy of the TAC scheme.

Dnmt3a-mediated inhibition of Wnt in cardiac progenitor cells improves differentiation and remote remodeling after infarction.

Adult cardiac progenitor cells (CPCs) display a low capacity to differentiate into cardiomyocytes in injured hearts, strongly limiting the regenerative capacity of the mammalian myocardium. To identify new mechanisms regulating CPC differentiation, we used primary and clonally expanded Sca-1+ CPCs from murine adult hearts in homotypic culture or coculture with cardiomyocytes. Expression kinetics analysis during homotypic culture differentiation showed downregulation of Wnt target genes concomitant with increased expression of the Wnt antagonist, Wnt inhibitory factor 1 (Wif1), which is necessary to stimulate CPC differentiation. We show that the expression of the Wif1 gene is repressed by DNA methylation and regulated by the de novo DNA methyltransferase Dnmt3a. In addition, miR-29a is upregulated early during CPC differentiation and downregulates Dnmt3a expression, thereby decreasing Wif1 gene methylation and increasing the efficiency of differentiation of Sca-1+ CPCs in vitro. Extending these findings in vivo, transient silencing of Dnmt3a in CPCs subsequently injected in the border zone of infarcted mouse hearts improved CPC differentiation in situ and remote cardiac remodeling. In conclusion, miR-29a and Dnmt3a epigenetically regulate CPC differentiation through Wnt inhibition. Remote effects on cardiac remodeling support paracrine signaling beyond the local injection site, with potential therapeutic interest for cardiac repair.