Methylated DNA Fragments Research Articles

Detection and Characterization of Methylated Circulating Tumor DNA in Gastric Cancer.

Gastric cancer is the fifth most common disease in the world and the fourth most common cause of death. It is diagnosed through esophagogastroduodenoscopy with biopsy; however, there are limitations in finding lesions in the early stages. Recently, research has been actively conducted to use liquid biopsy to diagnose various cancers, including gastric cancer. Various substances derived from cancer are reflected in the blood. By analyzing these substances, it was expected that not only the presence or absence of cancer but also the type of cancer can be diagnosed. However, the amount of these substances is extremely small, and even these have various variables depending on the characteristics of the individual or the characteristics of the cancer. To overcome these, we collected methylated DNA fragments using MeDIP and compared them with normal plasma to characterize gastric cancer tissue or patients' plasma. We attempted to diagnose gastric cancer using the characteristics of cancer reflected in the blood through the cancer tissue and patients' plasma. As a result, we confirmed that the consistency of common methylated fragments between tissue and plasma was approximately 41.2% and we found the possibility of diagnosing and characterizing cancer using the characteristics of the fragments through SFR and 5'end-motif analysis.

Methyl-Sensitive Amplification Polymorphism (MSAP) Analysis Provides Insights into the DNA Methylation Changes Underlying Adaptation to Low Temperature of Brassica rapa L.

DNA methylation can change rapidly to regulate the expression of stress-responsive genes. Previous studies have shown that there are significant differences in the cold resistance of winter rapeseed (Brassica rapa L.) after being domesticated in different selection environments; however, little is known about the epigenetic regulatory mechanisms of its cold resistance formation. Four winter rapeseed materials ('CT-2360', 'MXW-1', '2018-FJT', and 'DT-7') domesticated in different environments were selected to analyze the DNA methylation level and pattern changes under low temperature using methylation-sensitive amplified polymorphism technology with 60 primer pairs. A total of 18 pairs of primers with good polymorphism were screened, and 1426 clear bands were amplified, with 594 methylation sites, accounting for 41.65% of the total amplified bands. The total methylation ratios of the four materials were reduced after low-temperature treatment, in which the DNA methylation level of 'CT-2360' was higher than that of the other three materials; the analysis of methylation patterns revealed that the degree of demethylation was higher than that of methylation in 'MXW-1', '2018-FJT', and 'DT-7', which were 22.99%, 19.77%, and 24.35%, respectively, and that the methylation events in 'CT-2360' were predominantly dominant at 22.95%. Fifty-three polymorphic methylated DNA fragments were randomly selected and further analyzed, and twenty-nine of the cloned fragments were homologous to genes with known functions. The candidate genes VQ22 and LOC103871127 verified the existence of different expressive patterns before and after low-temperature treatment. Our work implies the critical role of DNA methylation in the formation of cold resistance in winter rapeseed. These results provide a comprehensive insight into the adaptation epigenetic regulatory mechanism of Brassica rapa L. to low temperature, and the identified differentially methylated genes can also be used as important genetic resources for the multilateral breeding of winter-resistant varieties.

An electrochemical biosensor for detecting DNA methylation based on AuNPs/rGO/g-C3N4 nanocomposite

DNA methylation as a ubiquitously regulation is closely associated with cell proliferation and differentiation. Growing data shows that aberrant methylation contributes to disease incidence, especially in tumorigenesis. The approach for identifying DNA methylation usually depends on treatment of sodium bisulfite, which is time-consuming and conversion-insufficient. Here, with a special biosensor, we establish an alternative approach for detecting DNA methylation. The biosensor is consisted of two parts, which are gold electrode and nanocomposite (AuNPs/rGO/g-C3N4). Nanocomposite was fabricated by three components, which are gold nanoparticles (AuNPs), reduced graphene oxide (rGO) and graphite carbon nitride (g-C3N4). For methylated DNA detection, the target DNA was captured by probe DNA immobilized on the gold electrode surface through thiolating process and subjected to hybrid with anti-methylated cytosine conjugated to nanocomposite. When the methylated cytosines in target DNA were recognized by anti-methylated cytosine, a change of electrochemical signals will be observed. With different size of target DNAs, the concentration and methylation level were tested. It is shown that in short size methylated DNA fragment, the linear range and LOD of concentration is 10−7M-10−15M and 0.74 fM respectively; in longer size methylated DNA, the linear range of methylation proportion and LOD of copy number is 3%–84% and 103 respectively. Also, this approach has a high sensitivity and specificity as well as anti-disturbing ability.

Physiological and DNA methylation analysis provides epigenetic insights into kenaf cadmium tolerance heterosis

Soil heavy metal pollution is one of the most challenging problems. Kenaf is an important natural fiber crop with strong heterosis and a higher tolerance to heavy metals. However, little is known about the molecular mechanisms of kenaf heavy metal tolerance, especially the mechanism of genomic DNA methylation regulating heterosis. In this study, kenaf cultivars CP085, CP089, and their hybrid F1 seedlings were subjected to 300 µM cadmium stress and found obvious heterosis of cadmium resistance in morphology and antioxidant enzyme activity of F1 hybrid seedlings. Through methylation-sensitive amplification polymorphism (MSAP) analysis, we highlighted that the total DNA methylation level under cadmium decreased by 16.9 % in F1 and increased by 14.0 % and 3.0 % in parents CP085 and CP089, respectively. The hypomethylation rate was highest (21.84 %), but hypermethylation was lowest (17.24 %) in F1 compared to parent cultivars. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between F1 and its parents under cadmium. Furthermore, 21 differentially methylated DNA fragments (DMFs) were analyzed. Especially, the expression of NPF2.7, NADP-ME, NAC71, TPP-D, LRR-RLKs, and DHX51 genes were changed due to cadmium stress and related to cytosine methylation regulation. Finally, the knocked-down of the differentially methylated gene NPF2.7 by virus-induced gene silencing (VIGS) resulted in increased sensitivity of kenaf seedlings under cadmium stress. It is speculated that low DNA methylation levels can regulate gene expression that led to the heterosis of cadmium tolerance in kenaf.

Consistent DNA Hypomethylations in Prostate Cancer.

With approximately 1.4 million men annually diagnosed with prostate cancer (PCa) worldwide, PCa remains a dreaded threat to life and source of devastating morbidity. In recent decades, a significant decrease in age-specific PCa mortality has been achieved by increasing prostate-specific antigen (PSA) screening and improving treatments. Nevertheless, upcoming, augmented recommendations against PSA screening underline an escalating disproportion between the benefit and harm of current diagnosis/prognosis and application of radical treatment standards. Undoubtedly, new potent diagnostic and prognostic tools are urgently needed to alleviate this tensed situation. They should allow a more reliable early assessment of the upcoming threat, in order to enable applying timely adjusted and personalized therapy and monitoring. Here, we present a basic study on an epigenetic screening approach by Methylated DNA Immunoprecipitation (MeDIP). We identified genes associated with hypomethylated CpG islands in three PCa sample cohorts. By adjusting our computational biology analyses to focus on single CpG-enriched 60-nucleotide-long DNA probes, we revealed numerous consistently differential methylated DNA segments in PCa. They were associated among other genes with NOTCH3, CDK2AP1, KLK4, and ADAM15. These can be used for early discrimination, and might contribute to a new epigenetic tumor classification system of PCa. Our analysis shows that we can dissect short, differential methylated CpG-rich DNA fragments and combinations of them that are consistently present in all tumors. We name them tumor cell-specific differential methylated CpG dinucleotide signatures (TUMS).

Identifying Transcription Factors That Prefer Binding to Methylated DNA Using Reduced G-Gap Dipeptide Composition.

Transcription factors (TFs) play an important role in gene expression and regulation of 3D genome conformation. TFs have ability to bind to specific DNA fragments called enhancers and promoters. Some TFs bind to promoter DNA fragments which are near the transcription initiation site and form complexes that allow polymerase enzymes to bind to initiate transcription. Previous studies showed that methylated DNAs had ability to inhibit and prevent TFs from binding to DNA fragments. However, recent studies have found that there were TFs that could bind to methylated DNA fragments. The identification of these TFs is an important steppingstone to a better understanding of cellular gene expression mechanisms. However, as experimental methods are often time-consuming and labor-intensive, developing computational methods is essential. In this study, we propose two machine learning methods for two problems: (1) identifying TFs and (2) identifying TFs that prefer binding to methylated DNA targets (TFPMs). For the TF identification problem, the proposed method uses the position-specific scoring matrix for data representation and a deep convolutional neural network for modeling. This method achieved 90.56% sensitivity, 83.96% specificity, and an area under the receiver operating characteristic curve (AUC) of 0.9596 on an independent test set. For the TFPM identification problem, we propose to use the reduced g-gap dipeptide composition for data representation and the support vector machine algorithm for modeling. This method achieved 82.61% sensitivity, 64.86% specificity, and an AUC of 0.8486 on another independent test set. These results are higher than those of other studies on the same problems.

Detection of transcription factors binding to methylated DNA by deep recurrent neural network.

Transcription factors (TFs) are proteins specifically involved in gene expression regulation. It is generally accepted in epigenetics that methylated nucleotides could prevent the TFs from binding to DNA fragments. However, recent studies have confirmed that some TFs have capability to interact with methylated DNA fragments to further regulate gene expression. Although biochemical experiments could recognize TFs binding to methylated DNA sequences, these wet experimental methods are time-consuming and expensive. Machine learning methods provide a good choice for quickly identifying these TFs without experimental materials. Thus, this study aims to design a robust predictor to detect methylated DNA-bound TFs. We firstly proposed using tripeptide word vector feature to formulate protein samples. Subsequently, based on recurrent neural network with long short-term memory, a two-step computational model was designed. The first step predictor was utilized to discriminate transcription factors from non-transcription factors. Once proteins were predicted as TFs, the second step predictor was employed to judge whether the TFs can bind to methylated DNA. Through the independent dataset test, the accuracies of the first step and the second step are 86.63% and 73.59%, respectively. In addition, the statistical analysis of the distribution of tripeptides in training samples showed that the position and number of some tripeptides in the sequence could affect the binding of TFs to methylated DNA. Finally, on the basis of our model, a free web server was established based on the proposed model, which can be available at https://bioinfor.nefu.edu.cn/TFPM/.

Physiological and DNA methylation analysis provides epigenetic insights into chromium tolerance in kenaf

As a toxic heavy metal, chromium (Cr) has become a major environmental concern in cultivated areas. Kenaf is a fast-growing fibre crop with high economic value and can potentially be used for phytoremediation. However, limited information is known regarding the mechanism of kenaf in response to Cr stress, especially from the perspective of epigenetics. The present study was conducted to explore the physiological and DNA methylation changes in kenaf seedlings grown under different concentrations (from 0 to 600 μM) of Cr stress. The results show that Cr reduced plant height, root length, biomass, and root cell viability compared with the control. The activities of antioxidant enzymes (SOD, POD, and CAT) increased under Cr stress and reached their peaks at Cr concentrations of 500, 300, and 400 μM respectively. Methylation-sensitive amplification polymorphism (MSAP) analysis revealed a significant positive correlation between an increase in the total DNA methylation level and Cr concentration, and the increasing rate ranged from 20.97% to 26.34%. Among the 205 loci examined, 40 differentially methylated DNA fragments (DMFs) were cloned and analysed. DMFs such as the ABC transport family (ABC) may play essential roles in kenaf Cr stress. In addition, the methylation status of three Cr stress- related DMFs (HcABC, HcRHP and HcTyDC) was investigated via the bisulfite sequencing polymerase chain reaction (BSP) method. Furthermore, knockdown of HcTyDC by virus-induced gene silencing (VIGS) led to increased sensitivity to Cr stress. Overall, our data indicate that higher antioxidant activity and increased cytosine methylation levels in the plant genome could be related to Cr tolerance mechanisms in kenaf, and these changes then affect the expression of specific genes involved in the Cr stress response.

Increased Sensitivity of Detection of RASSF1A and GSTP1 DNA Fragments in Serum of Prostate Cancer Patients: Optimisation of Diagnostics Using OBBPA-ddPCR.

Simple SummaryProstate carcinoma (PCa) incidence rates have continued to increase over the last 3 decades worldwide and PCa is the most common cancer in men in Germany. Classical tumour markers exhibit unsatisfactory sensitivity and specificity, leading to unnecessary prostate biopsies and associated clinical complications. New, minimally invasive biomarkers with high sensitivity and specificity are required for an early diagnosis and a better prognosis. In the present study, we evaluated the performance of a previously developed analytical method to detect trace amounts of aberrant methylated tumour DNA. The optimised method which was developed in this study evaluates DNA methylation changes of RASSF1A and GSTP1 in the blood of PCa patients at different stages compared to benign prostatic hyperplasia patients and healthy individuals. RASSF1A methylation analysis was found to be beneficial as a complementary biomarker for PCa diagnosis in combination with classical PCa markers. Both RASSF1A and GSTP1 exhibited pathological DNA methylation levels in all metastatic PCa patients, which may improve early detection of PCa metastases and therapeutic outcomes.Identification of aberrant DNA methylation is a promising tool in prostate cancer (PCa) diagnosis and treatment. In this study, we evaluated a two-step method named optimised bias-based preamplification followed by digital PCR (OBBPA-dPCR). The method was used to identify promoter hypermethylation of 2 tumour suppressor genes RASSF1A and GSTP1 in the circulating cell-free DNA (cfDNA) from serum samples of PCa patients (n = 75), benign prostatic hyperplasia (BPH, n = 58), and healthy individuals (controls, n = 155). The PCa cohort was further subdivided into subgroups comprising (I) patients with Gleason Scores (GS) ≤ 7 (n = 55), (II) GS ≥ 8 (n = 10), and (III) patients with metastatic PCa diagnosis (n = 10). We found that RASSF1A methylation levels were significantly increased in all 3 PCa subgroups compared to the controls and BPH cohorts (p < 0.01 for all comparisons). Fractional abundances of methylated GSTP1 DNA fragments were significantly increased in subgroup III of metastatic PCa patients (p < 0.001). RASSF1A methylation analysis was found to be beneficial as a complementary biomarker where further diagnostic validation is most crucial. In combination with free PSA, RASSF1A methylation status helps to identify PCa patients with GS ≥ 8 and grey-zone total PSA values between 2–10 ng/mL. In our study, PCR biases between 80–90% were sufficient to detect minute amounts of tumour DNA with high signal-to-noise ratios as well as high analytical sensitivity and specificity. Both RASSF1A and GSTP1 exhibited strongly increased DNA methylation levels in all metastatic PCa patients. Our data indicates a superior sensitivity of epigenetic biomarker analyses in early detection of PCa metastases that should also help to improve PCa therapy.

Tissue-specific DamID protocol using nanopore sequencing.

DNA adenine methylation identification (DamID) is a powerful method to determine DNA binding profiles of proteins at a genomic scale. The method leverages the fusion between a protein of interest and the Dam methyltransferase of E. coli, which methylates proximal DNA in vivo. Here, we present an optimized procedure, which was developed for tissue-specific analyses in Caenorhabditis elegans and successfully used to footprint genes actively transcribed by RNA polymerases and to map transcription factor binding in gene regulatory regions. The present protocol details C. elegans-specific steps involved in the preparation of transgenic lines and genomic DNA samples, as well as broadly applicable steps for the DamID procedure, including the isolation of methylated DNA fragments, the preparation of multiplexed libraries, Nanopore sequencing, and data analysis. Two distinctive features of the approach are (i) the use of an efficient recombination-based strategy to selectively analyze rare cell types and (ii) the use of Nanopore sequencing, which streamlines the process. The method allows researchers to go from genomic DNA samples to sequencing results in less than a week, while being sensitive enough to report reliable DNA footprints in cell types as rare as 2 cells per animal.

Detection of colorectal cancer in urine using DNA methylation analysis

Colorectal cancer (CRC) is the second leading cause for cancer-related death globally. Clinically, there is an urgent need for non-invasive CRC detection. This study assessed the feasibility of CRC detection by analysis of tumor-derived methylated DNA fragments in urine. Urine samples, including both unfractioned and supernatant urine fractions, of 92 CRC patients and 63 healthy volunteers were analyzed for DNA methylation levels of 6 CRC-associated markers (SEPT9, TMEFF2, SDC2, NDRG4, VIM and ALX4). Optimal marker panels were determined by two statistical approaches. Methylation levels of SEPT9 were significantly increased in urine supernatant of CRC patients compared to controls (p < 0.0001). Methylation analysis in unfractioned urine appeared inaccurate. Following multivariate logistic regression and classification and regression tree analysis, a marker panel consisting of SEPT9 and SDC2 was able to detect up to 70% of CRC cases in urine supernatant at 86% specificity. First evidence is provided for CRC detection in urine by SEPT9 methylation analysis, which combined with SDC2 allows for an optimal differentiation between CRC patients and controls. Urine therefore provides a promising liquid biopsy for non-invasive CRC detection.

DNA methylation changes stimulated by drought stress in ABA-deficient maize mutant vp10

Plants are constantly challenged with several biotic and abiotic stresses, and the adaptation to these stresses requires molecular and morphological changes. Epigenetic regulation provides effective control that enables plants to tolerate stress, which results in improved survivability. The distinct role of abscisic acid (ABA) in controlling numerous stress-responsive genes and enhancing respiration metabolism is well known; however, whether DNA methylation is associated with the regulation of ABA-dependent gene expression remains unclear. This study was conducted to identify the changes in DNA methylation induced by drought stress in ABA-deficient maize mutant vp10 using the amplified methylation polymorphism–polymerase chain reaction (AMP–PCR) technique. Differentially methylated DNA fragments were mapped to intragenic regions of zinc finger, amino acid catabolic enzymes, and other genes implicated in DNA repair and plant survival, in addition to several demethylated transposable elements.

Effects of cadmium stress on DNA methylation in soybean

Epigenetics plays an important role in plant resistance to biotic and abiotic stresses, although few studies have focused on soybean epigenetics associated with cadmium stress. In this study, hydroponically grown soybean seedlings were treated with hydroponic nutrient solution containing various concentrations of CdCl2 (0.5, 5, 15 mg L−1) to investigate the effects of cadmium exposure on soybean genomic DNA methylation levels and patterns using methylation-sensitive amplified polymorphism (MSAP) analysis. Overall, the results revealed alterations of genomic DNA methylation levels and patterns in soybean seedlings under cadmium stress, with methylation level and methylation polymorphism rate both increasing in a dose-dependent manner with increasing cadmium concentration. The total methylation rates for genomic DNA of control (CK), S1 (0.5 mg L−T CdCl2), S2 (5 mg L−1 CdCl2) and S3 (15 mg L−1 CdCl2) groups were 27.92%, 29.78%, 33.2% and 35.79%, respectively, and methylation polymorphism rates were 14.46%, 16.57% and 18.15%, respectively. Thus, soybean cadmium stress-induced methylation changes mainly reflected increased genomic DNA methylation. After polymorphic methylated genomic DNA fragments were recovered from polyacrylamide gels, cloned and sequenced, sequence homology comparisons revealed that among 30 detected differentially methylated fragments, 15 sequences were highly homologous to genes encoding functional proteins already known to be associated with plant stress responses. These results indicate that soybean resists cadmium stress by increasing both the level of genomic DNA methylation and the genomic DNA remethylation rate. This work provides a methodological foundation for investigating molecular mechanisms underlying the soybean response to cadmium stress.

Changes in DNA methylation profiles of myalgic encephalomyelitis/chronic fatigue syndrome patients reflect systemic dysfunctions

BackgroundMyalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a lifelong debilitating disease with a complex pathology not yet clearly defined. Susceptibility to ME/CFS involves genetic predisposition and exposure to environmental factors, suggesting an epigenetic association. Epigenetic studies with other ME/CFS cohorts have used array-based technology to identify differentially methylated individual sites. Changes in RNA quantities and protein abundance have been documented in our previous investigations with the same ME/CFS cohort used for this study.ResultsDNA from a well-characterised New Zealand cohort of 10 ME/CFS patients and 10 age-/sex-matched healthy controls was isolated from peripheral blood mononuclear (PBMC) cells, and used to generate reduced genome-scale DNA methylation maps using reduced representation bisulphite sequencing (RRBS). The sequencing data were analysed utilising the DMAP analysis pipeline to identify differentially methylated fragments, and the MethylKit pipeline was used to quantify methylation differences at individual CpG sites. DMAP identified 76 differentially methylated fragments and Methylkit identified 394 differentially methylated cytosines that included both hyper- and hypo-methylation. Four clusters were identified where differentially methylated DNA fragments overlapped with or were within close proximity to multiple differentially methylated individual cytosines. These clusters identified regulatory regions for 17 protein encoding genes related to metabolic and immune activity. Analysis of differentially methylated gene bodies (exons/introns) identified 122 unique genes. Comparison with other studies on PBMCs from ME/CFS patients and controls with array technology showed 59% of the genes identified in this study were also found in one or more of these studies. Functional pathway enrichment analysis identified 30 associated pathways. These included immune, metabolic and neurological-related functions differentially regulated in ME/CFS patients compared to the matched healthy controls.ConclusionsMajor differences were identified in the DNA methylation patterns of ME/CFS patients that clearly distinguished them from the healthy controls. Over half found in gene bodies with RRBS in this study had been identified in other ME/CFS studies using the same cells but with array technology. Within the enriched functional immune, metabolic and neurological pathways, a number of enriched neurotransmitter and neuropeptide reactome pathways highlighted a disturbed neurological pathophysiology within the patient group.

Genome-wide DNA methylation analysis using MethylCap-seq in canine high-grade B-cell lymphoma.

DNA methylation is a comprehensively studied epigenetic modification and plays crucial roles in cancer development. In the present study, MethylCap-seq was used to characterize the genome-wide DNA methylation patterns in canine high-grade B-cell lymphoma (cHGBL). Canine methylated DNA fragments were captured and the MEDIUM-HIGH and LOW fraction of methylated DNA was obtained based on variation in CpG methylation density. In the MEDIUM-HIGH and LOW fraction, 2144 and 1987 cHGBL-specific hypermethylated genes, respectively, were identified. Functional analysis highlighted pathways strongly related to oncogenesis. The relevant signaling pathways associated with neuronal system were also revealed, echoing recent novel findings that neurogenesis plays key roles in tumor establishment. In addition, 14 genes were hypermethylated in all the cHGBL cases but not in the healthy dogs. These genes might be potential signatures for tracing cHGBL, and some of them have been reported to play roles in various types of cancers. Further, the distinct methylation pattern of cHGBL showed a concordance with the clinical outcome, suggesting that aberrant epigenetic changes may influence tumor behavior. In summary, our study characterized genome-wide DNA methylation patterns using MethylCap-seq in cHGBL; the findings suggest that specific DNA hypermethylation holds promise for dissecting tumorigenesis and uncovering biomarkers for monitoring the progression of cHGBL.

Abstract 2761: Effect of DNA fragments and nucleotid supplementation on tumor development in mice model

Abstract Background: The balance of dNTP pool is needed for normal DNA synthesis. Nucleotide depletion can cause DNA damage thus it could lead to tumor development. The supplementation of substrates that are involved in DNA synthesis and methylation reactions could be a preventive step against oncogenic processes. Furthermore, tumor origin and heterogeneity could influence the cell response depend on experimental models (human SW480 versus animal-derived MCA38 colon adenocarcinoma). Aims: Our aims were to investigate the effect of elevated dNTP pool and modified DNA fragments on tumor growth, furthermore to detect the influence of different types of folates on tumor formation. Methods: C57BL/6 (n=35) mice with implanted MCA38 mouse colorectal adenocarcinoma cells were divided into 7 groups. Mice were injected subcutaneously on every 2nd days over 4 weeks in the doses of 10ug/animal with the following materials: dNTP (Group1) and methylated (d5mCTP) deoxynucleotide solution (Group2). In Group 3-5, mice were injected with artificially methylated and unmethylated spleen-derived DNA in different doses. Group 6 was supplemented with 20mg/kg folic acid orally, while saline (0.9% NaCl) was used as a vehicle in control group. In parallel, human-derived SW480 colon cancer cells (5 × 106) were injected subcutaneously on SCID mice (n=15). Xenograft mice were divided into 3 groups. 20mg/kg folic acid (Group A) and 20mg/kg L-methylfolate (Group B) were supplemented orally 5 times a week, as well as control mice were treated with saline. The length and width of the implanted tumors were measured with sliding calipers two times weekly by the same observer. Results: In the animal-derived (MCA38) tumor model, slower tumor developing tendency was observed in the dNTP supplementation group; d5mCTP supplementation moderately decreased tumor growth. Non-methylated DNA has also a concentration-dependent growth decreasing effect in MCA38 tumor development. However, folate supplementation has a different effect on tumor development based on the origin of the implanted colon tissue. Slower tumor developing tendency could be detected in the case of C57BL/6-C38 tumor model compared to controls, while folate supplementation stimulated the growth and proliferation of tumor cells in SW480-derived xenograft model (p&amp;lt;0.05). Conclusion: Our study described the effect of differently methylated DNA fragments and precursor molecules on the growth of tumor transplants. According to our results, tumor volume decrease was observed by providing sufficient DNA components to normal synthesis. Based on our results folate treatment has a different effect on cancer development regarding the tumor origin. Folate has a key role in nucleotide synthesis and also in DNA methylation therefore the selection of the appropriate dosing time is crucial. Citation Format: Zsófia Brigitta Nagy, Barbara Kinga Barták, Sára Zsigrai, Alexandra Kalmár, Krisztina Szigeti, Peter Igaz, Bela Molnár. Effect of DNA fragments and nucleotid supplementation on tumor development in mice model [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2761.

Alteration in global DNA methylation status following preconditioning injury influences axon growth competence of the sensory neurons

Preconditioning peripheral nerve injury primes the sensory neurons in the dorsal root ganglia (DRGs) to acquire axon regeneration competence. Transcription of a large set of regeneration-associated-genes (RAGs) contributes to the enhanced intrinsic axonal regeneration capacity. However, the mechanism underlying the coordinated upregulation of RAGs orchestrated by preconditioning injury is unclear. We sought to determine potential influence of DNA methylation change on transcriptional activation of RAGs in the L4-L6 DRGs following sciatic nerve injury. Genome-wide sequencing revealed that about 20% of the methylated DNA fragments were differentially methylated, and >3000 genes contained differentially methylated regions. Not only demethylation but also increased methylation was observed to a similar extent. The change in the global DNA methylation did not correlate with the gene expression level of most genes, including the well-documented RAGs. However, pharmacological inhibition or activation of DNA methylation markedly attenuated the axon growth capacity of the preconditioned DRG neurons. Pharmacological perturbation of DNA methylation resulted in simultaneous downregulation of many highly overlapping non-transcription factor RAGs, which was accompanied by a concurrent, robust upregulation of SOCS3 and Serpine1. Overexpression of SOCS3 and Serpine1 in the DRG neurons overrode injury-induced axon growth competence, corroborating their roles as the negative regulators of axon regeneration. We conclude that the injury-induced global alteration of DNA methylome strongly influences the axon growth competence in preconditioned DRG neurons. Our results also suggest a possibility that perturbing DNA methylome changes might lead to the upregulation of negative regulator RAGs thereby attenuating axon growth capacity.

GENOME-WIDE METHYLOME ANALYSIS USING METHYLCAP-SEQ UNCOVERS NOVEL METHYLATED MARKERS FOR CANINE LYMPHOMA CELL LINE CLBL-1

The most common type of tumor occurring in canine is the lymphomas. Epigenetic factors like DNA methylation is one of the important factors for causing cancer in canine. Cytology-based screening methods, such as fine needle aspiration (FNA) or immunocytochemistry (ICC), for canine lymphomas lack sensitivity. Despite an improvement in the detection of canine lymphomas by the DNA methylation analysis of lymph scrapings, only few methylation markers have been described. Even though technique like Digital Restriction Enzyme Analysis of Methylation (DREAM) can analyze the methylation profile of canine lymphoma cell line CLBL-1 at very low experimental cost without specific antibodies, the sensitivity and accuracy remain low in comparison to MethylCap-seq. In this study, a methylated DNA fragment of CLBL-1 was successfully captured by a specific protein for MethylCap-seq, and the methylation map of CLBL-1 was reconstructed by high throughput sequencing. For the first time, the genomic methylation region of canine CLBL-1 was analyzed. Using CLBL-1, we aimed to identify novel methylated markers for monitoring canine lymphomas. With the aid of methylation maps, several researchers have attempted to explore the relationship between DNA methylation and specific cancers. Additionally, the discovery of important biomarkers can predict or observe the course of canine cancer. Finally, the proposed advanced methylation information is likely to be suitable as a useful reference for clinical application of canine lymphoma research.

Identification of rare levels of methylated tumor DNA fragments using an optimized bias based pre-amplification-digital droplet PCR (OBBPA-ddPCR).

BackgroundThe analysis of aberrant DNA methylations is used for the diagnosis of cancer as significant changes in the gene methylation pattern are often detected during early carcinogenesis. In this study, we evaluated the performance of a two-step method that combines pre-amplification with ddPCR technique.ResultsBy using ddPCR, the dependence of amplification efficiency for methylated and unmethylated DNA fragments on the relevant MgCl2 concentration and the annealing temperature was established in addition to the primer design. We found that the efficiency can be adjusted toward methylated sequences by using primers covering one to four CpG sites under appropriately selected MgCl2 concentration and annealing temperature. Applying a PCR bias between 85% and 95%, five copies of methylated tumor DNA fragments were detected against a background of 700,000 copies of unmethylated DNA fragments with a high signal-to-noise ratio. The analysis of serum samples from patients with prostate cancer showed a significantly improved performance of the new method in comparison with the MS-HRM technique, ddPCR alone, or ddPCR in combination with an unbiased pre-amplification using methylation-independent primers.ConclusionsWe define this method as an optimized bias-based pre-amplification-digital droplet PCR (OBBPA-ddPCR) technique. This novel method is recommended for the early detection of cancer-specific DNA methylation biomarkers in the form of a liquid biopsy.

In vivo analysis of circulating cell-free DNA release and degradation

Cell-free DNA (cfDNA) was first detected in human plasma in the 1940s, but the knowledge on its regulation and rate of release is incomplete. CfDNA can originate from both normal and tumour cells. Our aims were to investigate the rate of cfDNA's release in SHO mice/HT-29 colorectal adenocarcinoma cell line xenograft model and to define the decay of methylated and non-methylated DNA fragments in C57BL/6 bloodstream. SHO mice were xenografted with human HT-29 cells, than blood samples were collected over 2 months. CfDNA was isolated, then quantified by real-time PCR with highly specific genomic and mitochondrial human and mouse primer sets. This method permitted to define the ratio of human/mouse DNA. To assess the degradation rate of cfDNA, 3000 bp sized methylated and non-methylated DNA fragments were injected into healthy and C38 tumour-cell vaccinated C57BL/6 mice's bloodstream. The decay of amplicons was measured with 19 PCR assays. The amount of human DNA until the 2nd week was below the limit of detection. From the third week, a continuous growth was experienced, which reached 18.26% by the 8th week. Moreover, it was found that in healthy animals the non-methylated DNA disappears from the plasma after 6 hours, while the methylated fragment was detectable even after 24 hours. In animals with tumour, both amplicons were detectable after 24 hours. The examination of the role and mechanism of cfDNA shows an increasing level of interest. This work can contribute to a better understanding of the release and degradation of cfDNA. Orv Hetil. 2018; 159(6): 223-233.