Long Interspersed Nucleotide Element-1 Methylation Research Articles

Seropositivity-Dependent Association between LINE-1 Methylation and Response to Methotrexate Therapy in Early Rheumatoid Arthritis Patients.

Methotrexate (MTX) is considered the first choice among disease-modifying anti-rheumatic drugs (DMARDs) for rheumatoid arthritis (RA) treatment. However, response to it varies as approximately 40% of the patients do not respond and would lose the most effective period of treatment time. Therefore, having a predictive biomarker before starting MTX treatment is of utmost importance. Methylation of long interspersed nucleotide element-1 (LINE-1) is generally considered a surrogate marker for global genomic methylation, which has been reported to associate with disease activity after MTX therapy. We performed a prospective study on 273 naïve early RA (ERA) patients who were treated with MTX, followed up to 12 months, and classified according to their therapy response. The baseline LINE-1 methylation levels in peripheral blood mononuclear cells (PBMC) of cases were assessed by bisulfite pyrosequencing. Baseline LINE-1 methylation level per se turned out not to predict the response to the therapy, nor did age, sex, body mass index, or smoking status. However, if cases were stratified according to positivity to rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) or seronegativity, we observed an opposite association between baseline LINE-1 methylation levels and optimal response to MTX therapy among responders. The best response to MTX therapy was associated with hypermethylated LINE-1 among double-positive ERA cases (p-value: 0.002) and with hypomethylated LINE-1 in seronegative ERA patients (p-value: 0.01). The LINE-1 methylation level in PBMCs of naïve ERA cases associates with the degree of response to MTX therapy in an opposite way depending on the presence of RF and ACPA antibodies. Our results suggest LINE-1 methylation level as a new epigenetic biomarker for predicting the degree of response to MTX in both double-positive and seronegative ERA patients.

LINE-1 and Alu methylation signatures in autism spectrum disorder and their associations with the expression of autism-related genes

Long interspersed nucleotide element-1 (LINE-1) and Alu elements are retrotransposons whose abilities cause abnormal gene expression and genomic instability. Several studies have focused on DNA methylation profiling of gene regions, but the locus-specific methylation of LINE-1 and Alu elements has not been identified in autism spectrum disorder (ASD). Here we interrogated locus- and family-specific methylation profiles of LINE-1 and Alu elements in ASD whole blood using publicly-available Illumina Infinium 450 K methylation datasets from heterogeneous ASD and ASD variants (Chromodomain Helicase DNA-binding 8 (CHD8) and 16p11.2del). Total DNA methylation of repetitive elements were notably hypomethylated exclusively in ASD with CHD8 variants. Methylation alteration in a family-specific manner including L1P, L1H, HAL, AluJ, and AluS families were observed in the heterogeneous ASD and ASD with CHD8 variants. Moreover, LINE-1 and Alu methylation within target genes is inversely related to the expression level in each ASD variant. The DNA methylation signatures of the LINE-1 and Alu elements in ASD whole blood, as well as their associations with the expression of ASD-related genes, have been identified. If confirmed in future larger studies, these findings may contribute to the identification of epigenomic biomarkers of ASD.

Smoking and colorectal cancer survival in relation to tumor LINE-1 methylation levels: a prospective cohort study

BackgroundCarcinogens in cigarette smoke may cause aberrant epigenomic changes. The hypomethylation of long interspersed nucleotide element-1 (LINE-1) in colorectal carcinoma has been associated with genomic instability and worse clinical outcome. We hypothesized that the association between smoking behavior and colorectal cancer mortality might be stronger in tumors with lower LINE-1 methylation levels.FindingsTo test our hypothesis, we examined the interaction of tumor LINE-1 methylation levels and smoking status at diagnosis using data of 1208 cases among 4420 incident colorectal cancer cases that were ascertained in two prospective cohort studies. We conducted multivariable Cox proportional hazards regression analyses, using inverse probability weighting with covariate data of the 4420 cases to control for potential confounders and selection bias due to data availability. The prognostic association of smoking status at diagnosis differed by tumor LINE-1 methylation levels (Pinteraction = 0.050 for overall mortality and 0.017 for colorectal cancer-specific mortality; with an alpha level of 0.005). In cases with <60% LINE-1 methylation, current smoking (vs. never smoking) was associated with worse overall mortality (multivariable hazard ratio, 1.80; 95% confidence interval, 1.19–2.73). In contrast, smoking status was not associated with mortality in cases with ≥60% LINE-1 methylation.ConclusionsOur findings suggest that the association between smoking status and mortality is stronger in colorectal cancer patients with lower tumor LINE-1 methylation levels. These results warrant further investigation into an interactive role of smoking and aberrant DNA methylation in colorectal cancer progression.

Individual and mixtures of metal exposures in associations with biomarkers of oxidative stress and global DNA methylation among pregnant women

BackgroundPrenatal exposure to metals has been linked with adverse pregnancy outcomes. Oxidative stress and epigenetic changes are potential mechanisms of action. ObjectivesWe aimed to examine the associations of individual and mixtures of metal exposures with oxidative stress and DNA methylation among pregnant women. MethodsWe measured a panel of 16 metals and 3 oxidative stress biomarkers including 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA) and 8-isoprostaglandin F2α (8-isoPGF2α) in urine from 113 pregnant women in a Chinese cohort. Biomarkers of global DNA methylation including Alu and long interspersed nucleotide element-1 (LINE-1) in cord blood were measured. Multivariable linear regression and Bayesian kernel machine regression (BKMR) models were separately applied to estimate the associations between individual and mixtures of metal exposures and biomarkers of oxidative stress and global DNA methylation. ResultsIn single-metal analyses, we observed positive associations between 11 metals [arsenic (As), cadmium (Cd), thallium (Tl), barium (Ba), nickel (Ni), vanadium (V), cobalt (Co), zinc (Zn), copper (Cu), selenium (Se) and molybdenum (Mo)] and at least one of oxidative stress biomarkers (all FDR-adjusted P-values < 0.05). In mixture analyses, we found positive overall associations of metal mixtures with 8-OHdG and 8-isoPGF2α, and Se was the most important predictor. There was no evidence on associations of urinary metals as individual chemicals and mixtures with Alu and LINE-1 methylation. ConclusionUrinary metals as individual chemicals and mixtures were associated with increased oxidative stress, especially Se.

Polyphenol-Rich Aronia melanocarpa Juice Consumption Affects LINE-1 DNA Methylation in Peripheral Blood Leukocytes in Dyslipidemic Women.

Cardiovascular disease (CVD) is associated with alterations in DNA methylation and polyunsaturated fatty acid (PUFA) profile, both modulated by dietary polyphenols. The present parallel, placebo-controlled study (part of the original clinical study registered as NCT02800967 at www.clinicaltrials.gov) aimed to determine the impact of 4-week daily consumption of polyphenol-rich Aronia melanocarpa juice (AMJ) treatment on Long Interspersed Nucleotide Element-1 (LINE-1) methylation in peripheral blood leukocytes and on plasma PUFAs, in subjects (n = 54, age range of 40.2 ± 6.7 years) at moderate CVD risk, including an increased body mass index, central obesity, high normal blood pressure, and/or dyslipidemia. The goal was also to examine whether factors known to affect DNA methylation (folate intake levels, MTHFR C677T gene variant, anthropometric and metabolic parameters) modulated the LINE-1 methylation levels upon the consumption of polyphenol-rich aronia juice. Experimental analysis of LINE-1 methylation was done by MethyLight method. MTHFR C677T genotypes were determined by the polymerase chain reaction–restriction fragment length polymorphism method, and folate intake was assessed by processing the data from the food frequency questionnaire. PUFAs were measured by gas–liquid chromatography, and serum lipid profile was determined by using Roche Diagnostics kits. The statistical analyses were performed using Statistica software package. In the comparison after vs. before the treatment period, in dyslipidemic women (n = 22), we observed significant decreases in LINE-1 methylation levels (97.54 ± 1.50 vs. 98.39 ± 0.86%, respectively; P = 0.01) and arachidonic acid/eicosapentaenoic acid ratio [29.17 ± 15.21 vs. 38.42 (25.96–89.58), respectively; P = 0.02]. The change (after vs. before treatment) in LINE-1 methylation directly correlated with the presence of MTHFR 677T allele, average daily folate intake, and the change in serum low-density lipoprotein cholesterol but inversely correlated with the change in serum triacylglycerols (R = 0.72, R2 = 0.52, adjusted R2 = 0.36, P = 0.03). The current results imply potential cardioprotective effects of habitual polyphenol-rich aronia juice consumption achieved through the modifications of DNA methylation pattern and PUFAs in subjects at CVD risk, which should be further confirmed. Hence, the precision nutrition-driven modulations of both DNA methylation and PUFA profile may become targets for new approaches in the prevention of CVD.

Correlation of LINE-1 Hypomethylation With Size and Pathologic Extent of Dysplasia in Colorectal Tubular Adenomas.

INTRODUCTION:Conventional adenomas (tubular adenoma [TA] or tubulovillous adenoma) and sessile serrated lesions (SSLs) are neoplastic precancerous lesions frequently detected in patients undergoing average risk screening colonoscopy and polyp surveillance. Metachronous risk stratification of adenomas is currently limited to histologic features and size of polyps. We report long interspersed nucleotide element-1 (LINE-1) methylation levels in SSL in comparison to TA and the impact of TA size and presence of high-grade dysplasia (HGD) on LINE-1 methylation.METHODS:LINE-1 methylation was assessed by pyrosequencing of bisulfite-converted DNA. We compared LINE-1 methylation between TA and SSL, among varying sizes of TA, and between TA with HGD and low-grade dysplasia (LGD).RESULTS:LINE-1 methylation declined with increasing polyp size in TA when comparing those <5 mm (72.31 ± 6.11), 5 to <10 mm (67.50 ± 7.00), and ≥10 mm (66.75 ± 11.89). There were lower LINE-1 methylation levels in TA with LGD (n = 119) compared with SSLs (n = 29) (69.11 ± 8.62 vs 81.41 ± 2.43, P < 0.001). TA containing HGD (n = 26) had lower LINE-1 methylation levels than those with LGD (n = 119) (59.86 ± 7.93 vs 69.11 ± 8.62, P < 0.001).DISCUSSION:HGD and increasing size of TA/tubulovillous adenoma were associated with lower LINE-1 methylation. This supports a hypothesis that LINE-1 hypomethylation in TAs indicates advancement along the CRC tumorigenesis pathway. Lower LINE-1 methylation and greater variance of global DNA methylation was seen in TA compared with SSL. LINE-1 methylation in adenomas correlates with polyp size and degree of dysplasia and deserves further study as a predictor of metachronous colorectal cancer risk.

Tumor Long Interspersed Nucleotide Element-1 (LINE-1) Hypomethylation in Relation to Age of Colorectal Cancer Diagnosis and Prognosis.

Simple SummaryThe rising incidence of early-onset cancers diagnosed before 50 years in many body sites (including the colorectum) is a growing concern. Despite the many studies of early-onset colorectal cancer, the characteristics of colorectal cancer diagnosed at age 50 or slightly after (close to age 50) have not been adequately examined. Although epigenetic alterations are considered to play a role in early-onset colorectal cancer, the association of LINE-1 hypomethylation (that reflects global DNA hypomethylation) with the age of colorectal cancer diagnosis has not been thoroughly clarified. Using a database of 1356 colorectal cancers, we found that tumor LINE-1 hypomethylation was increasingly more common with decreasing age of colorectal cancer diagnosis and was associated with higher colorectal cancer-specific mortality. Our findings support the “age continuum” model that has substantial implications in research on cancers in not only the colorectum but also many other body sites.Evidence indicates the pathogenic role of epigenetic alterations in early-onset colorectal cancers diagnosed before age 50. However, features of colorectal cancers diagnosed at age 50–54 (hereafter referred to as “intermediate-onset”) remain less known. We hypothesized that tumor long interspersed nucleotide element-1 (LINE-1) hypomethylation might be increasingly more common with decreasing age of colorectal cancer diagnosis. In 1356 colorectal cancers, including 28 early-onset and 66 intermediate-onset cases, the tumor LINE-1 methylation level measured by bisulfite-PCR-pyrosequencing (scaled 0 to 100) showed a mean of 63.6 (standard deviation (SD) 10.1). The mean tumor LINE-1 methylation level decreased with decreasing age (mean 64.7 (SD 10.4) in age ≥70, 62.8 (SD 9.4) in age 55–69, 61.0 (SD 10.2) in age 50–54, and 58.9 (SD 12.0) in age <50; p < 0.0001). In linear regression analysis, the multivariable-adjusted β coefficient (95% confidence interval (CI)) (vs. age ≥70) was −1.38 (−2.47 to −0.30) for age 55–69, −2.82 (−5.29 to −0.34) for age 50–54, and −4.54 (−8.24 to −0.85) for age <50 (Ptrend = 0.0003). Multivariable-adjusted hazard ratios (95% CI) for LINE-1 methylation levels of ≤45, 45–55, and 55–65 (vs. >65) were 2.33 (1.49–3.64), 1.39 (1.05–1.85), and 1.29 (1.02–1.63), respectively (Ptrend = 0.0005). In conclusion, tumor LINE-1 hypomethylation is increasingly more common with decreasing age of colorectal cancer diagnosis, suggesting a role of global DNA hypomethylation in colorectal cancer arising in younger adults.

Impact of Tumor LINE-1 Methylation Level and Neoadjuvant Treatment and Its Association with Colorectal Cancer Survival.

Recent studies suggest that long-interspersed nucleotide element-1 (LINE-1) hypomethylation is commonly found in colorectal cancer (CRC), and is associated with worse prognosis. However, the utility of LINE-1 methylation on the prognosis of CRC is still controversial, and may be due to the fact that some clinical and pathological features may affect LINE-1 methylation. Thus, the aim of this study was to assess the prognostic value of tumor LINE-1 methylation in CRC, through their association with the CRC clinical and pathological characteristics. Survival of sixty-seven CRC patients was evaluated according to the median of tumor LINE-1 methylation, as well as pathological and oncological variables. We also studied the association between LINE-1 methylation and pathological features, and finally, we assessed the overall and disease-free survival of LINE1 methylation, stratified by neoadjuvant treatment and further checked by multivariate Cox regression to assess the statistical interactions. LINE-1 was hypomethylated in the CRC tumor with respect to the tumor adjacent-free area (p < 0.05), without association with any other clinical and oncological features, nor with overall and disease-free survival rates for CRC. Relevantly, in neoadjuvant treatment, LINE-1 methylation was associated with survival rates. Thus, disease-free and overall survival rates of treated CRC patients were worse in the hypomethylated LINE-1 tumors than those with normal LINE-1 methylation (p = 0.004 and 0.0049, respectively). Indeed, LINE-1 was hypermethylated more in the treated patients than in the non-treated patients (p < 0.05). The present study showed that tumor LINE-1 hypomethylation was associated with worse survival rates in only treated patients. Our data suggest an interactive effect of neoadjuvant treatment and tumor LINE-1 methylation, which could be a specific-tissue biomarker to predict survival of the treated patients, and help to personalize treatment in CRC.

Sperm Ribosomal DNA Promoter Methylation Levels Are Correlated With Paternal Aging and May Relate With in vitro Fertilization Outcomes.

The impact of aging on reproductive outcomes has received considerable critical attention; however, there is much less information available on the effects of paternal age compared to the effects of maternal age. In this study, methylation levels of sperm rDNA promoter regions and Long Interspersed Nucleotide Element 1 (LINE-1) were measured using pyrosequencing and fertilization, day 3 good-quality embryo, pregnancies, and implantation results were assessed. We observed significantly increasing levels of DNA methylation in the sperm rDNA promoter regions with age based on stratifying the samples by age alone (P = 0.0001) and performing linear regression analysis (P < 0.0001). Meanwhile, no statistically significant correlations were observed between global LINE-1 methylation with age. No statistically significant correlations were observed between sperm rDNA promoter methylation levels and either the day 3 good-quality embryo rate or clinical pregnancy rate. In contrast, the correlation between sperm rDNA promoter methylation levels and fertilization (2 pronuclei) rate was nearly significant (P = 0.0707), especially the methylation levels of some individual CpG units (CpG_10, P = 0.0176; CpG_11, P = 0.0438; CpG_14, P = 0.0232) and rDNA promoter methylation levels measured using primerS2 (P = 0.0513). No significant correlation was found between sperm rDNA promoter methylation levels and fertilization rates (2 pronuclei, 1 pronuclei, and 1 polypronuclei). Our results demonstrate that sperm are susceptible to age-associated alterations in methylation levels of rDNA promoter regions, suggesting that sperm rDNA promoter methylation levels can be applied to DNA methylation-based age prediction, and that the aberrant methylation of rDNA promoters may be partially responsible for enhanced disease susceptibility of offspring sired by older fathers. Methylation levels of sperm rDNA promoter regions may correlate with polypronuclei rates of IVF programs.

Long interspersed nucleotide element‐1 hypomethylation in canine malignant mucosal melanoma

Canine malignant melanoma is a common cancer with a high mortality rate and is a clinically important disease. DNA methylation has been considered to be a potential tumorigenic mechanism through aberrant DNA methylation at promoter region which represses gene transcription. Global hypomethylation could also facilitate chromosome instability. There are few reports regarding DNA methylation in canine malignant melanoma; therefore, the purpose of this study was to examine DNA methylation status of long interspersed nucleotide element-1 (LINE-1) to be a surrogate marker of genome-wide methylation changes in this disease. We measured levels of DNA methylation of two adjacent cytosine-guanine sites on CpG island (CGI) at the putative promoter of canine LINE-1 sequence by bisulphite-pyrosequencing in 41 canine melanoma patient samples as well as six cell lines compared with normal mucosae. The survival rates were obtained from owners or medical records. We found DNA methylation levels of LINE-1 in normal mucosae were methylated. Interestingly, both melanoma cell lines and clinical melanoma samples showed remarkable hypomethylation. In addition, patients with lower LINE-1 methylation showed worse prognosis than those with higher LINE-1 methylation, though the difference did not reach statistical significance (P = .09). Here, we demonstrate that hypomethylation of LINE-1 is an epigenetically aberrant feature in canine melanoma with possible prognostic value.

Hypomethylation of LINE-1 repeat elements and global loss of DNA hydroxymethylation in vapers and smokers

ABSTRACT The outbreak of vaping-related severe lung injuries and deaths and the epidemic of teen vaping in the U.S. underscore the urgent need for determining the biological consequences of electronic cigarette (e-cig) use. We have investigated the association between vaping and epigenetic changes by quantifying DNA methylation levels in Long Interspersed Nucleotide Element 1 (LINE-1) and global DNA hydroxymethylation (5-hmC) levels and measuring the expression level of enzymes catalysing the respective processes in peripheral blood of exclusive vapers, smokers, and controls, matched for age, gender, and race (n = 45). Both vapers and smokers showed significant loss of methylation in LINE-1 repeat elements in comparison to controls (P = 0.00854 and P = 0.03078, respectively). Similarly, vapers and smokers had significant reductions in 5-hmC levels relative to controls (P = 0.04884 and P = 0.0035, respectively). Neither the LINE-1 methylation levels nor the global 5-hmC levels were different between vapers and smokers. There was a direct correlation between methylation levels in the LINE-1 elements and global 5-hmC levels in the study subjects (r = 0.31696, P = 0.03389). Inverse and statistically significant correlations were found between both the LINE-1 methylation levels and the global 5-hmC levels and various vaping/smoking metrics in the study subjects. There were modest but not statistically significant changes in transcription of DNA methyltransferases and ten-eleven translocation enzymes in both vapers and smokers relative to controls. Our findings support follow-up genome-wide investigations into the epigenetic effects of vaping, which may further clarify the health consequences of e-cig use. Abbreviations 5-mC: 5-methylcytosine; 5-hmC: 5-hydroxymethylcytosine; 8-OHdG: 8-hydroxy-2ʹ-deoxyguanosine; ACTIN: actin beta; ANOVA: Analysis of Variance; BER: base excision repair; BMI: body mass index; CO: carbon monoxide; COHb: carboxyhaemoglobin; COBRA: combined bisulphite restriction analysis; COPD: chronic obstructive pulmonary disease; DNMT1: DNA methyltransferase 1; DNMT3A: DNA methyltransferase 3A; DNMT3B: DNA methyltransferase 3B; e-cigs: electronic cigarettes; ELISA: enzyme-linked immunosorbent assay; ENDS: electronic nicotine delivery systems; FDA: Food and Drug Administration; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; HPLC: high-performance liquid chromatography; LINE-1: Long Interspersed Nucleotide Element 1; PBS: phosphate-buffered saline; RFU: relative fluorescence units; RT-qPCR: quantitative reverse-transcription polymerase chain reaction; ROS: reactive oxygen species; SAM, S-adenosylmethionine; SE: standard error; TET1: ten-eleven translocation 1; TET2: ten-eleven translocation 2; TET3: ten-eleven translocation 3.

Association of autophagy status with amount of Fusobacterium nucleatum in colorectal cancer.

Fusobacterium nucleatum (F. nucleatum), which has been associated with colorectal carcinogenesis, can impair anti-tumour immunity, and actively invade colon epithelial cells. Considering the critical role of autophagy in host defence against microorganisms, we hypothesised that autophagic activity of tumour cells might influence the amount of F. nucleatum in colorectal cancer tissue. Using 724 rectal and colon cancer cases within the Nurses' Health Study and the Health Professionals Follow-up Study, we evaluated autophagic activity of tumour cells by immunohistochemical analyses of BECN1 (beclin 1), MAP1LC3 (LC3), and SQSTM1 (p62) expression. We measured the amount of F. nucleatum DNA in tumour tissue by quantitative polymerase chain reaction (PCR). We conducted multivariable ordinal logistic regression analyses to examine the association of tumour BECN1, MAP1LC3, and SQSTM1 expression with the amount of F. nucleatum, adjusting for potential confounders, including microsatellite instability status; CpG island methylator phenotype; long-interspersed nucleotide element-1 methylation; and KRAS, BRAF, and PIK3CA mutations. Compared with BECN1-low cases, BECN1-intermediate and BECN1-high cases were associated with lower amounts of F. nucleatum with odds ratios (for a unit increase in three ordinal categories of the amount of F. nucleatum) of 0.54 (95% confidence interval, 0.29-0.99) and 0.31 (95% confidence interval, 0.16-0.60), respectively (Ptrend < 0.001 across ordinal BECN1 categories). Tumour MAP1LC3 and SQSTM1 levels were not significantly associated with the amount of F. nucleatum (Ptrend > 0.06). Tumour BECN1, MAP1LC3, and SQSTM1 levels were not significantly associated with patient survival (Ptrend > 0.10). In conclusion, tumour BECN1 expression is inversely associated with the amount of F. nucleatum in colorectal cancer tissue, suggesting a possible role of autophagy in the elimination of invasive microorganisms. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Low Maternal Dietary Folate Alters Retrotranspose by Methylation Regulation in Intrauterine Growth Retardation (IUGR) Fetuses in a Mouse Model.

BackgroundMaternal folate deficiency-mediated metabolic disruption is considered to be associated with the risk of intrauterine growth retardation (IUGR), but the exact mechanism remains unclear. The retrotransposon long interspersed nucleotide element-1 (LINE-1), which can induce birth defects via RNA intermediates, plays crucial roles during embryonic development. We investigated potential relationships between maternal folate and DNA methylation, and possible roles of LINE-1 in IUGR.Material/MethodsThe IUGR model was established by feeding female mice 1 of 3 diets – control diet (CD), folate-deficient diet for 2 weeks (FD2w), and folate-deficient diet for 4 weeks (FD4w) – prior to mating. Maternal serum folate, 5-methyltetrahydrofolate (5-MeTHF), S-adenosylmethionine (SAM), and S-adenosylhomocysteine (SAH) concentrations and global DNA methylation were assessed by LC/MS/MS method. LINE-1 methylation levels in fetuses were examined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. LINE-1 expression levels were validated by real-time PCR.ResultsMaternal folate deficiency caused plasma folate and 5-MeTHF levels to decrease and SAH level to increase in the FD4w group. Compared with the CD group, methylation levels of genomic DNA and LINE-1 decreased significantly in placenta and fetal tissues from the FD4w group. Expression of LINE-1 open reading frame 1 (ORF1) protein was elevated in fetal liver tissues. Furthermore, a strong correlation was found between methylation and disrupted one-carbon metabolism, implying that dietary folate plays important roles during embryogenesis.ConclusionsMaternal dietary folate deficiency impaired one-carbon metabolism, leading to global DNA and LINE-1 hypomethylation, and then increased retrotransposition in fetuses, which can lead to IUGR.

Tumor Long-interspersed Nucleotide Element-1 Methylation Level and Immune Response to Esophageal Cancer.

To examine the relationship between tumor long-interspersed nucleotide element-1 (LINE-1) methylation level and immune response to esophageal cancer. Evidence points to a correlation between the abundance of immune cells and a favorable prognosis in esophageal cancer patients. Accumulating evidence indicates a critical role of tumor LINE-1 hypomethylation in the aggressive behavior of esophageal cancer, which in turn leads to an unfavorable prognosis. Utilizing a nonbiased database of 292 resected esophageal cancers, we measured tumor LINE-1 methylation level by pyrosequencing assay, and examined the relationship between LINE-1 methylation and the density of T cells (CD8 and FOXP3) and the lymphocytic reaction patterns (follicle lymphocytic reaction, peritumoral lymphocytic reaction, stromal lymphocytic reaction, and tumor-infiltrating lymphocytes) in esophageal carcinoma tissue. LINE-1 hypomethylation was associated with male gender and advanced stage cancer (P = 0.03 and P = 0.048, respectively). Tumor LINE-1 methylation level was significantly positively associated with peritumoral lymphocytic reaction (P = 0.004), but not with others. Compared with LINE-1 hypermethylation group, LINE-1 hypomethylation group showed much lower level of peritumoral lymphocytic reaction (univariable odds ratio 0.32, 95% confidence interval 0.16-0.64, P = 0.002). In multivariable model to control for potential confounders including disease stage, the similar finding was observed (multivariable odds ratio 0.31, 95% confidence interval 0.14-0.66, P = 0.004). Tumor LINE-1 hypomethylation level is associated with a diminished peritumoral lymphocytic reaction, providing impetus for further investigations on potential interactive roles of tumor LINE-1 hypomethylation and host immunity in esophageal cancer development.

Soy Isoflavone Supplementation Increases Long Interspersed Nucleotide Element-1 (LINE-1) Methylation in Head and Neck Squamous Cell Carcinoma

Aim: Soy isoflavones have been suggested as epigenetic modulating agents with effects that could be important in carcinogenesis. Hypomethylation of LINE-1 has been associated with head and neck squamous cell carcinoma (HNSCC) development from oral premalignant lesions and with poor prognosis. To determine if neoadjuvant soy isoflavone supplementation could modulate LINE-1 methylation in HNSCC, we undertook a clinical trial.Methods: Thirty-nine patients received 2–3 weeks of soy isoflavone supplements (300 mg/day) orally prior to surgery. Methylation of LINE-1, and 6 other genes was measured by pyrosequencing in biopsy, resection, and whole blood (WB) specimens. Changes in methylation were tested using paired t tests and ANOVA. Median follow up was 45 months.Results: LINE-1 methylation increased significantly after soy isoflavone (P < 0.005). Amount of change correlated positively with days of isoflavone taken (P = 0.04). Similar changes were not seen in corresponding WB samples. No significant changes in tumor or blood methylation levels were seen in the other candidate genes.Conclusion: This is the first demonstration of in vivo increases in tissue-specific global methylation associated with soy isoflavone intake in patients with HNSCC. Prior associations of LINE-1 hypomethylation with genetic instability, carcinogenesis, and prognosis suggest that soy isoflavones maybe potential chemopreventive agents in HNSCC.

Genome-wide DNA hypermethylation and homocysteine increase a risk for myopia.

To test for the association between genome-wide methylation and myopia in human and mice. Long interspersed nucleotide element 1 (LINE-1) methylation levels were used to surrogate genome-wide methylation level. We first tested for the association between high myopia (<-6 D) and LINE-1 methylation in leukocytes in 220 cases and 220 control subjects. Secondly, we validated the results of LINE-1 methylation in eyes from the form deprivation myopia (FDM) mice. Furthermore, we calculated the correlation of LINE-1 methylation levels between leukocyte DNA and ocular DNA in the mice. We also tested whether dopamine can alter LINE-1 methylation levels. The LINE-1 methylation level was significantly higher in the myopic human subjects than controls. The upper and middle tertiles of the methylation levels increased an approximately 2-fold (P≤0.002) risk for myopia than the lower tertile. Similarly, FDM mice had high LINE-1 methylation levels in the leukocyte, retina and sclera, and furthermore the methylation levels detected from these three tissues were significantly correlated. Immunohistochemical staining revealed higher levels of homocysteine and methionine in the rodent myopic eyes than normal eyes. Dopamine treatment to the cells reduced both LINE-1 methylation and DNA methyltransferase levels. LINE-1 hypermethylation may be associated with high myopia in human and mice. Homocysteine and methionine are accumulated in myopic eyes, which may provide excess methyl group for genome-wide methylation.

Association of long interspersed nucleotide element-1 and interferon regulatory factor 6 methylation changes with nonsyndromic cleft lip with or without cleft palate.

To examine the possible associations between methylation changes in the promoter regions of long interspersed nucleotide element-1 (LINE-1) and interferon regulatory factor 6 gene (IRF6) and nonsyndromic cleft lip with or without cleft palate (NSCL/P). A case-control investigation was performed to compare 37 infants affected by NSCL/Ps with 60 babies without cleft malformations. Their genomic DNA samples were obtained, and the LINE-1 and IRF6 methylation levels were measured by using Sequenom MassArray. Unconditional logistic regression was adopted to estimate the odds ratio. Infants with NSCL/Ps had a higher methylation level at LINE-1 and IRF6 promoter regions than controls. High levels of LINE-1 (≥64.07%) and IRF6 (≥6.46%) methylation were associated with an increased risk of NSCL/P (LINE-1, OR=2.63, 95% CI: 1.07-6.57; IRF6, OR=4.73, 95% CI: 2.10-13.07), and the associations remained to be significant after adjusting for potential confounders. Similar associations were also found for cleft lip only, cleft lip, and palate. Our study suggested that aberrant methylation of LINE-1 and IRF6 might contribute to the development of NSCL/Ps. Further studies are needed to replicate the findings.

The Amount of Bifidobacterium Genus in Colorectal Carcinoma Tissue in Relation to Tumor Characteristics and Clinical Outcome

Evidence indicates a complex link between microbiota, tumor characteristics, and host immunity in the tumor microenvironment. In experimental studies, bifidobacteria appear to modulate intestinal epithelial cell differentiation. Accumulating evidence suggests that bifidobacteria may enhance the antitumor immunity and efficacy of immunotherapy. We hypothesized that the amount of bifidobacteria in colorectal carcinoma tissue might be associated with tumor differentiation and higher immune response to colorectal cancer. Using a molecular pathologic epidemiology database of 1313 rectal and colon cancers, we measured the amount of Bifidobacterium DNA in carcinoma tissue by a quantitative PCR assay. The multivariable regression model was used to adjust for potential confounders, including microsatellite instability status, CpG island methylator phenotype, long-interspersed nucleotide element-1 methylation, and KRAS, BRAF, and PIK3CA mutations. Intratumor bifidobacteria were detected in 393 cases (30%). The amount of bifidobacteria was associated with the extent of signet ring cells (P=0.002). Compared with Bifidobacterium-negative cases, multivariable odd ratios for the extent of signet ring cells were 1.29 (95% CI, 0.74-2.24) for Bifidobacterium-low cases and 1.87 (95% CI, 1.16-3.02) for Bifidobacterium-high cases (Ptrend=0.01). The association between intratumor bifidobacteria and signet ring cells suggests a possible role of bifidobacteria in determining distinct tumor characteristics or as an indicator of dysfunctional mucosal barrier in colorectal cancer.

Prognostic value of LINE-1 methylation level in 321 patients with primary liver cancer including hepatocellular carcinoma and intrahepatic cholangiocarcinoma.

BackgroundThe methylation level of long interspersed nucleotide element-1 (LINE-1) is a good surrogate marker of the global DNA methylation level. The relationship between LINE-1 methylation level and prognosis in primary liver cancer (PLC) patients remains unclear.ResultsLINE-1 methylation levels were significantly lower in HCC and cHCC-CC tissues, but not in ICC tissues, than those in noncancerous liver parenchyma (HCC: p < 0.0001; cHCC-CC: p < 0.001; and ICC: p = 0.053). HCC cases with hypomethylated LINE-1 had significantly shorter relapse-free survival (RFS) (log-rank, p = 0.008); however, this was not observed for the cHCC-CC or ICC cases. Multivariate Cox regression analysis revealed a significantly higher HCC recurrence rate in the group with hypomethylated LINE-1 (hazard ratio, 1.62; 95% confidence interval, 1.06–2.58; p = 0.025).ConclusionsThe genome-wide DNA hypomethylation status estimated via LINE-1 methylation levels might be indicative of poor RFS in patients with HCC but not ICC or cHCC-CC.MethodsWe evaluated the level of LINE-1 methylation in 321 cases of curatively resected PLC {231 hepatocellular carcinoma (HCC), 19 combined hepatocellular and cholangiocarcinoma (cHCC-CC) and 71 intrahepatic cholangiocarcinoma (ICC)} via pyrosequencing of formalin-fixed paraffin-embedded (FFPE) tissues and examined its prognostic value.

LINE-1 hypomethylation in colon cancer.

588 Background: Global hypomethylation of the genome is one of the causes of chromosomal instability and aneuploidy and is associated with an increased risk of colorectal cancer (CRC). The long interspersed nucleotide element-1 (LINE-1) is the main part of the mobile repeated elements of the genome, and the level of its methylation is concordant with genome-wide methylation. The aim of the study was to assess the methylation of LINE-1, as well as methylation of the promoter of six cancer-associated genes adjusting for the clinical and pathological characteristics in colorectal cancer (CRC). Methods: Using pyrosequencing (PyroMark Q24 analysis system, Qiagen, Germany) DNA methylation levels of LINE-1 and 6 genes ( APC, CDH13, MLH1, MGMT, P16 and RASSF1A) were measured in tumoral and normal tissues of 50 patients with CRC. Results: The level of LINE-1 methylation was 66.8 ± 0.89% in tumors, 72.5 ± 0.45% in normal tissues ( p = 0.0013). The differences in LINE-1 methylation level were not associated with age, differentiation grade, tumor localization, and the presence of activating mutation within the KRAS gene. At the same time, the MGMT methylation was positively associated with age ( p = 0.04), and the hypermethylation of the CDH13 gene was more common in tumors of the proximal colon ( p = 0.004). Methylation of the CDH13 promoter negatively correlated with methylation level of LINE1 ( r = -0.427, p &lt; 0.05). Hypomethylation of LINE-1 was associated with progression of TNM-stages of the tumors ( r = +0.606, p &lt; 0.001). The cancer-associated genes were hypermethylated in 70% of CRC cases when compared with normal epithelium, and hypomethylation of LINE-1 was observed in 90% of CRC cases. Conclusions: The present study demonstrated the association of LINE-1 methylation with the TNM tumor stage, which may be used as a biomarker for early diagnosis and prognosis of sporadic CRC, together with assessment of hypermethylation of tumor suppressors.