SFRP4 protein expression is reduced in high grade astrocytomas which is not caused by the methylation of its promoter.

Introduction: Epigenetics play a vital role in stratifying CNS tumors and gliomas. The importance of studying Secreted frizzled-related protein 4 (SFRP4) in gliomas is to improve diffuse glioma methylation profiling. Here we examined the methylation status of SFRP4 promoter and the level of its protein expression in diffuse gliomas WHO grades 2-4. Methods: SFRP4 expression was detected by immunohistochemistry and evaluated semi-quantitatively. In the tumor hot-spot area, the intensity of protein expression in 200 cells was determined using ImageJ (National Institutes of Health, United States). The assessment of immunopositivity was based on the IRS score (Immunoreactivity Score). Promoter methylation was examined by methylation specific-PCR (MSP) in fifty-one diffuse glioma samples and appropriate controls. Isolated DNA was treated with bisulfite conversion and afterwards used for MSP. Public databases (cBioPortal, COSMIC and LOVD) were searched to corroborate the results. Results and discussion: SFRP4 protein expression in glioblastomas was very weak or non-existent in 86.7% of samples, moderate in 13.3%, while strong expression was not observed. The increase in astrocytoma grade resulted in SFRP4 protein decrease (p = 0.008), indicating the loss of its antagonistic role in Wnt signaling. Promoter methylation of SFRP4 gene was found in 16.3% of cases. Astrocytomas grade 2 had significantly more methylated cases compared to grade 3 astrocytomas (p = 0.004) and glioblastomas (p < 0.001), which may indicate temporal niche of methylation in grade 2. Furthermore, the expression levels of SFRP4 were high in samples with methylated SFRP4 promoter and low or missing in unmethylated cases (Pearson's R = -0.413; p = 0.003). We also investigated the association of SFRP4 changes to key Wnt regulators GSK3ß and DKK3 and established a positive correlation between methylations of SFRP4 and GSK3ß (Pearson's R = 0.323; p = 0.03). Furthermore, SFRP4 expression was correlated to unmethylated DKK3 (Chi square = 7.254; p = 0.027) indication that Wnt signaling antagonist is associated to negative regulator's demethylation. Conclusion: The study contributes to the recognition of the significance of epigenetic changes in diffuse glioma indicating that restoring SFRP4 protein holds potential as therapeutic avenue. Reduced expression of SFRP4 in glioblastomas, not following promoter methylation pattern, suggests another mechanism, possible global methylation, that turns off SFRP4 expression in higher grades.

Evaluation of silica spin­column and magnetic bead formats for rapid DNA methylation analysis in clinical and point­of­care settings.

Late-stage cancers lack effective treatment, underscoring the need for early diagnosis to improve prognosis and decrease mortality rates. Molecular markers, such as DNA methylation, offer promise in early cancer detection. The present study compared commercial kits for analyzing DNA from cervical liquid cytology samples in cancer screening. Rapid bisulfite conversion kits using silica spin-columns and magnetic beads were assessed against standard DNA extraction and bisulfite conversion methods for profiling DNA methylation using quantitative methylation-specific PCR. ß-actin amplification indicated the suitability of small sample volumes for methylation studies using either the pellet or supernatant (cell-free DNA) parts. Comparison of Bisulfite Conversion Kit-Whole Cell (Abcam), Methylamp Bisulfite Modification (Epigentek), EpiTect Fast LyseAll Bisulfite Kit (Qiagen GmbH) and EZ DNA Methylation-Direct Kit (Zymo Research Corp.) showed no significant differences in ß-actin cycle threshold values. EZ-96 DNA Methylation-Lightning MagPrep (Zymo Research Corp.), a hybrid kit in a 96-well plate format, exhibited swift turnaround time and similar amplification efficiency. Automation with magnetic bead kits increased throughput without compromising amplification efficiency in open PCR systems. Cost analysis favored direct kits over the gold standard manual protocol. This comparison aids in selecting cost-effective DNA methylation diagnostic tests. The present study confirmed comparable kit performance in methylation-based analysis, highlighting the adequacy of cytology samples and the potential of bodily fluids as alternatives for liquid biopsy.

Methylation Analysis to Detect CIN3+ in High-Risk Human Papillomavirus-Positive Self-Samples From the Population-Based Cervical Cancer Screening Program.

Since 2017, a self-sampling device has been introduced to the Dutch population-based screening program to enable higher participation rates. However, routine triage cytology cannot be performed on self-sampling material. Methylation is an alternative triage method that can be performed directly on DNA extracted from self-samples. Recently, we tested a set of 15 published cervical intraepithelial neoplasia grade 3 or worse (CIN3+)-specific methylation markers and found a panel of 3 markers with a sensitivity of 82% and a specificity of 74%. In this study, we determined the sensitivity and specificity of 2 commercial assays using quantitative methylation-specific PCR. DNA from the same cohort of high-risk human papillomavirus-positive self-sampled material obtained through the population-based screening program in the North of the Netherlands from women with CIN2 or less (

O6 -methylguanine methyltransferase promoter methylation status of glioblastoma cell line clonal population.

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

Study of the Methylation of Bovine GSTP1 Gene under the Influence of Pesticide Mospilan 20SP Alone and in Combination with Pesticide Orius 25EW.

DNA methylation, one of the most studied epigenetic mechanisms, when present in the promoter region of genes, causes inhibition of gene expression, and conversely, hypomethylation of these regions enables gene expression. DNA methylation is susceptible to nutritional and environmental influences, and undesirable alterations in methylation patterns manifested in changes in the expression of relevant genes can lead to pathological consequences. In the present work, we studied the methylation status of the bovine GSTP1 gene under the influence of pesticide Mospilan 20SP alone and in combination with pesticide Orius 25EW in in vitro proliferating bovine lymphocytes. We employed methylation-specific PCR, and when studying the effect of pesticide combinations, we also used its real-time version followed by a melting procedure. Our results showed that Mospilan 20SP alone at 5, 25, 50, and 100 µg.ml-1 and 5, 10, 25, and 50 µg.ml-1 for the last 4 and 24 hours of culture with in vitro proliferating bovine lymphocytes, respectively, did not induce methylation of the bovine GSTP1 gene. The same results were revealed when studying the effect of the combination of the pesticides added to the lymphocyte cultures for the last 24 hours of cultivation in the following amounts: 1.25, 2.5, 5, 10, and 25 µg.ml-1 of Mospilan 20SP and 1.5, 3, 6, 15, and 30 µg.ml-1 of Orius 25EW. We have also revealed that the less laborious real-time MSP followed by a melting procedure may replace MSP for studying the methylation status of the GSTP1 gene.

Ras Association Domain Family 1A Gene Promoter Methylation as a Biomarker for Chronic Viral Hepatitis C-related Hepatocellular Carcinoma.

Background One of the most prevalent aberrant epigenetic modifications found in hepatocellular carcinoma (HCC) is abnormal DNA methylation. Our study aimed to evaluate serum Ras association domain family 1A (RASSF1A) gene promoter methylation in patients with chronic viral hepatitis C (HCV)-associated liver cirrhosis with and without HCC as a potential new marker for the early detection of HCC. Methodology The 60 participants who participated in the trial were divided into the following three groups: 20 patients with newly diagnosed primary HCC on top of HCV-related liver cirrhosis, 20 patients with HCV-related liver cirrhosis, and 20 age- and sex-matched healthy individuals as a control group. All participants underwent methylation-specific polymerase chain reaction testing to detect the blood level of the RASSF1A gene's methylated promoter. Results Methylated RASSF1A was found in 30% of patients with liver cirrhosis caused by HCV and in 65% of patients with HCC, but not in any of the controls. It was discovered that the serum methylation RASSF1A had an accuracy of 82.50% and an area under the curve (AUC) of 0.825 for separating HCC patients from healthy controls. With an AUC of 0.675 and an accuracy of 67.50%, it was able to differentiate patients with HCC from those with HCV-related liver cirrhosis. Additionally, there was no statistically significant association between RASSF1A methylation status and HCC mass size (p = 0.449). Conclusions Serum RASSF1A promoter methylation status detection could be useful for detecting HCC early, especially in high-risk individuals such as those with HCV.

BRCA1 Promoter Hypermethylation in Malignant Breast Tumors and in the Histologically Normal Adjacent Tissues to the Tumors: Exploring Its Potential as a Biomarker and Its Clinical Significance in a Translational Approach.

The hypermethylation status of the promoter region of the breast cancer 1 (BRCA1), a well-known tumor suppressor gene, has been extensively investigated in the last two decades as a potential biomarker for breast cancer. In this retrospective study, we investigated the prevalence of BRCA1 promoter methylation in 84 human breast tissues, and we correlated this epigenetic silencing with the clinical and histopathological parameters of breast cancer. We used methylation-specific PCR (MSP) to analyze BRCA1 promoter hypermethylation in 48 malignant breast tumors (MBTs), 15 normal adjacent tissues (NATs), and 21 benign breast lesions (BBLs). The results showed that BRCA1 promoter hypermethylation was higher in MBTs (20/48; 41.67%) and NATs (7/15; 46.67%) compared to BBLs (4/21; 19.05%). The high percentage of BRCA1 hypermethylation in the histologically normal adjacent tissues to the tumors (NATs) suggests the involvement of this epigenetic silencing as a potential biomarker of the early genomic instability in NATs surrounding the tumors. The detection of BRCA1 promoter hypermethylation in BBLs reinforces this suggestion, knowing that a non-negligible rate of benign breast lesions was reported to evolve into cancer. Moreover, our results indicated that the BRCA1 promoter hypermethylated group of MBTs exhibited higher rates of aggressive features, as indicated by the SBR III grade (14/19; 73.68%), elevated Ki67 levels (13/16; 81.25%), and Her2 receptor overexpression (5/20; 25%). Finally, we observed a concordance (60%) in BRCA1 promoter hypermethylation status between malignant breast tumors and their paired histologically normal adjacent tissues. This study highlights the role of BRCA1 promoter hypermethylation as a potential useful biomarker of aggressiveness in MBTs and as an early marker of genomic instability in both histological NATs and BBLs.

Genome-wide DNA methylation profiles analysis in primary warm autoimmune hemolytic anemia patients.

BACKGROUND: Autoimmune hemolytic anemia (AIHA) is caused by auto-antibodies, secreted by overactivated B cells, directed against self-red blood cells, resulting in hemolysis. It found that aberrant DNA methylation in B cells can induce the production of autoantibodies. Therefore, we attempted to explore if similar aberrant DNA methylation occur in AIHA patients. METHODS: A 49-year-old female wAIHA patient and a 47-year-old female healthy control (HC) were enrolled. Peripheral blood (PB) B cells DNA was extracted. After constructing genomic libraries, bisulfite genomic sequencing (BSP) and DNA methylation profiles were analyzed. BSP was verified using PB B cells from 10 patients with hemolysis, 10 patients with hemolytic remission, and 10 healthy controls (HCs) by Methylation-specific PCR. RESULTS: Total DNA methylation of whole-genome C bases (4.8%) and CG type bases (76.8%) in wAIHA patient were lower than those in the HC (5.3 and 82.5%, respectively) (p = 0.022 and p < 0.001). DNA methylation of C bases and CG type bases in whole-genome regulatory elements, such as coding sequence, up2Kb and down2Kb in the patient were also lower than those in the HC (p = 0.041, p = 0.038, and p = 0.029). 30,180 DNA-methylated regions (DMRs) on all 23 chromosomes were identified. DMR-related genes were mainly involved in the Rap1, phospholipase D, HIF-1, calcium, vascular endothelial growth factor (VEGF) and Ras signaling pathways. CONCLUSION: The DNA methylation spectrum of B cells in AIHA patients is different from that of HC, and the proportion of hypo-methylation regions is higher than that of HC. DMR-related genes are mainly related to some signaling pathways.

Identification of a methylation panel as an alternative triage to detect CIN3+ in hrHPV-positive self-samples from the population-based cervical cancer screening programme.

BACKGROUND: The Dutch population-based cervical cancer screening programme (PBS) consists of primary high-risk human papilloma virus (hrHPV) testing with cytology as triage test. In addition to cervical scraping by a general practitioner (GP), women are offered self-sampling to increase participation. Because cytological examination on self-sampled material is not feasible, collection of cervical samples from hrHPV-positive women by a GP is required. This study aims to design a methylation marker panel to detect CIN3 or worse (CIN3+) in hrHPV-positive self-samples from the Dutch PBS as an alternative triage test for cytology. METHODS: Fifteen individual host DNA methylation markers with high sensitivity and specificity for CIN3+ were selected from literature and analysed using quantitative methylation-specific PCR (QMSP) on DNA from hrHPV-positive self-samples from 208 women with CIN2 or less (< CIN2) and 96 women with CIN3+. Diagnostic performance was determined by area under the curve (AUC) of receiver operating characteristic (ROC) analysis. Self-samples were divided into a train and test set. Hierarchical clustering analysis to identify input methylation markers, followed by model-based recursive partitioning and robustness analysis to construct a predictive model, was applied to design the best marker panel. RESULTS: QMSP analysis of the 15 individual methylation markers showed discriminative DNA methylation levels between < CIN2 and CIN3+ for all markers (p < 0.05). The diagnostic performance analysis for CIN3+ showed an AUC of ≥ 0.7 (p < 0.001) for nine markers. Hierarchical clustering analysis resulted in seven clusters with methylation markers with similar methylation patterns (Spearman correlation> 0.5). Decision tree modeling revealed the best and most robust panel to contain ANKRD18CP, LHX8 and EPB41L3 with an AUC of 0.83 in the training set and 0.84 in the test set. Sensitivity to detect CIN3+ was 82% in the training set and 84% in the test set, with a specificity of 74% and 71%, respectively. Furthermore, all cancer cases (n = 5) were identified. CONCLUSION: The combination of ANKRD18CP, LHX8 and EPB41L3 revealed good diagnostic performance in real-life self-sampled material. This panel shows clinical applicability to replace cytology in women using self-sampling in the Dutch PBS programme and avoids the extra GP visit after a hrHPV-positive self-sampling test.

DNA Methylation Analysis.

DNA methylation of promoter CpG islands silences their downstream genes, and enhancer methylation can be associated with decreased or increased gene expression. DNA methylation alterations in normal and diseased cells provide rich information, such as tissue origin, disease risk, patient response, and prognosis. DNA methylation status is detected by bisulfite conversion, which converts unmethylated cytosines into uracils but methylated cytosines very inefficiently. A genome-wide DNA methylation analysis is conducted by a BeadChip microarray or next-generation sequencing (NGS) of bisulfite-treated DNA. A region-specific DNA methylation analysis can be conducted by various methods, such as methylation-specific PCR (MSP), quantitative MSP, and bisulfite sequencing. This chapter provides protocols for bisulfite-mediated conversion, a BeadChip array-based method (Infinium), quantitative MSP, and bisulfite sequencing.

Identification of DNA methylation change in TCF7L2 gene in the blood of type 2 diabetes mellitus as a predictive biomarker in Iraq Kurdistan region by using methylation-specific PCR.

Objective. Nowadays, type 2 diabetes mellitus (T2D) is the most common chronic endocrine disorder affecting an estimated 5-10% of adults worldwide, and this disease also rapidly increased among the population in the Kurdistan region. This research aims to identify DNA methylation change in the TCF7L2 gene as a possible predictive T2D biomarker. Methods. One hundred and thirteen participants were divided into three groups: diabetic (47), prediabetic (36), and control (30). The study was carried out in patients who visited the private clinical sector between August and December 2021 in Koya city (Iraq Kurdistan region) to determine DNA methylation status using a methylation-specific PCR (MSP) with paired primers for each methylated and non-methylated region. In addition, the X2 Kruskal-Wallis statistical and Wilcoxon signed-rank tests were used, p<0.05 was considered significant. Results. The results showed hypermethylation of DNA in the promoter region in diabetic and prediabetic groups compared to the healthy controls. Different factors affected the DNA methylation level, including body max index, alcohol consumption, family history, and physical activity with the positive Coronavirus. Conclusion. The results obtained indicate that DNA methylation changes in the TCF7L2 promoter region may be used as a potential predictive biomarker of the T2D diagnosis. However, the findings obtained in this study should be supported by additional data.

Targeting miR-5088-5p attenuates radioresistance by suppressing Slug.

Radiotherapy is widely used for cancer treatment, but paradoxically, it has been reported that surviving cancer cells can acquire resistance, leading to recurrence or metastasis. Efforts to reduce radioresistance are required to increase the effectiveness of radiotherapy. miRNAs are advantageous as therapeutic agents because it can simultaneously inhibit the expression of several target mRNAs. Therefore, this study discovered miRNA that regulated radioresistance and elucidated its signaling mechanism. Our previous study confirmed that miR-5088-5p was associated with malignancy and metastasis in breast cancer. As a study to clarify the relationship between radiation and miR-5088-5p identified as onco-miRNA, it was confirmed that radiation induced hypomethylation of the promoter of miR-5088-5p and its expression increased. On the other hand, miR-5088-5p inhibitors were confirmed to reduce radiation-induced epithelial-mesenchymal transition, stemness, and metastasis by reducing Slug. Therefore, this study showed the potential of miR-5088-5p inhibitors as therapeutic agents to suppress radioresistance.

Molecular aspects of ABCB1 and ABCG2 in Gallbladder cancer and its clinical relevance.

The function of ABC transporters in the body is manifold; such as maintenance of homeostasis, effect on multi-drug resistance and their role in tumor initiation & progression. Evidence pointing towards the direct or indirect role of ABC transporter genes in particular; ABCB1 and ABCG2 in cancer genesis is increasing. However, their role in gallbladder cancer is unexplored. Therefore, we investigated the methylation status and expression pattern of ABCB1 and ABCG2in gallbladder carcinogenesis. The methylation and expression study of ABCB1/MDR1 and ABCG2/BCRP was performed in tumour and normal fresh tissue samples collected from 61 histopathologically diagnosed gallbladder cancer patients. The methylation status was analysed by Methylation-Specific PCR and expression was determined by Real-Time PCR and Immunohistochemistry. Hypomethylation of ABCB1 and ABCG2 was found in 44 (72.13%) and 48 (78.6%) cases, respectively. ABCB1 hypomethylation pattern showed association with female patients (p = 0.040) and GradeII tumors (p = 0.036) while, ABCG2 hypomethylation was more frequent in early tumors (T1-T2). The mRNA expression ofABCB1 and ABCG2 was up-regulated in 33 (54.10%) and 41 (67.21%) patients with fold change of 4.7 and 5.5, respectively. The mRNA expression of both genes showed association with Grade II tumours and the increased fold change of ABCG2 was higher in (T1-T2) depth of invasion (p = 0.02) and Stage I-II disease (p = 0.08). The protein expression on IHC was strongly positive for ABCB1/MDR1and ABCG2/BCRP in 32 (52.46%) and 45 (73.77%) patients, respectively. The protein expression in ABCG2 showed association with patients age > 50 years (p = 0.04) and GradeII differentiation (p = 0.07). Interestingly, the hypomethylation of both the genes showed significant correlation with increased expression. ABCB1/MDR1 and ABCG2/BCRP hypomethylation and overexpression could have a potential role in gallbladder cancer tumorigenesis especially in early stages. The epigenetic change might be a plausible factor for altered gene expression of ABCB1 and ABCG2 in gallbladder cancer.

Diagnostic value of molecular approach in screening for fragile X premutation cases.

BACKGROUND: Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, caused by CGG-repeats expansion (> 200 repeats). Premutation alleles (PM) (55-200 CGG repeats) are associated with tremor ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and autistic problems. AIM: To screen the frequency of premutation carriers using molecular diagnostic assays, in a cohort of Egyptian males with suspected clinical features of (FXS) checking for the presence of premutation alleles. METHODS: The current study comprised 192 Egyptian male children, 92 participants presented with intellectual disability, delayed language development, autistic-like features, behavioral difficulties, anxiety, seizures, and depression compared to 100 healthy males. All cases were subjected to clinical and neuroimaging assessments, when indicated as well as molecular analysis using methylation-specific PCR (MS-PCR) and quantitative real-time PCR (qRT-PCR). RESULTS: Thirty-four premutation carriers out of 92 Egyptian males (37%) of CGG repeats (55 to 200) were illustrated with elevated FMR1 mRNA expression level (p-value < 0.001). Additionally, 2 intermediate (IM) cases (0.03%) (45-55 CGG repeats) showed poor increase in expression level (p-value = 0.02838) plus 6 full mutation (FM) patients (0.07%) with (> 200 CGG repeats) (p-value < 0.001) resulted in FMR1 gene silence. CONCLUSION: Molecular diagnostic assay including (MS-PCR) and (qRT-PCR) proved to be a sensitive and rapid screening tool for the detection of premutation cases. Furthermore, the presence of positive correlation between FMR1 mRNA expression levels with CGG repeats in premutation cases could serve as a potential diagnostic marker. Application of these diagnostic tools on larger number clinically suspected cases is recommended.

DNA Methylation Analysis of Cnr1 Gene Promoter.

DNA methylation pattern could be considered a biomarker to be exploited for the study and management of several human diseases. In this chapter, detailed protocols are provided for two experimental approaches used for quantitative methylation analysis of bisulfite converted DNA: methylation-specific PCR (MSP) and pyrosequencing.

Promoter methylation of transient receptor potential melastatin-related 7 (TRPM7) predicts a better prognosis in patients with Luminal A breast cancers.

Breast cancer is the most common female tumors arising worldwide, and genetic and epigenetic events are constantly accumulated in breast tumorigenesis. The melastatin-related transient receptor potential 7 channel (TRPM7) is a nonselective cation channel, mainly maintaining Zn2+, Ca2+ and Mg2+ homeostasis. It is also involved in regulating proliferation and migration in various cancers including breast cancer. However, epigenetic alterations (such as promoter methylation) of TRPM7 and their correlation with clinical outcomes in breast cancer patients remain largely unclear. In this study, we found that TRPM7 was highly expressed in the luminal A subtype of breast cancers but no other subtypes compared with GTEx (Genotype-Tissue Expression Rad) or normal samples by analyzing the TCGA database. Correspondingly, TRPM7 was methylated in 42.7% (93 of 219) of breast cancers. Further studies found that promoter methylation of TRPM7 were significantly associated with better clinical outcomes in breast cancer patients, especially in the Luminal A subtype. Besides, methylated TRPM7 was correlated with less number of metastatic lymph nodes and longer local failure free survival time in this subtype. In summary, our data indicate that promoter methylation of TRPM7 may predict poor prognosis in patients with luminal A breast cancer.

Cohesin RAD21 Gene Promoter Methylation Correlated with Better Prognosis in Breast Cancer Patients.

RAD21 plays multiple roles in numerous cancers. In breast cancer (BC), a high level of RAD21 correlates with poor disease outcomes and resistance to chemotherapy. However, data regarding RAD21 promoter methylation in BC tissue and its correlation with clinical outcomes in patients with BC remain limited. Here, we investigated the clinicopathological features associated with the methylation status of RAD21 in BC to figure out its possible role in pathogenesis and the formation of breast carcinogenesis. The methylation status of the RAD21 gene was significantly associated with better clinical outcomes in patients with BC.

Effects of fluoride on the proliferation and activation of osteoblasts by regulating methylation of the DNA repair genes MGMT and MLH1.

INTRODUCTION: Fluoride can induce the proliferation and activation of osteoblasts, resulting in skeletal fluorosis progression; however, the specific mechanism is unclear. METHODS: Cell proliferation was examined using the MTT assay. Flow cytometry was performed to detect the cell cycle distribution. Alkaline phosphatase (ALP) was calculated to evaluate bone formation and turnover. Gene methylation was examined using the MSP assay. mRNA and protein expression levels were assessed using qRT-PCR and Western blot assays. RESULTS: Low-concentration NaF treatment promoted the cell cycle progression of osteoblasts to S-phase, thus accelerating cell proliferation and activation in a concentration-dependent manner. In addition, the methylation of the MGMT and MLH1 genes was increased, and their mRNA expression was reduced. Furthermore, the DNA methyltransferase inhibitor 5-AZA-dC suppressed cell viability, cell number in S-phase, ALP activity and osteogenesis-related protein levels in osteoblasts treated with low doses of NaF. Meanwhile, 5-AZA-dC suppressed the increase in MGMT and MLH1 gene methylation in osteoblasts treated with low doses of NaF, leading to enhanced expression of MGMT and MLH1 mRNA. CONCLUSION: NaF treatment led to methylation of the DNA repair genes MGMT and MLH1 in osteoblasts, resulting in cell proliferation and activation and causing the development of skeletal fluorosis.

Droplet Magnetofluidic Assay Platform for Quantitative Methylation-Specific PCR.

Early cancer detection requires identification of cellular changes resulting from oncogenesis. Abnormal DNA methylation patterns occurring early in tumor development have been widely identified as early biomarkers for multiple types of cancer tumors. Methylation-Specific PCR (MSP) has permitted highly sensitive detection of these methylation changes at known biomarker locations. MSP requires multiple sample preparation steps including protein digestion, DNA isolation, and bisulfite conversion prior to detection. In this work, we present a streamlined assay platform and instrumentation for integration of all sample processing steps required to obtain quantitative MSP signal from raw biological samples through the use of droplet magnetofluidic principles. In conjunction with this platform, we present a streamlined protocol for solid-phase DNA extraction from cells and bisulfite conversion of genomic DNA, minimizing the processing steps and reagent volume for implementation on a compact assay platform.

Computational challenges in detection of cancer using cell-free DNA methylation.

Cell-free DNA(cfDNA) methylation profiling is considered promising and potentially reliable for liquid biopsy to study progress of diseases and develop reliable and consistent diagnostic and prognostic biomarkers. There are several different mechanisms responsible for the release of cfDNA in blood plasma, and henceforth it can provide information regarding dynamic changes in the human body. Due to the fragmented nature, low concentration of cfDNA, and high background noise, there are several challenges in its analysis for regular use in diagnosis of cancer. Such challenges in the analysis of the methylation profile of cfDNA are further aggravated due to heterogeneity, biomarker sensitivity, platform biases, and batch effects. This review delineates the origin of cfDNA methylation, its profiling, and associated computational problems in analysis for diagnosis. Here we also contemplate upon the multi-marker approach to handle the scenario of cancer heterogeneity and explore the utility of markers for 5hmC based cfDNA methylation pattern. Further, we provide a critical overview of deconvolution and machine learning methods for cfDNA methylation analysis. Our review of current methods reveals the potential for further improvement in analysis strategies for detecting early cancer using cfDNA methylation.