SFRP2 Methylation Research Articles

Frequent Inactivation of Secreted Frizzled-Related Protein 2 during the Development of Cervical Carcinoma: Identification of Susceptible Alleles and Clinical Implications

ABSTRACT Background: In this study, importance of SFRP2, wnt stem cell renewal pathway antagonist, in the development of cervical cancer (CACX) was evaluated Aims and Objectives: Alterations (expression/ methylation/ deletion) of SFRP2 were analysed in primary cervical lesions of different clinical stages followed by their correlation with different clinicopathological parameters. Then, susceptible allele(s) of SFRP2 was identified through case control study followed by and in vitro validation. Results: The mRNA expression of SFRP2 was gradually reduced with progression of CACX. In immunohistochemistry, SFRP2 membrane expression was mainly present in the spinous layers of normal cervical epithelium and its reduced protein expression in CACX samples showed concordance with mRNA expression. Frequent deletion/ methylation of SFRP2 were seen to be associated with development of cervical cancer. Methylation of SFRP2 was prevalently associated with early invasive lesions (stage I/II) while, deletion with late invasive lesions (stage III/IV). Overall alterations (deletion/ methylation) of SFRP2 were significantly increased from premalignant CIN to stage-I/II samples followed by comparable change to the next stage (stage III/IV) samples. Moreover, deletion and/or methylation of SFRP2 were associated with poor prognosis of the patients. In a case control study, out of its seven microsatellite alleles infrequent SFRP_CA15/16 alleles along with frequent SFRP_CA17 allelewere found to be associated with CACX development. Comparatively reduced expression (mRNA/ protein) of SFRP2 was seen in the tumor adjacent normal cervical epithelium having SFRP_CA15/16 alleles than the other alleles. This has been further validated in in vitro luciferase promoter activity assay where SFRP_CA16 repeat showed high reduced activity followed by SFRP_CA15 repeat than the other repeats. Conclusion: Thus, our data showed that presence of the infrequent susceptible alleles along with deletion/methylation might have synergistic effect on frequent inactivation of SFRP2 during development of CACX.

EVALUATION OF SECRETED FRIZZLED-RELATED PROTEIN-2 METHYLATION STATUS IN PATIENTS WITH CHRONIC MYELOID LEUKEMIA

BackgroundChronic Myeloid Leukemia (CML) is a myeloproliferative disorder defined by the chromosomal translocation (t) (9;22) encoding for the BCR-ABL fusion gene. Tumor development and progression may be affected by hypermethylation of promoter regions of certain genes. Secreted Frizzled-Related Proteins (SFRPs) genes produce Wnt signaling antagonists, and their epigenetic silencing has been detected in multiple types of leukaemia. While epigenetic silencing has been broadly reported for SFRPs, SFRP2 is a family member less studied in leukaemia.
 ObjectivesTo evaluate SFRP2 gene methylation patterns in CML patients compared with healthy individuals.
 Materials and MethodsSodium bisulfite was applied to the extracted DNA from blood samples of 75 CML patients and 25 healthy subjects who served as the control group. The DNA was then analyzed by methylation-specific high-resolution melting (MS-HRM) using specific primers for the SFRP2 gene in promoter sequence.
 ResultsForty-five out of 75 CML patients investigated were shown to have SFRP2 methylation percentages between 50% to 100%, whereas 19 out of 25 healthy subjects had a methylation level of less than 50%.
 ConclusionThe current study demonstrated that SFRP2 promoter hypermethylation exists in CML, as in several other solid tumours. Hence, methylation of this gene may play a part in initiating leukemogenesis.

Promoter hypermethylation of SFRP1 as a prognostic and potentially predictive blood-based biomarker in patients with stage III or IV pancreatic ductal adenocarcinoma

BackgroundPancreatic ductal adenocarcinoma remains one of the major causes of cancer-related mortality globally. Unfortunately, current prognostic biomarkers are limited, and no predictive biomarkers exist. This study examined promoter hypermethylation of secreted frizzled-related protein 1 (phSFRP1) in cfDNA as a prognostic biomarker and predictor of treatment effect in patients with metastatic FOLFIRINOX-treated PDAC and locally advanced PDAC. MethodsWe performed methylation-specific PCR of the SFRP1 genes’ promoter region, based on bisulfite treatment. Survival was assessed as time-to-event data using the pseudo-observation method and analyzed with Kaplan-Meier curves and generalized linear regressions. ResultsThe study included 52 patients with FOLFIRINOX-treated metastatic PDAC. Patients with unmethylated (um) SFRP1 (n = 29) had a longer median overall survival (15.7 months) than those with phSFRP1 (6.8 months). In crude regression, phSFRP1 was associated with an increased risk of death of 36.9% (95% CI 12.0%–61.7%) and 19.8% (95% CI 1.9–37.6) at 12 and 24-months, respectively. In supplementary regression analysis, interaction terms between SFRP1 methylation status and treatment were significant, indicating reduced benefit of chemotherapy. Forty-four patients with locally advanced PDAC were included. phSFRP1 was associated with an increased risk of death at 24-months ConclusionsThis indicates that phSFRP1 is a clinically useful prognostic biomarker in metastatic PDAC and possibly in locally advanced PDAC. Together with existing literature, results could indicate the value of cfDNA-measured phSFRP1 as a predictive biomarker of standard palliative chemotherapy in patients with metastatic PDAC. This could facilitate personalized treatment of patients with metastatic PDAC.

Identification and Validation of Potentially Clinically Relevant CpG Regions within the Class 2 Tumor Suppressor Gene SFRP1 in Pancreatic Cancer.

In pancreatic cancer treatment, tumor stage-dependent chemotherapies are used to prolong overall survival. By measuring DNA promoter hypermethylation in the plasma of patients with stage IV pancreatic cancer, it was recently shown that promoter DNA methylation of the tumor suppressor gene SFRP1 has a high value for predicting failure of drug treatment with gemcitabine. In this study, we therefore aimed to identify as precisely as possible the region in the SFRP1 promoter that is frequently hypermethylated in pancreatic cancer tissue. First, we used the TCGA data set to define CpG-rich regions flanking the SFRP1 transcription start site that were significantly more methylated in pancreatic cancer compared to normal pancreatic acinar tissue. A core CpG island was identified that exhibited abundant tumor DNA methylation and anti-correlation of SFRP1 mRNA expression. To validate our in silico results, we performed bisulfide conversion followed by DNA pyrosequencing of 28 matched formalin-fixed, paraffin-embedded (FFPE) pancreatic cancer cases and six pancreatic cancer cell lines. A defined block of seven CpG sites within the core CpG island was identified, which confirmed our in silico results by showing significantly higher SFRP1 methylation in pancreatic cancer specimens than in normal pancreatic tissue. By selecting this core CpG island, we were able to determine a median overall survival benefit for the low SFRP1 methylation group compared to the high SFRP1 methylation group (702 versus 517 days, p = 0.01) in the TCGA pancreatic cancer cohort. We propose a compact pyrosequencing assay that can be used in the future to further investigate the prognostic value of SFRP1 promoter hypermethylation in predicting pancreatic cancer chemoresistance. Therefore, instead of DNA analysis from blood (liquid biopsy), DNA easily extractable from cancer tissue blocks (FFPE material) could be used.

M6A Methyltransferase METTL3 Reduces Hippocampal Neuron Apoptosis in a Mouse Model of Autism Through the MALAT1/SFRP2/Wnt/β-catenin Axis.

Hippocampal neuron apoptosis contributes to autism, while METTL3 has been documented to possess great potentials in neuron apoptosis. Our study probed into the role of METTL3 in neuron apoptosis in autism and to determine the underlying mechanism. Bioinformatics analysis was used to analyze expressed genes in autism samples. Institute of Cancer Research mice were treated with valproic acid to develop autism models. The function of METTL3 in autism-like symptoms in mice was analyzed with behavioral tests and histological examination of their hippocampal tissues. Primary mouse hippocampal neurons were extracted for in vitro studies. Downstream factors of METTL3 were explored and validated. METTL3, MALAT1, and Wnt/β-catenin signaling were downregulated, while SFRP2 was upregulated in the hippocampal tissues of a mouse model of autism. METTL3 stabilized MALAT1 expression by promoting m6A modification of MALAT1. MALAT1 promoted SFRP2 methylation and led to reduced SFRP2 expression by recruiting DNMT1, DNMT3A, and DNMT3B to the promoter region of SFRP2. Furthermore, SFRP2 facilitated activation of the Wnt/β-catenin signaling. By this mechanism, METTL3 suppressed autism-like symptoms and hippocampal neuron apoptosis. This research suggests that METTL3 can reduce autism-like symptoms and hippocampal neuron apoptosis by regulating the MALAT1/SFRP2/Wnt/β-catenin axis.

MiR-26a-5p Suppresses Wnt/β-Catenin Signaling Pathway by Inhibiting DNMT3A-Mediated SFRP1 Methylation and Inhibits Cancer Stem Cell-Like Properties of NSCLC

Background Lung cancer is a malignant cancer which results in the most cancer incidence and mortality worldwide. There is increasing evidence that the pattern of DNA methylation affects tumorigenesis and progression. However, the molecules and mechanisms regulating DNA methylation remain unclear. Methods The expression of miR-26a-5p in NSCLC cell lines was detected by qPCR and verified in NSCLC tissues from TCGA using Limma R package. CCK-8 assay, plate clone formation assay, flow cytometry, and sphere formation assay were used to detect the cell proliferation, colony formation, cell cycle, and cancer stem cell- (CSC-) like property in NSCLC cell lines. The immunoblotting was used to detect the protein levels of DNMT3A, SFRP1, and Ki67. Global DNA methylation levels and DNA methylation levels of SFRP1 promoter were examined using ELISA and MSP-PCR assay, respectively. The distribution of β-catenin was examined using immunofluorescence (IF). Besides, xenograft mouse model was used to investigate the antitumor effects of miR-26a-5p in vivo. The pathology and protein levels were, respectively, detected by hematoxylin and eosin (H&E) and immunocytochemistry (IHC). Results The expression of miR-26a-5p was downregulated in the tumor tissues comparted to adjacent normal tissues as well as NSCLC cell lines compared to normal lung epithelial cell (BEAS2B). The overexpression of miR-26a-5p inhibited cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase. DNMT3A was a target of miR-26a-5p and upregulated DNA methylation on SFRP1 promoter. Mechanistically, miR-26a-5p repressed cell proliferation, colony formation, CSC-like property, and arrested cell cycle at G1 phase by binding DNMT3A to reduce DNA methylation levels of SFRP1 then upregulated SFRP1 expression. Moreover, miR-26a-5p exerted antitumor effects in vivo. Conclusion Our results revealed that miR-26a-5p acted as a tumor suppressor through targeting DNMT3A to upregulate SFRP1 via reducing DNMT3A-dependent DNA methylation.

A Liquid Biopsy-Based Approach for Monitoring Treatment Response in Post-Operative Colorectal Cancer Patients.

Monitoring the therapeutic response of colorectal cancer (CRC) patients is crucial to determine treatment strategies; therefore, we constructed a liquid biopsy-based approach for tracking tumor dynamics in non-metastatic (nmCRC) and metastatic (mCRC) patients (n = 55). Serial blood collections were performed during chemotherapy for measuring the amount and the global methylation pattern of cell-free DNA (cfDNA), the promoter methylation of SFRP2 and SDC2 genes, and the plasma homocysteine level. The average cfDNA amount was higher (p < 0.05) in nmCRC patients with recurrent cancer (30.4 ± 17.6 ng) and mCRC patients with progressive disease (PD) (44.3 ± 34.5 ng) compared to individuals with remission (13.2 ± 10.0 ng) or stable disease (12.5 ± 3.4 ng). More than 10% elevation of cfDNA from first to last sample collection was detected in all recurrent cases and 92% of PD patients, while a decrease was observed in most patients with remission. Global methylation level changes indicated a decline (75.5 ± 3.4% vs. 68.2 ± 8.4%), while the promoter methylation of SFRP2 and SDC2 and homocysteine level (10.9 ± 3.4 µmol/L vs. 13.7 ± 4.3 µmol/L) presented an increase in PD patients. In contrast, we found exact opposite changes in remission cases. Our study offers a more precise blood-based approach to monitor the treatment response to different chemotherapies than the currently used markers.

Tumor molecular differences associated with outcome disparities of Black patients with head and neck cancer.

Numerous studies of head and neck squamous cell carcinoma (HNSCC) have demonstrated disparate outcomes by race and ethnicity. Beyond known associations with socioeconomic variables, whether these are also associated with differences in tumor molecular composition has thus far been poorly explored. We downloaded clinical and multiplatform molecular data from The Cancer Genome Atlas and other published studies. These were compared between non-Hispanic Black (n=43) and White (n=354) patients with non-HPV-related tumors, using multivariable models. Publicly available validation cohorts were used. Black patients had poorer progression-free survival than White patients. Tumors of Black patients had greater copy number aberrations, and increased SFRP1 methylation and miRNA-mediated PRG4 silencing associated with poor survival. PI3K/AkT/mTOR pathway proteins were differentially expressed. There are molecular differences between tumors of Black and White patients that may partially account for differences in survival. These may inform targeted treatment decisions to achieve equitable outcomes.

Multiple Biomarker-Combined Screening for Colorectal Cancer Based on Bisulfate Conversion-Free Detection of Fecal DNA Methylation

To evaluate the applicability of bisulfate conversion-free methylation assay based on enzyme digestion in fecal screening for colorectal cancer (CRC). Stool samples were collected from a total of 1142 participants with intestinal abnormalities, including 180 positive cases, 60 advanced adenomas, and 902 negative cases. DNA from reference cell lines and clinical samples was extracted and digested with an enzyme to detect the methylation of CRC markers SEPT9, SDC2, NDRG4, SFRP2, and BMP3 genes. Statistical analysis was then used to determine the ability of the markers, both individually and in combination, to detect CRC and adenoma. Our results showed that the enzyme digestion method could suitably detect DNA marker methylation in as low as 1% of the cell lines. BMP3 had a considerably low detection rate in all clinical samples, with only 6 positive cases detected out of 180 cancer samples. Our findings showed that the combination of SEPT9, SDC2, and SFRP2 had an area under the receiver operation curve of 0.937, sensitivity of 94.11%, and specificity of 89.21% for detecting CRC. Moreover, the detection sensitivity of adenoma can also reach 38.33%. After innovatively utilizing bisulfate conversion-free methylation assay for CRC screening, this study verified the potential clinical applicability of combining multiple biomarkers for CRC screening in a large number of samples.

Value of combined detection of ITGA4 and SFRP2 gene methylation in stool DNA in diagnosis and prognostic evaluation of colorectal tumors

To investigate the value of quantitative detection of ITGA4 and SFRP2 gene methylation in stool DNA for the early diagnosis and prognostic evaluation of colorectal tumors. Real-time PCR was used for quantitative assessment of ITGA4 and SFRP2 gene methylation levels in stool samples of 85 patients with colorectal cancer, 65 patients with colorectal adenoma and 40 healthy subjects. The 3 groups were comparable for age and gender composition. Methylated ITGA4 and SFRP2 promoters were detected in 48.2% and 62.4% of patients with colorectal cancer, respectively, with a combined positivity of 81.2%. ITGA4 and SFRP2 promoter methylation was detected in 23.1% and 43.1% of patients with colorectal adenoma, respectively, with a combined positivity of 69.2%. The positivity rates of ITGA4 and SFRP2 methylation were significantly higher in patients with colorectal cancer than in those with colorectal adenoma (P < 0.001; P= 0.001) and healthy subjects (P < 0.001; P < 0.001). In colorectal cancer group, ITGA4 and SFRP2 promoter methylation levels were correlated with postoperative tumor recurrence in colorectal cancer group, and the relapse-free survival rate was significantly lower in positive patients for ITGA4 and SFRP2 promoter methylation than in the negative patients (P=0.0002; P=0.007). Multivariate analysis with the COX proportional hazard regression model showed that methylation of ITGA4 and SFRP2 gene promoters (P=0.01) and the degree of tumor differentiation (P=0.03) were associated with the recurrence of colorectal cancer, and were independent risk factors for the recurrence of colorectal cancer. Combined detection of ITGA4 and SFRP2 gene methylation levels in stool DNA can improve the early diagnosis rate of colorectal tumor. ITGA4 and SFRP2 promoter methylation and the degree of tumor differentiation are independent risk factors for colorectal cancer recurrence.

Development of a prognostic risk model for clear cell renal cell carcinoma by systematic evaluation of DNA methylation markers

BackgroundCurrent risk models for renal cell carcinoma (RCC) based on clinicopathological factors are sub-optimal in accurately identifying high-risk patients. Here, we perform a head-to-head comparison of previously published DNA methylation markers and propose a potential prognostic model for clear cell RCC (ccRCC).Patients and methodsPromoter methylation of PCDH8, BNC1, SCUBE3, GREM1, LAD1, NEFH, RASSF1A, GATA5, SFRP1, CDO1, and NEURL was determined by nested methylation-specific PCR. To identify clinically relevant methylated regions, The Cancer Genome Atlas (TCGA) was used to guide primer design. Formalin-fixed paraffin-embedded (FFPE) tissue samples from 336 non-metastatic ccRCC patients from the prospective Netherlands Cohort Study (NLCS) were used to develop a Cox proportional hazards model using stepwise backward elimination and bootstrapping to correct for optimism. For validation purposes, FFPE ccRCC tissue of 64 patients from the University Hospitals Leuven and a series of 232 cases from The Cancer Genome Atlas (TCGA) were used.ResultsMethylation of GREM1, GATA5, LAD1, NEFH, NEURL, and SFRP1 was associated with poor ccRCC-specific survival, independent of age, sex, tumor size, TNM stage or tumor grade. Moreover, the association between GREM1, NEFH, and NEURL methylation and outcome was shown to be dependent on the genomic region. A prognostic biomarker model containing GREM1, GATA5, LAD1, NEFH and NEURL methylation in combination with clinicopathological characteristics, performed better compared to the model with clinicopathological characteristics only (clinical model), in both the NLCS and the validation population with a c-statistic of 0.71 versus 0.65 and a c-statistic of 0.95 versus 0.86 consecutively. However, the biomarker model had limited added prognostic value in the TCGA series with a c-statistic of 0.76 versus 0.75 for the clinical model.ConclusionIn this study we performed a head-to-head comparison of potential prognostic methylation markers for ccRCC using a novel approach to guide primers design which utilizes the optimal location for measuring DNA methylation. Using this approach, we identified five methylation markers that potentially show prognostic value in addition to currently known clinicopathological factors.

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.

DNA methylation analysis with methylation-sensitive high-resolution melting (MS-HRM) reveals gene panel for glioma characteristics.

IntroductionLocal DNA hypermethylation is a potential source of cancer biomarkers. While the evaluation of single gene methylation has limited value, their selected panel may provide better information.AimsThis study aimed to analyze the promoter methylation level in a 7‐gene panel in brain tumors and verifies the usefulness of methylation‐sensitive high‐resolution melting (MS‐HRM) for this purpose.MethodsForty‐six glioma samples and one non‐neoplastic brain sample were analyzed by MS‐HRM in terms of SFRP1, SFRP2, RUNX3, CBLN4, INA, MGMT, and RASSF1A promoter methylation. The results were correlated with patients’ clinicopathological features.ResultsDNA methylation level of all analyzed genes was significantly higher in brain tumor samples as compared to non‐neoplastic brain and commercial, unmethylated DNA control. RASSF1A was the most frequently methylated gene, with statistically significant differences depending on the tumor WHO grade. Higher MGMT methylation levels were observed in females, whereas the levels of SFRP1 and INA promoter methylation significantly increased with patients’ age. A positive correlation of promoter methylation levels was observed between pairs of genes, for example, CBLN4 and INA or MGMT and RASSF1A.ConclusionsOur 7‐gene panel of promoter methylation can be helpful in brain tumor diagnosis or characterization, and MS‐HRM is a suitable method for its analysis.

Association between variation of circulating 25-OH vitamin D and methylation of secreted frizzled-related protein 2 in colorectal cancer

BackgroundsColorectal cancer (CRC) results from the accumulation of epigenetic and genetic changes in colon cells during neoplasic transformation, which the activation of Wingless (Wnt) signaling pathway is a common mechanism for CRC initiation. The Wnt pathway is mainly regulated by Wnt antagonists, as secreted frizzled-related protein (SFRP) family. Indeed, SFRP2 is proposed as a noninvasive biomarker for CRC diagnosis. Vitamin D also antagonizes Wnt signaling in colon cancers cells. Several studies showed that vitamin D was able to alter DNA methylation, although this mechanism is not yet clear. Therefore, the aim of this study was to find an association between circulating 25-OH vitamin D (30th percentile of vitamin D) and the SFRP2 methylation.MethodsA total of 67 CRC patients were included in the study. These patients were subdivided into two groups based on their 30th percentile vitamin D (20 patients were below, and 47 participants were above the 30th percentile of vitamin D). We investigated the SFRP2 methylation in peripheral blood mononuclear cells (PBMCs), visceral adipose tissue (VAT), CRC tumor tissue, and adjacent tumor-free area. We also determined the relationship between SFRP2 methylation and methylation of carcinogenic and adipogenic genes. Finally, we tested the effect of vitamin D on the SFRP2 methylation in human colorectal carcinoma cell lines 116 (HCT116) and studied the association of neoadjuvant therapy under the 30th percentile vitamin D with SFRP2 promoter methylation.ResultsSFRP2 methylation in tumor area was decreased in patients who had higher levels of vitamin D. SFRP2 promoter methylation was positively correlated in tumor area with insulin and homeostasis model assessment of insulin resistance (HOMA-IR) but negatively correlated with HDL-c. SFRP2 methylation was also correlated with T cell lymphoma invasion and metastasis 1 (TIAM1) methylation in tumor area and CCAAT/enhancer-binding protein alpha (C/EBPα) in VAT. Treatment with vitamin D did not affect SFRP2 methylation in HCT116 cell line. Finally, neoadjuvant treatment was correlated with higher circulating 25-OH vitamin D and SFRP2 methylation under linear regression model.ConclusionOur results showed that higher circulating vitamin D is associated with low SFRP2 promoter methylation. Therefore, our results could suggest that vitamin D may have an epigenetic effect on DNA methylation. Finally, higher vitamin D could contribute to an improvement response to neoadjuvant treatment.

The Prognostic Value of DNA Methylation Markers in Renal Cell Cancer: A Systematic Review

Introduction: DNA methylation is an epigenetic event that occurs in normal tissues but changes in both the early and late stages of multiple tumor entities, including renal cell cancer (RCC). Many studies have shown that DNA methylation markers are relevant to RCC prognosis, but none of these markers have entered into clinical routine. Furthermore, because of its potential reversibility, DNA methylation might provide a new target for RCC therapy strategies. Materials and methods: Following PRISMA guidelines, we performed a systematic literature search up to February 2019. After selection for eligibility, a total of 56 studies were identified for analysis. Each study was categorized and the level of evidence assessed. Only articles reporting on DNA methylation markers and their association with survival were included. Descriptive statistical analyses were conducted with R statistical software. Results: We identified promoter methylation of SFRP1, GATA5, NEFH, GREM1, and BCN1 as associated with survival in RCC. Moreover, we found evidence that methylation signatures, i.e., grouping of different potential gene markers, might be of better prognostic value than single gene marker investigations. Nevertheless, because of the heterogeneous features of the studies in terms of design, methodology, patient cohorts, and statistics, the true clinical impact of these methylation markers for prognosis in RCC patients remains uncertain. Conclusion: This systematic review elucidates the potential impact of DNA methylation on survival of patients with RCC. Several promising prognostic markers, especially methylation signatures, were identified, which is encouraging, but prospective validations are necessary to establish their true clinical value.

LUCAT1 Epigenetically Downregulates the Tumor Suppressor Genes CXXC4 and SFRP2 in Gastric Cancer.

PurposeThe mechanisms of Wnt/β-catenin pathway signaling and abnormal expression of tumor suppressor genes is not well known in gastric cancer (GC). Long non-coding RNA (lncRNA) has recently been identified as a possible link therein. In this study, we investigated the role of lung cancer associated transcript 1 (LUCAT1) in GC.Materials and MethodsThe expression of LUCAT1 in GC cell lines and 100 tissue samples was examined by qRT-PCR. Two different siRNAs were used for knockdown of LUCAT1 expression. Cell viability was assessed by MTT assay. To analyze metastasis, scratch wound-healing assay, a Matrigel invasion assay, and colony formation assay were performed. Apoptosis was analyzed by PI/Annexin-V staining. To check the methylation status in tumor suppressor genes, methylation-specific PCR was carried out. Western blot was performed to detect epithelial-mesenchymal transition and apoptosis markers upon silencing of LUCAT1 (siLUCAT1).ResultsLUCAT1 expression in GC cell lines and tissues was significantly elevated, compared to that in normal gastric cells and adjacent non-tumor tissues (p<0.001). Two different siRNAs for LUCAT1 reduced cell proliferation, invasion, and migration, compared to siCT (p<0.05), and these reductions were restored by pcDNA-LUCAT1 (p<0.05). siLUCAT1 elicited upregulation of the expression of CXXC4 and SFRP2. The expression of H3K27me3 was reduced by siLUCAT1, and this reduction was correlated with methylation of CXXC4 and SFRP2. Inhibition of LUCAT1 up-regulated EZH2 expression and resulted in demethylation of CXXC4 and SFRP2 through the Wnt/β-catenin signaling pathway.ConclusionWe concluded that LUCAT1 induces methylation of CXXC4 and SFRP2, thereby regulating Wnt/β-catenin signaling in GC.

DNA methylation of SFRP1, SFRP2, and WIF1 and prognosis of postoperative colorectal cancer patients

BackgroundAs biomarkers, DNA methylation is used to detect colorectal cancer (CRC) and make assessment of CRC prognosis. The published findings showed the association between the methylation of SFRP1, SFRP2, and WIF1, located in the Wnt signaling pathway, and the prognosis of CRC were not consistent. Our study aimed to explore the potential possibility of SFRP1, SFRP2, and WIF1 concomitant promoter methylation as prognostic biomarkers of postoperative CRC patients.MethodsAs a total of 307 sporadic postoperative CRC patients were followed up, we detected SFRP1, SFRP2, and WIF1 methylation obtained from tumor tissues and adjacent non-tumor tissues respectively on the basis of methylation-sensitive high resolution melting analysis. Univariate and multivariate Cox regressions were carried out so as to assess the potential possibility of SFRP1, SFRP2, and WIF1 promoter methylation as predictors of prognosis. Confounders in our study were controlled by Propensity Score (PS) analysis.ResultsThe SFRP1, SFRP2, and WIF1 methylation levels in tumor tissues were significantly higher than that in adjacent non-tumor tissues (P < 0.001). SFRP2 hypermethylation was significantly associated with a favorable clinical outcome at the hazard ratio (HR) of 0.343 [95% confidence intervals (CI): 0.164–0.718, P = 0.005] and 0.410 (95% CI: 0.200–0.842, P = 0.015) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1 and SFRP2 was significantly associated with a favorable clinical outcome at the HR of 0.333 (95% CI: 0.159–0.694, P = 0.003) and 0.398 (95% CI: 0.192–0.821, P = 0.013) in multivariate Cox regression and PS analysis, respectively. Co-hypermethylation of SFRP1, SFRP2 and WIF1 was significantly associated with a favorable clinical outcome at the HR of 0.326 (95% CI: 0.117–0.908, P = 0.032) and 0.401 (95% CI: 0.146–1.106, P = 0.077) in multivariate Cox regression and PS analysis, respectively.ConclusionsSFRP1, SFRP2, and WIF1 were frequently hypermethylated in CRC tumor tissues. It was apparent that the promoter hypermethylation of SFRP2 and co-hypermethylation of SFRP1 and SFRP2 might be considered as independent prognostic predictors for survival advantage of postoperative CRC patients.

RETRACTED: Silencing of Long Non-coding RNA HOTTIP Reduces Inflammation in Rheumatoid Arthritis by Demethylation of SFRP1

Accumulating evidence suggests long non-coding RNAs (lncRNAs) play crucial roles in the pathogenesis of rheumatoid arthritis (RA). Here, we aimed to define the role of HOXA transcript at the distal tip (HOTTIP) in RA pathogenesis in relation to SFRP1 methylation and Wnt signaling pathway. HOTTIP was found highly expressed, and SFRP1 was hypermethylated in RA synovial fibroblasts (RASFs). Next, gain- or loss-of-function experiments were conducted in RASFs to explore the effects of HOTTIP on the biological behaviors of RASFs. Silencing of HOTTIP or overexpression of SFRP1 inhibited RASF proliferation, invasion, and migration, while enhancing apoptosis. The relationship among HOTTIP, SFRP1, and Dnmt3b was determined using methylation-specific PCR (MSP), bisulfite sequencing PCR (BSP), RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) assays. The regulatory mechanisms of HOTTIP/Dnmt3b/SFRP1 were explored by altering their expression in RASFs. It was noted that HOTTIP could induce SFRP1 promoter methylation through recruitment of Dnmt3b and activate the Wnt signaling pathway. Finally, a rat RA model was established in order to evaluate the in vivo effects of HOTTIP and SFRP1, which suggested that HOTTIP silencing or SFRP1 elevation inhibited the progression of RA in vivo. Our key findings demonstrate the anti-inflammatory ability of HOTTIP silencing in RA through SFRP1 promoter demethylation. These findings support HOTTIP as a candidate anti-arthritis target.

A novel panel of stool-based DNA biomarkers for early screening of colorectal neoplasms in a Chinese population

The mortality of colorectal cancer ranked fifth in China according to cancer statistics in 2015. Cancer screening had been repeatedly proved to play a vital role in decreasing the incidence and mortality of colorectal cancer, but the existing screening methods could not meet the requirements. So it is of urgent need to develop a non-invasive, convenient and accurate screening method. In this study, stool samples were collected from 102 colorectal cancer, 20 colorectal adenoma, 6 hyperplastic polyps patients and 105 normal controls, and stool DNA was extracted for detection of methylation (BMP3, NDRG4, SDC2 and SFRP2) and KRAS mutations. Meanwhile, hemoglobin in stool samples was detected by immunoassays. Then, the logistic regression model used for classification was built with these biomarkers, and a ROC curve was drawn to evaluate the performance of each biomarker and the panel of them. Meanwhile, conventional serum biomarkers weredetected for the comparison of positive rate in colorectal cancer between serum biomarkers and stool DNA biomarkers. As a result, a classification model built with methylation of SDC2 and SFRP2, KRAS mutations and hemoglobin showed a sensitivity of 91.4% for colorectal cancer and 60% for adenoma with the specificity of 86.1%. Compared with it, most of the conventional serum biomarkers showed a sensitivity of less than 20% for colorectal cancer which was significantly lower than stool DNA biomarkers. A novel panel comprised of stool DNA biomarkers was of much higher sensitivity and specificity in early screening of colorectal neoplasms than conventional serum biomarkers.

Association between SFRP promoter hypermethylation and different types of cancer: A systematic review and meta-analysis.

Abnormal methylation of secreted frizzled-related proteins (SFRPs) has been observed in various human cancer types. The loss of SFRP gene expression induces the activation of the Wnt pathway and is a vital mechanism for tumorigenesis and development. The aim of the present systematic review was to assess the association between SFRP methylation and cancer risk. A meta-analysis was systematically conducted to assess the clinicopathological significance of SFRP methylation in cancer risk. The Cochrane Library, PubMed and Web of Science databases were comprehensively searched, and 83 publications with a total of 21,612 samples were selected for the meta-analysis. The pooled odds ratios (ORs) and corresponding 95% confidence intervals (CIs) were calculated to evaluate the degree of associations between SFRP promoter methylation and cancer risk. Subgroup analysis, meta regression and sensitivity analysis were used to identify the potential sources of heterogeneity. SFRP1, SFRP2, SFRP4 and SFRP5 hypermethylation was significantly associated with cancer risk, with ORs of 8.48 (95% CI, 6.26–11.49), 8.21 (95% CI, 6.20–10.88), 11.41 (95% CI, 6.42–20.30) and 6.34 (95% CI, 3.86–10.42), respectively. SFRP2 methylation was significantly associated with differentiation in colorectal cancer (OR, 2.16; 95% CI, 1.02–4.56). The results of the present study demonstrated that SFRP methylation may contribute to carcinogenesis, especially in certain cancer types, including hepatocellular carcinoma and colorectal cancer.