Hypermethylation Levels Research Articles

Silencing DNMT1 Attenuates the Effect of WIF-1 Gene Promoter Methylation on the Biological Behavior of Chronic Myeloid Leukemia K562 Cells

To investigate the effect of silencing DNA methyltransferase 1(DNMT1) to the methylation of the promoter of the tumor suppressor gene wnt-1 (WIF-1) in human chronic myeloid leukemia (CML) cells. DNMT1 siRNAi plasmid was constructed and DNMT1 siRNAi was transfected into CML K562 cells. RT-PCR and Western blot were used to detect the expression of DNMT1 gene and related protein, and methylation PCR was used to detect WIF-1 gene promoter methylation level. The trypan blue exclusion and MTT assay were used to detect the cell proliferation, flow cytometry were used to detect the cell apoptosis rate, colony formation assay was used to detect cell colony formation ability. Expression of Wnt/β- catenin and its downstream signaling pathway proteins were detected by Western blot after DNMT1 gene was silenced. The expression level of DNMT1 mRNA and its related protein in the experimental group were significantly lower than those in the control group and negative control group (P<0.05). After 72 hours of successful transfection, the WIF-1 gene in the control group and negative control group were completely methylated, while in the experimental group, the methylation level significantly decreased. The results of MSP showed that the PCR product amplified by the unmethylated WIF-1 primer in the experimental group increased significantly,while by the methylated WIF-1 primer decreased significantly. After 48 h of transfection, the OD value, viable cell number and colony formation of the cells in experimental group were significantly lower than those in the negative control group and the control group (P<0.05). The apoptosis rate of the cells in experimental group was significantly higher than those in the negative control group and control group (P<0.05). The expression levels of β- actin, myc, cyclin D1 and TCF-1 in K562 cells in the experimental group were significantly lower than those in the negative control group and control group (P<0.05). Silencing DNMT1 gene can inhibit the proliferation and promote the apoptosis of K562 cells. The mechanism may be related to reverse the hypermethylation level of the WIF-1 gene promoter, thereby inhibit the activity of the Wnt/β- catenin signaling pathway.

Correlation Analysis of DNA Methylation Level and Clinical Characteristics in JMML Patients

Objective: As a rare, aggressive pediatric myeloproliferative disease, juvenile myelomonocytic leukemia (JMML) encompassed both biological features of myelodysplastic syndrome and myeloproliferative neoplasm. Studies have shown that the methylation level in JMML patients is closely related to prognosis, and patients with high methylation level have poor prognosis. This study aimed to find clinical indicators that were associated with different methylation levels and prognosis.Methods: The clinical information of 24 JMML patients with DNA samples admitted to our center from December 2013 to May 2020 was retrospectively analyzed, and the DNA methylation level of their whole genome was detected.Results: The median age of onset was 14.5 months (0.1-153 months) among the 24 cases, including 17 males and 7 females. At diagnosis, the median WBC count was 27.1×10 9/L (6.2-98.1×10 9/L), and the median platelet count was 38×10 9/L (10-277×10 9/L). Chromosome karyotype abnormalities were found in 12.5% (3/24) of patients. Next-generation sequencing results showed that 79.2% (19/24) patients had at least one Ras pathway-related classical gene mutation, and 41.7% (10/24) patients had two or more somatic mutations. Genomic DNA methylation levels were divided into three groups: 10 cases in the hypomethylation group, 4 cases in the moderate methylation group, and 10 cases in the hypermethylation group. There were significant differences in age, platelets, PTPN11 gene mutation and the number of somatic mutations ≥2 in different methylation groups (P<0.05). The age of hypomethylated group was significantly lower than that of hypermethylated group (P<0.05), and the platelets of hypomethylated group was significantly higher than that of hypermethylated group (P<0.05). Patients ≤12m and platelets＞32×10 9/L had lower DNA methylation level (P<0.0001). The number of patients with PTPN11 gene mutation in the hypomethylated group was significantly lower than that in the hypermethylated group (P<0.05), and the number of patients with ≥2 mutations in the low and medium methylated groups was significantly lower than that in the hypermethylated group (P<0.05). Correlation analysis showed that hypermethylation level was significantly correlated with PTPN11 gene mutation and ≥2 somatic mutations (P<0.001).Conclusions: JMML patients with high methylation level in the DNA genome at diagnosis were older and with lower platelet levels, and hypermethylation were significantly correlated with high-risk prognostic factors such as PTPN11 gene mutation and ≥2 somatic mutations. [Display omitted] DisclosuresNo relevant conflicts of interest to declare. OffLabel Disclosure:Decitabine for treatment of children with JMML

DHA-rich n-3 PUFAs intake from the early- and mid-pregnancy decreases the weight gain by affecting the DNA methylation status among Chinese Han infants

Background: Maternal exogenous docosahexaenoic acid (DHA)-rich n-3 polyunsaturated fatty acids (PUFAs) intake during the pregnancy, especially DHA, has inconsistent effects on reducing the fat storage of the infants in different clinical studies.&#x0D; Objectives: We sought to determine the effects of maternal exogenous DHA-rich n-3 PUFAs capsule intake from the different pregnancy periods on the weight gain of their infants through modifying the DNA methylation status of obesity-associated genes in the umbilical cord blood.&#x0D; Design: A prospective 3-year follow-up study after the pregnancy was enrolled in this cohort from May to October 2016. They were divided into different groups according to the initial time of exogenous DHA capsule intake through the questionnaires (S1 – early trimester, S2 – mid-trimester, S3 – late trimester, and control – without). The concentrations and compositions of DHA were determined by gas chromatography. We applied quantitative DNA methylation states of the obesity-associated genes in the umbilical cord blood. The growth outcomes and relevant Z-scores were recorded at birth and 1 and 2 years. The correlations between DNA methylation status of the obesity-associated genes with the consents of DNA and body mass index (BMI) values were investigated as the measures.&#x0D; Results: In total, 205 pregnant women and their infants were eligible for this follow-up study. The concentrations and compositions of DHA in the colostrum and umbilical cord blood were higher in the S1 and S2 groups than those in the control and S3 groups as well as the decreased weight, BMI, weight for age Z-score (WAZ) and BMI for age Z-score (BMI Z) at birth and 1 and/or 2 years, and higher levels of global DNA methylation and many CpG sites in the obesity-associated genes, such as CpG2, CpG9, CpG11, and CpG16 of PPAR-γ; CpG2,3, CpG4-6, CpG8, CpG9,10, CpG11, CpG15,16, and average of CCAAT/enhancer binding protein α (C/EBP-α); CpG1 and average of adiponectin; CpG1, CpG2, CpG3, CpG5, CpG6, CpG7, and average of insulin-like growth factor 2 (IGF-2); CpG6, CpG7, CpG9, CpG16, CpG23, and CpG24 of leptin, which were more obvious in the S1 group when compared with those in the S2 group. These above hypermethylation levels of CpG sites were negatively correlated with the BMI and positively related with the changes of DHA in the colostrum and umbilical cord blood.&#x0D; Conclusions: Maternal exogenous DHA-rich n-3 PUFAs intake from early- and mid- trimesters of the pregnancy may avoid the development of obesity among Chinese Han infants until 2 years by modulating DNA methylation states of obesity-associated genes, which could provide attractive targets for prenatal prevention of the metabolic disorders.

GCM2 Silencing in Parathyroid Adenoma Is Associated With Promoter Hypermethylation and Gain of Methylation on Histone 3.

Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of the parathyroid glands. We sought to identify whether the epigenetic alterations in GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. Messenger RNA (mRNA) and protein expression of GCM2 were analyzed by reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of the GCM2 promoter were measured by bisulfite sequencing and chromatin immunoprecipitation-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in parathyroid (PTH)-C1 cells by treating with 5-aza-2'-deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P < .001) and higher H3K9me3 levels in the GCM2 promoter (P < .04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a nonsignificant change in GCM2 transcription. These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be a promising avenue of research for parathyroid adenoma therapeutics.

Aberrant DNA Methylation in Bladder Cancer among Saudi Arabia Population

AbstractTumor biomarkers developed based on the aberrant deoxyribonucleic acid (DNA) methylation patterns in bladder cancer (BC) hold great promise due to their stability, specificity, and known associations with the disease. No study has investigated DNA methylation patterns in BC patients from Saudi population. We analyzed DNA methylation levels of 48 tumor suppressor genes loci in 24 bladder tissues (19 BC and 5 control samples) using Human Tumour Suppressor Genes EpiTect Methyl II Complete PCR Array (Qiagen, Hilden, Germany). We identified significant difference in DNA hypermethylation levels at E2F1, ERBB2, HIC1, OPCML, SFN, SFRP1, SFRP2, SPARC, and TERT gene loci between controls and cancerous samples. SCGB3A1 was differentially methylated in nonmuscle invasive versus muscle invasive BC samples. Results suggest that these aberrant DNA methylation patterns in BC are disease and population specific and can be developed as distinct DNA methylation-based biomarkers for BC detection.

Gestational high-fat diet impaired demethylation of Pparα and induced obesity of offspring.

Gestational and postpartum high‐fat diets (HFDs) have been implicated as causes of obesity in offspring in later life. The present study aimed to investigate the effects of gestational and/or postpartum HFD on obesity in offspring. We established a mouse model of HFD exposure that included gestation, lactation and post‐weaning periods. We found that gestation was the most sensitive period, as the administration of a HFD impaired lipid metabolism, especially fatty acid oxidation in both foetal and adult mice, and caused obesity in offspring. Mechanistically, the DNA hypermethylation level of the nuclear receptor, peroxisome proliferator‐activated receptor‐α (Pparα), and the decreased mRNA levels of ten‐eleven translocation 1 (Tet1) and/or ten‐eleven translocation 2 (Tet2) were detected in the livers of foetal and adult offspring from mothers given a HFD during gestation, which was also associated with low Pparα expression in hepatic cells. We speculated that the hypermethylation of Pparα resulted from the decreased Tet1/2 expression in mothers given a HFD during gestation, thereby causing lipid metabolism disorders and obesity. In conclusion, this study demonstrates that a HFD during gestation exerts long‐term effects on the health of offspring via the DNA demethylation of Pparα, thereby highlighting the importance of the gestational period in regulating epigenetic mechanisms involved in metabolism.

Metformin alleviates intervertebral disc degeneration by upregulating MMP-1 expression via the KDM6A/SOX9/miR-202-3p/MMP-1 signaling pathway

IntroductionMiR-202-3p is involved in the pathogenesis of intervertebral disc degeneration (IDD) via regulating MMP-1 expression in neuronal precursor cells (NPCs). As an activator of miR-202-3p and KDM6A expression, metformin (MET) may regulate the expression of SOX9 via regulating the methylation status of SOX9 promoter. However, the role of MET in the treatment of IDD remains to be explored.Material and methodsQuantitative real-time PCR was performed to analyze the expression of KDM66, SOX9, MMP-1 and miR-202-3p in NPCs and IDD rabbits. Western blot was carried out to evaluate the expression of KDM6A, SOX9 and MMP-1 protein under different conditions. Bisulfite sequencing PCR was used to analyze the DNA methylation of SOX9 promoter. Luciferase assay was carried out to explore the inhibitory effect of miR-202-3p upon MMP-1.ResultsThe expression of KDM6A, SOX9 and MMP-1 was abnormal in IDD cells and rabbits, while the MET treatment restored the normal expression of KDM6A, SOX9 and MMP-1 and miR-202-3p. Mechanistically, MET treatment reduced the level of hypermethylation of SOX9 promoter, thus restoring the expression of SOX9 in IDD cells and rabbits. The elevation in SOX9 expression promoted the expression of miR-202-3p, therefore inhibiting the expression of MMP-1, a downstream target of miR-202-3p.ConclusionsIn this study, we set up cellular and animal models of IDD and treated them with MET to probe the effect of MET on IDD and the signaling pathway of KDM6A/SOX9/miR-202-3p/MMP-1. Our work provided deep insight into the molecular mechanism underlying the therapeutic role of MET in the treatment of IDD.

Methylation Profiling of Specific Genes in Ependymomas.

Ependymomas are neuroepithelial tumors of the central nervous system with heterogeneous biology and clinical course. The aim of the present study is to investigate the relationship between the methylation status and clinicopathological parameters in ependymomas. DNA methylation status of CDKN2A, RASSF1A, KLF4 and ZIC2 genes were quantitatively analyzed with pyrosequencing in 44 ependymoma tumor tissues. The relationship of methylation profiles with tumor subtype, histological grade and patient age was statistically analyzed. DNA methylation analyses for CDKN2A revealed no difference in methylation levels. Of the 31 included samples for optimal ZIC2 methylation analysis, 10 were hypermethylated (32.3%) and this change was significantly found in the adult spinal ependymomas (p=0.01). KLF4 hypermethylation was observed in 6 of the overall included 35 samples (17.1%); however, there was no statistically significant relation of the methylation status with tumor subtype, histological grade or age group. RASSF1A hypermethylation was observed in overall 40 included samples with varying methylation levels. Higher levels of hypermethylation were significantly related to the grade 3 histology (p=0.01) and spinal ependymomas (p=0.006). The pediatric cases with grade 3 ependymomas and ependymomas of adulthood showed significantly increased RASSF1A hypermethylation levels (p < 0.001 and p=0.001, respectively). DNA methylation changes are likely to have biological importance in ependymomas. Both ZIC2 and RASSF1A methylation status may be useful parameters in the subclassification of these tumors.

Formation of Cisplatin Adducts with the Epigenetically Relevant Nucleobase 5‐Methylcytosine

With about 4 % abundance in the genome 5‐methylcytosine (5mC) is one of the most important epigenetic modifications. Change in DNA hypermethylation levels has even been linked to resistances to cisplatin treatment of cancer cells. This work aimed at the synthesis and full characterization of 5mC‐cisplatin adducts as well as for the examination of possible side‐reactions and transformations. We report the first X‐ray crystal structure of a cis‐[PtCl(5mC)(NH3)2]Cl complex and the formation of [PtCl(5mC)(NH3)2]+ and [PtCl(5mC)2(NH3)]+ with HR‐ESI mass spectrometry. Further, we explore complex formation and dynamics using 1H, 195Pt, and DOSY NMR spectroscopy. UV/Vis absorption and EPR spectroscopy also confirmed the formation of trace amounts of paramagnetic Pt‐blue species. In the process, a hemiprotonated 5mC dimer, 5mC‐5mCH+, reminiscent of the “i‐motif” in DNA tetramers, was structurally characterized.

Aging of Preleukemic Thymocytes Drives CpG Island Hypermethylation in T-Cell Acute Lymphoblastic Leukemia

During the last decade, aberrant DNA methylation has been identified as a hallmark of human cancer and several studies have highlighted the potential of DNA methylation as a clinically or diagnostically relevant biomarker. In comparison to their normal healthy counterparts, cancer cells generally display DNA hypermethylation at specific CpG islands, but the actual mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. To profile the DNA methylation landscape of human T-cell acute lymphoblastic leukemia (T-ALL), we analyzed 109 T-ALLs together with 10 stages of normal T cell development, which are considered the normal human counterparts of this disease, by 850 EPIC arrays. Here, we show that CpG islands are hypermethylated in all human T- ALLs compared to their normal counterparts. We designed a DNA methylation signature that can distinguish two types of T-ALL, with low or high levels of CpG island hypermethylation. This profile is dominated by CpGs in promoters of PRC2 target genes. T-ALLs with high levels of CpG island hypermethylation show low levels of H3K27me3 and vice versa, resulting in gene repression in both subtypes of T-ALL by different mechanisms. Furthermore, we found that aberrant CpG island hypermethylation shows a strong correlation with the epigenetic age of the leukemic T cells. By investigating the DNA methylation profile of two distinct mouse T-ALL models, the Lck-Cre Ptenfl/fland the CD2-Lmo2 transgenic mouse model, by Reduced Representation Bisulfite Sequencing, we could indeed recapitulate the DNA methylation features of the two human T-ALL subtypes in mice (Fig.1). The aggressive, fast-transforming Ptenfl/flmouse model displays low levels of CpG island hypermethylation, which correlated with human T-ALLs that have a shorter proliferative history and a worse prognosis. In contrast, murine CD2-Lmo2 T-ALLs have a longer disease latency and display a CpG island hypermethylation phenotype that is similar to human T-ALLs with a longer proliferative history. In CD2-Lmo2 mice, a pre-leukemic phase is present with self-renewing thymocytes. We elucidate that the CpG island methylation signature is gradually established in aging pre-leukemic thymocytes of 8, 16 and 24 weeks old CD2-Lmo2 mice (Fig.1). Of note, this hypermethylation phenotype is completely absent in age matched Lck-Cre Ptenfl/flmice that did not yet develop leukemia, suggesting that the proliferative history is responsible for aberrant CpG island DNA methylation observed in human T-ALL. Notably, this provides the first evidence that a pre-leukemic phase might be present in a large subset of human T-ALLs, and that epigenetic aberrations, either in the DNA methylation or histone methylation machinery are one of the first detectable alterations during T-ALL development. Finally, using patient derived xenografts (PDX), we show that DNA hypomethylation by the FDA-approved hypomethylating agent Decitabine is very effective in treating T-ALL. Gene expression profiling revealed that the anti-leukemic effect is exerted by down-regulation of the oncogenic MYC pathway. However, by profiling these PDX T-ALLs by EPIC arrays, we unexpectedly uncover that the age-related CpG island hypermethylation signature is completely resistant to Decitabine treatment. Altogether, our work demonstrates that DNA methylation reflects the epigenetic history of leukemic T cells and suggests that methylation-based subtypes of human T-ALL have followed a different trajectory towards T-cell transformation, possibly mediated by differences in the self-renewing capacity of the putative T-ALL cell-of-origin. Given that the concept of preleukemic thymocytes has only been reported in T-ALL mouse models so far, we here provide, for the first time, conceptual evidence that a pre-leukemic phase might also be involved in the pathogenesis of the human disease. Disclosures No relevant conflicts of interest to declare.

Abstract IA05: Microenvironmental signatures reveal biologic and clinical subtypes of diffuse large B-cell lymphoma

Abstract Diffuse large B-cell lymphoma (DLBCL) is a biologically and clinically heterogenous disease almost invariably treated with combinatorial chemoimmunotherapy resulting in a ~65% cure rate in first line. Further improvement of treatment outcome relies on elucidating the biology that underlies the clinical behavior of this heterogenous disease. Transcriptomic and genetic characterization of DLBCL have increased our understanding of its pathogenesis and provided potential novel therapeutic targets. Over the past few years, the role of the microenvironment in DLBCL biology has been increasingly recognized. A guiding framework is necessary to effectively and safely translate research evidence into clinical practice. Towards this goal, we characterized the DLBCL microenvironment based on the transcriptional footprint of microenvironment cells and processes in a large cohort of patients. We first developed transcriptional signatures reflecting distinct cellular subtypes of the microenvironment, biologic processes, and canonical signaling pathway activation. We then used these signatures to virtually reconstruct the lymphoma microenvironment (LME) by applying an unsupervised community detection algorithm in 4,656 DLBCL cases. As a result, we described four classes of LMEs that reflect distinct enrichment of associated signatures. 1. “Germinal center-like” (GC-like) due to the presence of signatures from cell types commonly found in germinal centers including follicular dendritic cells, lymphatic endothelial cells, total T cells, and several CD4+ T-cell subpopulations. 2. “Mesenchymal” (MS) for the abundance of signatures from stromal cells such as cancer-associated fibroblasts, fibroblastic reticular cells, vascular endothelial cells, and extracellular matrix pathways. The MS-LME class was enriched in mutations affecting antigen presentation. These lymphomas also presented with high activity of the TGFB/SMAD and HIF pathways, previously associated with favorable prognosis in DLBCL. 3. “Inflammatory” (IN) for the presence of signatures from macrophages, neutrophils, NK cells, and cytotoxic T cells. The IN-LME class was enriched for immune suppressive/pro-lymphoma cytokines, expression of the immune checkpoint molecules PD-L1 and IDO1, and higher activity of the NF-kB, JAK/STAT, and TNF signaling pathways. 4. A “depleted” (DP) form that, contrasting with the other LMEs, was characterized by an overall lower presence of microenvironment-derived signatures. We analyzed the association of LME classes with overall survival (OS) and progression-free survival (PFS) in patient cohorts of, respectively, 2,646 and 2,189 DLBCLs treated with rituximab-based chemoimmunotherapy. Analysis of OS by LME indicated significant differences in prognosis from better to poor as follows: GC-like, MS (p =0.03, vs. GC-like), IN (p = 0.006, vs. MS), and DP (p = 0.008, vs. IN) LMEs, whereas GC-like and MS have similarly favorable PFS curves (p = 0.9) followed by IN (p &amp;lt; 0.01) and DP (p = 0.03, vs. IN), which presented with the poorest PFS. In multivariate Cox-proportional hazard analysis controlled for C.O.O. and I.P.I., the LME subtypes remained informative with GC-like and MS LMEs associated with better outcome. When segregated by C.O.O., the DP-LME retained the poorest OS and PFS in both C.O.O. subtypes. However, in ABC-DLBCL, the LME with the best PFS and OS was MS, and in GCB-DLBCL, the best LME was GC-like, suggesting that the biologic impact of the microenvironment may be different depending on the lymphoma subtype. DHL and high-grade lymphomas (HGL) modified their outcome when LME was considered. DHL/HGL with the favorable prognosis LMEs GC-like and MS had significantly better PFS and OS than DHL/HGL with the unfavorable prognosis LMEs IN and DP. DLBCLs with a DP-LME were characterized by a minimal presence of LME signatures and tumor cells exhibited higher clonality, were enriched for mutations affecting TP53 and cell proliferation genes, as well as presented higher levels of aberrant hypermethylation of immune-related and TGFB/SMAD1 genes. Preclinical murine models and patient data demonstrated that some of these features can be pharmacologically reversed with hypomethylating agents, indicating that cytosine methylation patterning of lymphoma cells may contribute to a depleted LME. In summary, we categorized the DLBCL LME into four major transcriptionally defined subtypes with distinct biologic properties and clinical behavior, which complement genetically defined subtypes of DLBCL in guiding the development of rational therapeutic approaches for these patients. Citation Format: Leandro Cerchietti. Microenvironmental signatures reveal biologic and clinical subtypes of diffuse large B-cell lymphoma [abstract]. In: Proceedings of the AACR Virtual Meeting: Advances in Malignant Lymphoma; 2020 Aug 17-19. Philadelphia (PA): AACR; Blood Cancer Discov 2020;1(3_Suppl):Abstract nr IA05.

3-Aminobenzamide alleviates elevated DNA damage and DNA methylation in a BTBR T+Itpr3tf/J mouse model of autism by enhancing repair gene expression

Little is known about genetic and epigenetic alterations in autism spectrum disorder. Moreover, the efficiency of DNA repair in autism must be improved to correct these alterations. We examined whether 3-aminobenzamide (3-AB) could reverse these alterations. We conducted experiments to clarify the molecular mechanism underlying these ameliorations. An assessment of genetic and epigenetic alterations by a modified comet assay showed elevated levels of oxidative DNA strand breaks and DNA hypermethylation in BTBR T+Itpr3tf/J (BTBR) mice used as a model of autism. Oxidative DNA strand breaks and DNA methylation were further quantified fluorometrically, and the results showed similar changes. Conversely, 3-AB treated BTBR mice showed a significant reduction in these alterations compared with untreated mice. The expressions of 43 genes involved in DNA repair were altered in BTBR mice. RT2 Profiler PCR Array revealed significantly altered expression of seven genes, which was confirmed by RT-PCR analyses. 3-AB treatment relieved these disturbances and significantly improved Ogg1 and Rad1 up-regulation. Moreover, autism-like behaviors were also mitigated in BTBR animals by 3-AB treatment without alterations in locomotor activities. The simultaneous effects of reduced DNA damage and DNA methylation levels as well as the regulation of repair gene expression indicate the potential of 3-AB as a therapeutic agent to decrease the levels of DNA damage and DNA methylation in autistic patients. The current data may help in the development of therapies that ultimately provide a better quality of life for individuals suffering from autism.

DNMT1 and DNMT3B regulate tumorigenicity of human prostate cancer cells by controlling RAD9 expression through targeted methylation.

Prostate cancer is the second most common type of cancer and the second leading cause of cancer death in American men. RAD9 stabilizes the genome, but prostate cancer cells and tumors often have high quantities of the protein. Reduction of RAD9 level within prostate cancer cells decreases tumorigenicity of nude mouse xenographs and metastasis phenotypes in culture, indicating that RAD9 overproduction is essential for the disease. In prostate cancer DU145 cells, CpG hypermethylation in a transcription suppressor site of RAD9 intron 2 causes high-level gene expression. Herein, we demonstrate that DNA methyltransferases DNMT1 and DNMT3B are highly abundant in prostate cancer cells DU145, CWR22, LNCaP and PC-3; yet, these DNMTs bind primarily to the transcription suppressor in DU145, the only cells where methylation is critical for RAD9 regulation. For DU145 cells, DNMT1 or DNMT3B shRNA reduced RAD9 level and tumorigenicity, and RAD9 ectopic expression restored this latter activity in the DNMT knockdown cells. High levels of RAD9, DNMT1, DNMT3B and RAD9 transcription suppressor hypermethylation were significantly correlated in prostate tumors, and not in normal prostate tissues. Based on these results, we propose a novel model where RAD9 is regulated epigenetically by DNMT1 and DNMT3B, via targeted hypermethylation, and that consequent RAD9 overproduction promotes prostate tumorigenesis.

Maternal ageing causes changes in DNA methylation and gene expression profiles in mouse oocytes.

Although it is well known that maternal ageing causes reduced oocyte quality and fertility, little information is known about its effect on germ cell epigenetics. In the present study, we compared the gene expression and DNA methylation profiles in germinal vesicle oocytes from young (8-week-old) and aged (18-month-old) mice using single-cell RNA-sequencing and single-cell whole-genome DNA methylation sequencing. We found significant differences in the data from the two groups. Oocytes from aged mice showed significant changes in the expression of some metabolism-related genes, such as mitochondria-associated genes, that was in line with our expectations. Expression of some genes associated with reproduction also showed significant differences. DNA methylation levels were also changed in oocytes from aged mice. The two groups had significant gaps in hypermethylation and hypomethylation levels on each chromosome. These data provide useful information for further understanding the mechanisms of oocyte ageing.

Analysis of mRNA expression and DNA methylation level of RAC1 gene encoding focal adhesion molecule in endometrial and peritoneal endometriosis

Focal adhesion molecules involve in cellular migration, attachment, and play a role in endometriosis pathomechanisms. Recent studies showed that the expression of RAC1, a gene encoded focal adhesion molecule, was predominantly found in endometriosis. As gene expression may be regulated by DNA methylation. Therefore, this study aimed to analyze promoter methylation level of RAC1 gene and mRNA expression in endometrial and peritoneal endometriosis tissues. This study using 20 endometrial and 9 peritoneal tissues from the same patients and 20 normal endometrial. The DNA and RNA from samples were isolated, DNA was converted using sodium bisulfite and amplified using Methyl Specific Polymerase Chain Reaction (MSP) method. The methylation level was determined by the intensity measurement of the bands that arose in gel electrophoresis using ImageJ software, whereas mRNA expression level was measured by Reverse Transcription-quantitative PCR (RT-qPCR) method. The mRNA expression level of RAC1 gene in peritoneal endometriosis increased compared to normal endometrium, as well as compared to endometrial endometriosis, but there was no significant difference in endometrial endometriosis compared to normal. Promoter hypermethylation level of RAC1 gene in peritoneal endometriosis was significantly different compared to normal endometrium, however not significant to endometrial endometriosis. Methylation level of its gene in endometrial endometriosis shown no significant difference compared to normal. There was association between promoter hypermethylation level and its mRNA expression in endometrial endometriosis (R= 0.014; p=0.952). The elevation of mRNA expression of RAC1 gene plays a role in endometrial cell migration to peritoneum, and associated with promoter hypermethylation level of its gene.

Intranasal perillyl alcohol therapy improves survival of patients with recurrent glioblastoma harboring mutant variant for MTHFR rs1801133 polymorphism

BackgroundPolymorphisms in MTHFR gene influence risk and overall survival of patients with brain tumor. Global genomic DNA (gDNA) methylation profile from tumor tissues is replicated in peripheral leukocytes. This study aimed to draw a correlation between rs1801133 MTHFR variants, gDNA methylation and overall survival of patients with recurrent glioblastoma (rGBM) under perillyl alcohol (POH) treatment.MethodsgDNA from whole blood was extracted using a commercially available kit (Axygen) and quantified by spectrophotometry. Global gDNA methylation was determined by ELISA and rs1801133 polymorphism by PCR-RFLP. Statistical analysis of gDNA methylation profile and rs1801133 variants included Mann-Whitney, Kruskal-Wallis, Spearman point-biserial correlation tests (SPSS and Graphpad Prism packages; significant results for effect size higher than 0.4). Prognostic value of gDNA methylation and rs1801133 variants considered survival profiles at 25 weeks of POH treatment, having the date of protocol adhesion as starting count and death as the final event.ResultsMost rGBM patients showed global gDNA hypomethylation (median = 31.7%) and a significant, moderate and negative correlation between TT genotype and gDNA hypomethylation (median = 13.35%; rho = − 0.520; p = 0.003) compared to CC variant (median = 32.10%), which was not observed for CT variant (median = 33.34%; rho = − 0.289; p = 0.06). gDNA hypermethylated phenotype (median = 131.90%) exhibited significant, moderate and negative correlations between TT genotype (median = 112.02%) and gDNA hypermethylation levels when compared to CC (median = 132.45%; rho = − 0,450; p = 0.04) or CT (median = 137.80%; rho = − 0.518; p = 0.023) variants. TT variant of rs1801133 significantly decreased gDNA methylation levels for both patient groups, when compared to CC (d values: hypomethylated = 1.189; hypermethylated = 0.979) or CT (d values: hypomethylated = 0.597; hypermethylated = 1.167) variants. Positive prognostic for rGBM patients may be assigned to gDNA hypermethylation for survivors above 25 weeks of treatment (median = 88 weeks); and TT variant of rs1801133 regardless POH treatment length.ConclusionrGBM patients under POH-based therapy harboring hypermethylated phenotype and TT variant for rs1801133 had longer survival. Intranasal POH therapy mitigates detrimental effects of gDNA hypomethylation and improved survival of patients with rGBM harboring TT mutant variant for MTHFR rs1801133 polymorphism.Trial registrationCONEP -9681- 25,000.009267 / 2004. Registered 12th July, 2004.

Loss‐of‐Function Aldolase C Hypermethylation Cause Serotonin Production in Glioblastoma

Aldolase involves the glycolysis procedure to maintain glucose homeostasis and further produce some intermediates or energy synthesis. Aldolase C (ALDOC) is specific to the brain region and is activated when the brain develops, is injured or traumatized. Recently, we investigate that ALDOC had independent prognostic role in GBM, which was suppressed in low‐grade glioma and glioblastomas (GBM) and correlated with IDH1 mutant status, MGMT methylation and survival rate. We further found that ALDOC has CpG island in promoter region and was hypermethylated in GBM cells. We collected mRNA level and methylation state of ALDOC by RNA‐sequencing and bisulfite‐specific PCR from Cancer cell line encyclopedia (CCLE) database, respectively. The scatter plots performed the inverse correlation trends between methylation with expression level in GBM cells (n=39, spearman rho=−0.774, P=7.68e‐09). We further dissected the metabolomics pattern and compared with methylation status of ALDOC. The results showed serotonin as one of significant change in GBM cells, the production rate was increased when ALDOC hypermethylation or low expression level. Several malignant GBM cells treated 5‐aza‐2′‐deoxycytidine (5‐aza‐dC) to suppress DNA methyltransferase activity. We found that ALDOC expression levels were restored and migration/invasion ability was reduced. Similarly, the serotonin concentration has attenuated, depending on the ALDOC methylation status. We further computational analyzed the mRNA level of ALDOC correlated with IC50 of temozolomide (TMZ). T98G cells have been selected for ALDOC overexpression and the IC50 of TMZ is reduced by ALDOC induction. In contrast, we transfected shRNAs to knock down ALDOC expression levels in U‐87MG cells with an increase in IC50. The combination of selective serotonin inhibitor and 5‐aza treatment has a synergistic effect on IC50 of TMZ in GBM cells.

DNA hypermethylation of sirtuin 1 (SIRT1) caused by betel quid chewing\u2014a possible predictive biomarker for malignant transformation

BackgroundDNA hypermethylation of tumor suppressor genes is observed in precancerous lesions and oral cancer of individuals with the habits of betel quid (BQ) chewing. SIRT1 has been identified as playing a role in the maintenance of epithelial integrity, and its alteration is often related to carcinogenesis. However, the methylation and transcription status of SIRT1 in patients with BQ chewing-related oral cancer has not been investigated. We examined the methylation status of SIRT1 in paraffin-embedded tissue samples of oral squamous cell carcinoma (OSCC) obtained from BQ chewing and non-chewing patients and in tissue samples from healthy control subjects. In addition, we examined whether the hypermethylation of SIRT1 followed by its transcriptional downregulation in the human gingival epithelial cells could be caused by arecoline, a major component of BQ. Furthermore, we investigated the methylation status of SIRT1 in smear samples of macroscopically healthy buccal mucosa from subjects with a habit of BQ chewing.ResultsSIRT1 was significantly hypermethylated in tissue samples of OSCC from BQ chewers and non-chewers than in oral mucosa from healthy control subjects. Results also showed that the hypermethylation level of SIRT1 was significantly higher in OSCC of patients with BQ chewing habits than in those of non-chewing habits (p < 0.05). Our in vitro model showed that hypermethylation is followed by downregulation of the transcriptional level of SIRT1 (p < 0.05). The methylation levels of SIRT1 in the smear samples obtained from BQ chewing individuals were significantly higher than those in the samples obtained from individuals that did not chew BQ. The duration of BQ chewing habits was correlated positively to the frequency of SIRT1 hypermethylation (p < 0.05).ConclusionsOur results suggest that DNA hypermethylation of SIRT1 is involved in the occurrence of oral cancer in BQ chewing patients and that hypermethylation in the oral mucosa of BQ chewers could be a predictive marker for the occurrence of malignant transformation. This is the first report that showed DNA hypermethylation in clinically healthy oral epithelium of BQ chewers. Our study shows evidence that DNA hypermethylation may be an early event of oral carcinogenesis prior to observable clinical changes.

Hypermethylation status of DAPK, MGMT and RUNX3 in HPV negative oral and oropharyngeal squamous cell carcinoma.

Squamous cell carcinoma of the oral cavity and oropharynx is the sixth most common type of cancer in the world. During tumorigenesis, gene promoter hypermethylation is considered an important mechanism of transcription silencing of tumor suppressor genes, such as DAPK, MGMT and RUNX3. These genes participate in signaling pathways related to apoptosis, DNA repair and proliferation whose loss of expression is possibly associated with cancer development and progression. In order to investigate associations between hypermethylation and clinicopathological and prognostic parameters, promoter methylation was evaluated in 72 HPV negative oral and oropharyngeal tumors using methylation-specific PCR. Hypermethylation frequencies found for DAPK, MGMT and RUNX3 were 38.88%, 19.44% and 1.38% respectively. Patients with MGMT hypermethylation had a better 2-year overall survival compared to patients without methylation. Being MGMT a repair gene for alkylating agents, it could be a biomarker of treatment response for patients who are candidates for cisplatin chemotherapy, predicting drug resistance. In view of the considerable levels of hypermethylation in cancer cells and, for MGMT, its prognostic relevance, DAPK and MGMT show potential as epigenetic markers, in a way that additional studies may test its viability and efficacy in clinical management.

Comparison of the Validity of Quantitative and Qualitative Methods of RASSF1A Gene Hypermethylation Molecular Test in the Diagnosis of Thyroid Malignancies.

Within the past two decades, the incidence of thyroid cancer has increased across the globe. In the differential diagnosis of benign tumors from malignant tumors in the thyroid gland, hypermethylation of the RASSF1A gene is used. The aim of this study is comparing the validity of quantitative and qualitative methods of the RASSF1A gene hypermethylation molecular test in the diagnosis of thyroid malignancies. One hundred sixty samples from patients with malignant thyroid tumors (80 samples) and benign thyroid tumors (80 samples) were entered into this study from all patients referred to Ahvaz Medical Centers. First DNA was extracted from samples embedded in paraffin. After DNA extraction, hypermethylation of the gene was done by COBRA method. Finally, to calculate the effects of sensitivity, specificity, predictive value and accuracy of the test, epidemiological calculations were done. The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of the qualita-tive method of the RASSF1A gene hypermethylation molecular test in the diagnosis of thyroid malignancies are: 91.25%, 15%, 51.77%, 63.15%, and 53.12%, respectively and for the qualitative method are: 27.5%, 98.75%, 95.65%, 57.66%, and 6.12%, respectively. The quantitative level of hypermethylation differentiated groups of benign tumors from malignant groups better than the qualitative evaluation but the qualitative studies are not applicable for this purpose.