DNA Methylation-related Enzymes Research Articles

In ovo betaine injection improves breast muscle growth in newly hatched goslings through FXR/IGF-2 pathway

Betaine has been shown to enhance growth performance and increase breast muscle yield in ducks and broilers through various mechanisms, including the modification of DNA methylation. However, the impact of in ovo betaine injection on muscle growth in newly hatched goslings remains unclear. In this study, fifty eggs were injected with saline or betaine at 7.5 mg/egg prior to incubation, and the subsequent effects on breast muscle growth in the newly hatched goslings were investigated. Betaine significantly increased (P < 0.05) the hatch weight, breast muscle weight, and breast muscle index, accompanied by an augmentation in muscle bundle cross-sectional area. Concurrently, betaine significantly upregulated (P < 0.05) the expression levels of myogenic regulatory factors, including myogenin (MyoG) and paired box 7 (Pax7) both mRNA and protein, while downregulating (P < 0.05) the mRNA and protein levels of myostatin (MSTN). Histological analysis revealed a higher abundance of proliferating cell nuclear antigen (PCNA) and Pax7 immune-positive cells in the breast muscle of the betaine group, consistent with elevated PCNA and Pax7 mRNA and protein levels. Additionally, significantly increased (P < 0.05) contents of insulin-like growth factor 1 (IGF-1) and insulin-like growth factor 2 (IGF-2) were observed in the breast muscle of the betaine group, so was mRNA expression of IGF-1, IGF-2, and insulin-like growth factor 1 receptor (IGF-1R). Betaine also significantly in8creased (P < 0.05) global DNA methylation of the breast muscle, accompanied by enhanced mRNA and protein levels of methionine cycle and DNA methylation-related enzymes, Interestingly, the promoter regions of IGF-1, IGF-2, and IGF-1R genes were significantly hypomethylated (P < 0.05). Moreover, in ovo betaine injection significantly upregulated (P < 0.05) the protein level of farnesoid X receptor (FXR) in breast muscle and FXR binding to the promoter of IGF-2 gene. These findings suggest that in ovo betaine injection promotes breast muscle growth during embryonic development in goslings through the FXR-mediated IGF-2 pathway, ultimately improving hatch weight and breast muscle weight.

Alterations in DNA methylation machinery in a rat model of osteoarthritis of the hip

BackgroundThis study aimed to validate alterations in the gene expression of DNA methylation-related enzymes and global methylation in the peripheral blood mononuclear cell (PBMC) and synovial tissues of animal hip osteoarthritis (OA) models.MethodsAnimals were assigned to the control (no treatment), sham (25 µL of sterile saline), and OA (25 µL of sterile saline and 2 mg of monoiodoacetate) groups. Microcomputed tomography scan, histopathological assessment and pain threshold measurement were performed after induction. The mRNA expression of the DNA methylation machinery genes and global DNA methylation in the PBMC and hip synovial tissue were evaluated.ResultsThe OA group presented with hip joint OA histopathologically and radiologically and decreased pain threshold. The mRNA expression of DNA methyltransferase (Dnmt 3a), ten–eleven translocation (Tet) 1 and Tet 3 in the synovial tissue of the OA group was significantly upregulated. Global DNA methylation in the synovial tissue of the OA group was significantly higher than that of the control and sham groups.ConclusionsThe intra-articular administration of monoiodoacetate induced hip joint OA and decreased pain threshold. The DNA methylation machinery in the synovial tissues of hip OA was altered.

Global DNA hypomethylation of colorectal tumours detected in tissue and liquid biopsies may be related to decreased methyl-donor content

BackgroundHypomethylation of long interspersed nuclear element 1 (LINE-1) is characteristic of various cancer types, including colorectal cancer (CRC). Malfunction of several factors or alteration of methyl-donor molecules’ (folic acid and S-adenosylmethionine) availability can contribute to DNA methylation changes. Detection of epigenetic alterations in liquid biopsies can assist in the early recognition of CRC. Following the investigations of a Hungarian colon tissue sample set, our goal was to examine the LINE-1 methylation of blood samples along the colorectal adenoma-carcinoma sequence and in inflammatory bowel disease. Moreover, we aimed to explore the possible underlying mechanisms of global DNA hypomethylation formation on a multi-level aspect.MethodsLINE-1 methylation of colon tissue (n = 183) and plasma (n = 48) samples of healthy controls and patients with colorectal tumours were examined with bisulfite pyrosequencing. To investigate mRNA expression, microarray analysis results were reanalysed in silico (n = 60). Immunohistochemistry staining was used to validate DNA methyltransferases (DNMTs) and folate receptor beta (FOLR2) expression along with the determination of methyl-donor molecules’ in situ level (n = 40).ResultsSignificantly decreased LINE-1 methylation level was observed in line with cancer progression both in tissue (adenoma: 72.7 ± 4.8%, and CRC: 69.7 ± 7.6% vs. normal: 77.5 ± 1.7%, p ≤ 0.01) and liquid biopsies (adenoma: 80.0 ± 1.7%, and CRC: 79.8 ± 1.3% vs. normal: 82.0 ± 2.0%, p ≤ 0.01). However, no significant changes were recognized in inflammatory bowel disease cases. According to in silico analysis of microarray data, altered mRNA levels of several DNA methylation-related enzymes were detected in tumours vs. healthy biopsies, namely one-carbon metabolism-related genes—which met our analysing criteria—showed upregulation, while FOLR2 was downregulated. Using immunohistochemistry, DNMTs, and FOLR2 expression were confirmed. Moreover, significantly diminished folic acid and S-adenosylmethionine levels were observed in parallel with decreasing 5-methylcytosine staining in tumours compared to normal adjacent to tumour tissues (p ≤ 0.05).ConclusionOur results suggest that LINE-1 hypomethylation may have a distinguishing value in precancerous stages compared to healthy samples in liquid biopsies. Furthermore, the reduction of global DNA methylation level could be linked to reduced methyl-donor availability with the contribution of decreased FOLR2 expression.

Curcumin increases crizotinib sensitivity through the inactivation of autophagy via epigenetic modulation of the miR-142-5p/Ulk1 axis in non-small cell lung cancer.

Drug resistance is a critical factor responsible for the recurrence of non-small cell lung cancer (NSCLC). Previous studies suggest that curcumin acts as a chemosensitizer and radiosensitizer in human malignancies, but the underlying mechanism remains elusive. In the present study, we explored how curcumin regulates the expression of miR-142-5p and sensitizes NSCLC cells to crizotinib. We found that miR-142-5p is significantly downregulated in NSCLC tissue samples and cell lines. Curcumin could increase crizotinib cytotoxicity by epigenetically restoring the expression of miR-142-5p. Furthermore, curcumin treatment suppressed the expression of DNA methylation-related enzymes, including DNMT1, DNMT3A, and DNMT3B, in NSCLC cells. In addition, the upregulation of miR-142-5p expression increased crizotinib cytotoxicity and induced apoptosis in tumor cells in a similar manner to that of curcumin. Strikingly, miR-142-5p overexpression suppressed crizotinib-induced autophagy in A549 and H460 cells. Mechanistically, miR-142-5p inhibited autophagy in lung cancer cells by targeting Ulk1. Overexpression of Ulk1 abrogated the miR-142-5p-induced elevation of crizotinib cytotoxicity in A549 and H460 cells. Collectively, our findings demonstrate that curcumin sensitizes NSCLC cells to crizotinib by inactivating autophagy through the regulation of miR-142-5p and its target Ulk1.

Effects of triclosan exposure on placental extravillous trophoblast motility, relevant IGF2/H19 signaling and DNA methylation-related enzymes of HTR-8/SVneo cell line

Triclosan (TCS) is an antimicrobial agent widely used in personal care products and a potential endocrine disruptor chemical (EDC). TCS can pass through the placental barrier. Any influence of EDCs on epigenetic changes of placenta and embryo may bring profound impact on later health. This study aimed to investigate the effects of TCS exposure on cell proliferation and migration, and the expression of imprinted genes IGF2/H19 and DNA methylation-related enzymes in human placental extravillous trophoblast cell line HTR-8/SVneo. After exposure to TCS levels of 0 (DMSO Control), 10−11, 10−10, 10−9, 10−8, 10−7, 10−6, 10−5, 3 × 10−5, 6 × 10−5, 10−4 M and incubated for up to 36 h, cell proliferation and migration were examined by CCK-8, EdU incorporation assay and wound healing assay; the mRNA levels of IGF2, H19, DNA methyltransferases (Dnmt3a, Dnmt3b and Dnmt1), ten-eleven translocation enzymes (Tet1, Tet2 and Tet3) and IGF2 Receptor (IGF2R) were analyzed by qRT-PCR. The protein levels of IGF2 were measured by Western blot and ELISA. The cell viability turned to decline at TCS treatment of 3 × 10−5 M and above (all p < 0.05, compared to the DMSO Control). The cell migration decreased at TCS 10−5 and 3 × 10−5 M treatment (p < 0.05), but consistently unchanged at low dose of TCS from 10−9 to 10−7 M (p > 0.05). TCS treatments below cytotoxicity doses (< 10−5 M) did not significantly alter the mRNA levels of IGF2, Dnmt1, Dnmt3a, Dnmt3b, Tet1, Tet2 and Tet3, compared to DMSO Control treatment (all p > 0.05). The transcription level of H19 was up-regulated by TCS at 3 × 10−5 M. TCS at 10−7 and 6 × 10−5 M increased the protein level of IGF2 in cell supernatant. Our data suggest that high TCS exposure may suppress HTR-8/SVneo cells viability and migration, increase H19 gene expressions and IGF2 protein secretion. The exact mechanism of TCS action in human trophoblast needs further studies.

Abstract 155: Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression

Abstract Backgrounds: Global DNA hypomethylation is characteristic in various cancer types including colorectal cancer. Alterations of DNA methylation related enzymes expression and decreased level of methyl-donor molecules (folic acid (FA), S-adenosylmethionine (SAM)) can lead to aberrant DNA methylation pattern and elevated homocysteine level. Aims: Our aim was to examine global DNA methylation changes during aging and colorectal normal-adenoma-carcinoma sequence and in inflammatory bowel disease in tissue and liquid biopsy samples for diagnostic purposes. Moreover, we aimed to explore the reasons of global hypomethylation on gene expression level and methyl-donor molecule content. Methods: Bisulfite treatment was performed on DNA isolated from 30 normal (N), 10 adenoma (Ad), 10 colorectal carcinoma (CRC), 10 colitis ulcerosa (UC) tissue samples and on 11 N, 10 Ad, 15 CRC, 12 UC plasma specimens. 30 N samples contained different age groups derived from under 20 to 70 years old healthy controls for examination of aging process. LINE-1 PCR product was generated and pyrosequenced. Whole genome expression level of 60 biopsy samples was evaluated by HTA 2.0 RNA microarraychip (Affymetrix). In situ tissue appearance of 5-methylcytosine, FA, SAM, homocysteine, and expression of DNA methyltransferases (DNMTs) were analyzed by immunohistochemistry staining (IHC). Results: According to LINE-1 bisulfite sequencing results, DNA methylation was 72.6±1% in samples of healthy controls under 50 years old and 71.6±1.8% in specimens of patients over 50 years old. Significant DNA hypomethylation was found in CRC (62.9±8.7%; p&amp;lt;0.001, early stages: 67.4±7.8%, late stages: 58.3±0.1%), Ad (66.7±5.1%; p&amp;lt;0.001) tissue samples in comparison with N samples (72±1.4%). Significant decrease of DNA methylation was observed in CRC (78.8±1.7%; p&amp;lt;0.02), and Ad (80.1±1.7%; p&amp;lt;0.02) plasma samples compared to N specimens (82.2±1.8%). Global DNA hypomethylation was not detected in UC samples. Significantly elevated RNA expression of enzymes connected to nucleotide synthesis was observed in Ad and CRC samples compared to N (p&amp;lt;0.05), while no changes were detected in the RNA levels of DNA methylation-related proteins. The intensity of 5-mC labeling of CRC and Ad samples was lower than in N tissue samples. Decreased FA, SAM, and increased homocysteine levels were noticed in CRC compared to N specimens; however no expression changes of DNMT enzymes were observed. Conclusion: Significant decrease in DNA methylation level was found in tissue and liquid biopsy samples of colorectal normal-adenoma-carcinoma sequence, but not in UC specimens. Our results suggest that determination of global DNA hypomethylation could have prognostic and diagnostic value as well, and reduction of DNA methylation level could be linked to decreased FA and SAM availability and not to methylation related ezymes activity. Citation Format: Krisztina A. Szigeti, Orsolya Galamb, Alexandra Kalmár, Gábor Valcz, Sára Zsigrai, Barbara K. Barták, Zsófia B. Nagy, Zsolt Tulassay, Péter Igaz, Béla Molnár. Global DNA hypomethylation can be caused by decreased methyl-donor content in tissue and liquid biopsy samples in colorectal cancer progression [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 155.

Comprehensive analysis of miRNA-gene regulatory network with clinical significance in human cancers.

microRNAs (miRNAs), particularly the exosomal miRNAs have been widely used as biomarkers and promising therapeutic targets in cancer. However, a comprehensive analysis of miRNA-gene regulatory network with clinical significance remains scarce. The emergence of high-throughput multi-omics data over large, well-characterized patient cohorts provides an unprecedented opportunity to address this problem. Herein, we performed a clinic-centered analysis to identify cancer-associated miRNAs, miRNA-target axis. We first calculated the correlation among miRNA, mRNA and 75 unique clinico-pathological characteristics (CPCs) in 26 cancer types, and established an online resource (4CR). Interestingly, we found that the high expression of several DNA methylation-related enzymes was associated with adverse outcomes of cancer patients, and these genes were regulated by a cluster of miRNAs. Furthermore, by integrating exosomal miRNA and mRNA databases, we identified exosomal miRNA biomarkers for non-invasive cancer surveillance and therapy monitoring. Finally, we explored the role of CPC-related miRNAs for therapeutic effect prediction of drugs based on their shared targets. Our analysis pipeline illustrated the significance of clinic-centered analysis in miRNA-gene pair identification and provided helpful clues for future cancer studies.

Dysregulation of Placental Functions and Immune Pathways in Complete Hydatidiform Moles.

Gene expression studies of molar pregnancy have been limited to a small number of candidate loci. We analyzed high-dimensional RNA and protein data to characterize molecular features of complete hydatidiform moles (CHMs) and corresponding pathologic pathways. CHMs and first trimester placentas were collected, histopathologically examined, then flash-frozen or paraffin-embedded. Frozen CHMs and control placentas were subjected to RNA-Seq, with resulting data and published placental RNA-Seq data subjected to bioinformatics analyses. Paraffin-embedded tissues from CHMs and control placentas were used for tissue microarray (TMA) construction, immunohistochemistry, and immunoscoring for galectin-14. Of the 14,022 protein-coding genes expressed in all samples, 3,729 were differentially expressed (DE) in CHMs, of which 72% were up-regulated. DE genes were enriched in placenta-specific genes (OR = 1.88, p = 0.0001), of which 79% were down-regulated, imprinted genes (OR = 2.38, p = 1.54 × 10−6), and immune genes (OR = 1.82, p = 7.34 × 10−18), of which 73% were up-regulated. DNA methylation-related enzymes and histone demethylases were dysregulated. “Cytokine–cytokine receptor interaction” was the most impacted of 38 dysregulated pathways, among which 17 were immune-related pathways. TMA-based immunoscoring validated the lower expression of galectin-14 in CHM. In conclusion, placental functions were down-regulated, imprinted gene expression was altered, and immune pathways were activated, indicating complex dysregulation of placental developmental and immune processes in CHMs.

PGK1 facilities cisplatin chemoresistance by triggering HSP90/ERK pathway mediated DNA repair and methylation in endometrial endometrioid adenocarcinoma

BackgroundEndometrial carcinoma represents one of the most common cancer types of the female reproductive tract. If diagnosed at an early stage, the 5-year survival rate is promising. However, recurrence and chemoresistance remain problematic for at least 15% of the patients. In the present study, we aim to reveal the mechanism by which PGK1 regulates chemoresistance in endometrial carcinoma.MethodsqPCR was performed to detect expression of PGK1 in clinical tissue samples of endometrial carcinoma. Specific shRNAs were employed to knockdown PGK1 expression in endometrial cancer cell lines. MTT assay was used to evaluate cell viability and cisplatin sensitivity of endometrial carcinoma cell lines. Western blot was performed to assess the effects of PGK1 knockdown on the expression levels of HSP90, DNA repair-associated proteins (c-JUN, FOSL1, and POLD1), and DNA methylation-related enzymes (DNMT1, DNMT3A and DNMT3B). Immunoprecipitation was performed to verify direct binding between PGK1 and HSP90.ResultsWe first showed that PGK1 expression is elevated in tumor tissues of endometrial cancer, and high PGK1 levels are associated with clinical stages and metastasis. Knockdown of PGK1 inhibits proliferation of endometrial cancer cells, and enhances the inhibitory effect of cisplatin on cell viability. In addition, knockdown of PGK1 down-regulates the expression of DNA repair-related proteins, methylation-related enzymes, and total cellular methylation level. PGK1 was next shown to interact directly with HSP90 and exhibit pro-tumor effects by modulating the ATPase activity of HSP90.ConclusionsWe propose that PGK1 mediates DNA repair and methylation through the HSP90/ERK pathway, and eventually enhances the chemoresistance to cisplatin. The results provide new insights on functions of PGK1 and HSP90, which might make them as promising targets for endometrial cancer chemotherapy.

Global DNA 5-Hydroxymethylcytosine and 5-Formylcytosine Contents Are Decreased in the Early Stage of Hepatocellular Carcinoma.

Methylation of the fifth position of cytosine (5mC) is an important epigenetic modification of DNA. It has been shown that the oxidized derivatives of 5mC, namely 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), are in dynamic existence and have distinct regulatory functions. In the current study, we investigated whether there are changes in the contents of all three 5mC-oxidized derivatives in the hepatocellular carcinoma (HCC) genome and further explored the underlying mechanisms. We showed that both global genomic 5hmC and 5fC contents were decreased significantly in the very early stage (stage 0, Barcelona Clinic Liver Cancer [BCLC] staging) of HCC compared with those of paratumor tissues. Noteworthily, 5fC content continued to decrease in the late stage (BCLC staging from 0 to A) of HCC. The 5caC content in HCC tissues was below the detection threshold. Hepatitis B virus (HBV) infection was associated with 5mC, 5hmC, or 5fC decrease in HCC; and measurements in cell lines integrated with or without HBV DNA showed consistent results. On the other hand, both the expression level of ten-eleven translocation enzyme 2 (TET2) and α-ketoglutarate content were decreased significantly in HCC. The significantly positive correlations among the expression levels of DNA methylation-related enzymes in paratumor tissues were generally attenuated or even disappeared in HCC tumor tissues. The decreases of both 5hmC and 5fC contents in genomic DNA were associated with poor prognosis of HCC patients. Conclusion: Global 5hmC and 5fC contents were decreased significantly in the very early stage of HCC; the decrease of 5hmC and 5fC was mainly due to the decrease of 5mC and associated with HBV infection, decreased TET enzyme activity, and uncoordinated expression of DNA methylation-related enzymes.

Uncoordinated expression of DNA methylation-related enzymes in human cancer

BackgroundIn addition to the important roles played by 5-methylcytosine (5mC), emerging evidence suggests that 5mC derivatives, such as 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), also exhibit regulatory functions in physiological and pathological processes. Four cytosine modifications (5mC, 5hmC, 5fC and 5caC) are produced and erased by a cyclic enzymatic cascade mediated by DNA methyltransferases (DNMTs), ten-eleven translocation (TET) family enzymes and thymine DNA glycosylase (TDG). Stable maintenance of the DNA methylation profile is important for normal cell homeostasis, but its underlying mechanisms are largely unknown.MethodsThe expression levels of 7 DNA methylation-related enzymes from normal mouse tissues were assessed using quantitative real-time RT-PCR (qRT-PCR). The gene expression data and related information of human normal tissues and tumor tissues were obtained from the Genotype-Tissue Expression (GTEx) and the Cancer Genome Atlas (TCGA), respectively.ResultsWe observed significant positive correlations among the expression levels of DNA methylation-related enzymes in various mice and human normal tissues. By contrast, we found significantly decreased correlations in various tumor tissues compared with their corresponding normal tissues. Furthermore, we also found that alterations in these correlations are associated with several clinicopathological characteristics of cancer patients.ConclusionsThese observations suggest that uncoordinated expression of DNA methylation-related enzymes is another epigenetic hallmark of cancer. Our work provides important insights into an additional regulatory layer of the DNA methylation maintenance machinery.

DUSP23 is over-expressed and linked to the expression of DNMTs in CD4+ T cells from systemic lupus erythematosus patients.

We evaluated the transcriptional expression of dual-specificity protein phosphatase 23 (DUSP23) in CD4+ T cells from 30 systemic lupus erythematosus (SLE) patients and 30 healthy controls. DUSP23 mRNA levels were considerably higher in the patient group: 1490 ± 1713 versus 294·1 ± 204·2. No association was found between DUSP23 mRNA expression and the presence of typical serological and clinical parameters associated with SLE. Meaningful statistical values were obtained in the patient group between the levels of DUSP23 and integrin subunit alpha L (ITGAL), perforin 1 (PRF1) and CD40L. Similarly, transcript levels of different DNA methylation-related enzymes [DNA methylation-related enzymes (DNMT1, DNMT3A, DNMT3B, MBD2, and MBD4)] were also correlated positively with the expression of DUSP23. In an attempt to counteract the hypomethylation status of the promoters of certain genes known to be over-expressed in SLE, it is possible that DUSP23 acts as a negative regulatory mechanism which ultimately silences the transcription of these epigenetically regulated genes by triggering an increase in the expression of different DNMTs.

Preliminary Study on the Abnormal DNA Methylation in T Cells from the Patients with Immune Related Pancytopenia

Objective To explore the global DNA methylation and the expression of regulatory genes for methylation in CD4 + T cells of the patients with immune related pancytopenia (IRP) and explore the role of methylation in pathogenesis of IRP.Methods Thirty IRP patients (untreated, n=15; remission, n=15) and 15 healthy donors as controls were enrolled from December 2012 to December 2013. CD4+ T cells were sorted by immunomagnetic separation. The global DNA methylation was tested with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of DNA methylation-related regulating genes, DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs), were measured by real-time quantitative polymerase chain reaction (RT-PCR).Results The level of global DNA methylation in peripheral blood CD4+ T cells of untreated IRP patients (3.525%±2.046%)and remission patients (4.790%±1.471%) were significantly lower than that of normal controls (10.101%±3.449%) respectively (both P<0.05). DNMT3b mRNA level of untreated IRP patients (1.332±0.785) was significantly lower than that of normal controls (2.077±1.059) in CD4+T cells (P<0.05). The mRNA expression of MBD2 was significantly higher in CD4+ T cells from untreated and remission IRP patients (2.999±1.601, 2.055±1.576) than that in controls (0.581±0.247) (both P<0.05). The MBD4 mRNA level was significantly higher in CD4+ T cells from untreated IRP patients (2.736±2.719) compared to that in normal controls (1.167±1.006) (p<0.05). DNMT3b mRNA expression and CD4+ T cell DNA methylation to be positive correlated within IRP patients (r=0.569, p<0.01). The MBD2 mRNA expression negatively correlated with CD4+ T cell DNA methylation and the ratio of Th1/Th2 (r=-0.763, p<0.001; r = -0.652, p<0.05). The global methylation of CD4+ T cells negatively related to the ratio of CD5+ B cells (r= -0.439, p<0.05).Conclusions The globe DNA hypomethylation and abnormal expression of DNA methylation-related enzymes in peripheral blood CD4+ T cells may be related with the pathogenesis of IRP. DisclosuresNo relevant conflicts of interest to declare.

Abnormal DNA methylation in CD4⁺ T cells from patients with immune related pancytopenia: a preliminary study

To explore the global DNA methylation and the expression of regulatory genes for methylation in CD4⁺ T cells of the patients with immune related pancytopenia (IRP) and explore the role of methylation in the pathogenesis of IRP. Thirty IRP patients (untreated, n = 15; remission, n = 15) and 15 healthy donors as controls were enrolled from December 2012 to December 2013. CD4⁺ T cells were sorted by immunomagnetic separation. The global DNA methylation was tested with enzyme-linked immunosorbent assay (ELISA). The mRNA levels of DNA methylation-related regulating genes, DNA methyltransferases (DNMTs) and methylated CpG binding proteins (MBDs), were measured by real-time quantitative-polymerase chain reaction (RT-PCR). The level of global DNA methylation in peripheral blood CD4⁺ T cells of untreated IRP patients (3.525% ± 2.046%) and remission patients (4.790% ± 1.471%) were significantly lower than that of normal controls (10.101% ± 3.449%) respectively (both P < 0.05). DNMT3b mRNA level of untreated IRP patients (1.332 ± 0.785) was significantly lower than that of normal controls (2.077 ± 1.059) in CD4⁺ T cells (P < 0.05). The mRNA expression of MBD2 was significantly higher in CD4⁺ T cells from untreated and remission IRP patients (2.999 ± 1.601, 2.055 ± 1.576) than that in controls (0.581 ± 0.247) (both P < 0.05). The MBD4 mRNA level was significantly higher in CD4⁺ T cells from untreated IRP patients (2.736 ± 2.719) compared to that in normal controls (1.167 ± 1.006) (P < 0.05). DNMT3b mRNA expression and CD4⁺ T cell DNA methylation to be positive correlated within IRP patients (r = 0.569, P < 0.01). The MBD2 mRNA expression was negatively correlated with CD4⁺ T cell DNA methylation and the ratio of Th1/Th2 (r = -0.763, P < 0.01; r = -0.652, P < 0.05) . The global methylation of CD4⁺ T cells negatively related to the ratio of CD5⁺ B cells (r = -0.439, P < 0.05). The globe DNA hypomethylation and abnormal expression of DNA methylation-related enzymes in peripheral blood CD4⁺ T cells may be related with the pathogenesis of IRP.

Associations between the Expression of Epigenetically Regulated Genes and the Expression of DNMTs and MBDs in Systemic Lupus Erythematosus

ObjectivesWe determined the expression of ITGAL, PRF1, KIR2DL4, CD70, and CD40LG in patients with SLE and performed correlations with the global DNA methylation status and the levels of three DNA methylation enzymes and two methyl CpG-binding domain (MBD) proteins.Patients and MethodsCD4+ T cells were isolated from 35 SLE patients and 30 healthy controls. DNA deoxymethylcytosine content was measured by an enzyme-linked immunosorbent assay (ELISA). Transcript levels of ITGAL, PRF1, KIR2DL4, CD70, CD40LG, DNMT1, DNMT3A, DNMT3B, MBD2, and MBD4 were quantified by real-time reverse-transcription polymerase chain reaction (RT-PCR).ResultsSLE patients had significantly elevated transcript levels of ITGAL (18.61±22.17 vs. 7.33±9.17, p = 0.042), PRF1 (21.67±26.34 vs. 10.67±11.65, p = 0.039), and CD70 (1.45±1.63 vs. 0.67±0.28, p = 0.011). A positive correlation was observed between transcript levels of CD40LG and ITGAL (r = 0.477, p = 0.004) as well as between CD40LG and PRF1 (r = 0.557, p = 0.001). Transcript levels of KIR2DL4 were higher than controls' but it did not reach statistical significance (1.36±3.52 vs. 0.22±0.79, p = 0.560). A tight relationship with global DNA hypomethylation as well as with the expression of most of the DNA methylation-related genes was observed, especially for ITGAL, PRF1, and CD40LG.ConclusionsITGAL, PRF1, and CD70 are overexpressed in SLE CD4+ T cells. The tight association of CD40LG with ITGAL and PRF1 leads us to infer that it probably contributes to the pathogenesis of the disease. The apparent simultaneous regulation between their expression and the global DNA hypomethylation as well as with the transcription of many DNA methylation-related enzymes, reinforces the idea that epigenetic mechanisms are responsible for the deregulation of ITGAL, PRF1, and CD40LG.

Clinical Significance of Genetic Mutations of CD79B, CARD11, MYD88, and EZH2 Genes in Diffuse Large B-Cell Lymphoma Patients

Abstract 2633 Background:Recently, single nucleotide polymorphism array and next generation DNA sequencing techniques have been used to identify several genetic mutations in diffuse large B-cell lymphoma (DLBCL). The mutations seem to be localized in the B-cell receptor and NF-k B signaling pathway and histone- and DNA methylation-related enzymes. However, the relationship between the genetic mutations and the clinical features, such as outcome and disease progression, and pathological findings, has not yet been elucidated. Aims:To confirm frequency of genetic mutations in Japanese DLBCL patients, and to examine the relationship between genetic mutations and clinical and pathological features of the disease, including prognosis, responsiveness to chemotherapy with/without rituximab, and to examine the correlation between mutations and patient clinical symptoms. Methods:Genomic DNA was extracted from lymphoma cells harvested from 138 DLBCL patients enrolled in this study at Nagoya University Hospital. Genetic mutations in the coding regions of CD79B, CARD11, MYD88, and EZH2 were confirmed using pyrosequencing and direct DNA sequencing analyses. DLBCL subtype, germinal center B (GCB; 37 cases) or non-GCB cell (40 cases), were confirmed by immuno-pathological analysis. Overall survival rate was calculated by Kaplan-Meier analysis, and statistical differences were detected by the log-rank test. Results and Discussion:Mutations in CD79B leading to disruption of the immunoreceptor tyrosine-based activation motif (ITAM) were observed in 15 cases [10.5%; GCB, 2 cases (5.4%) and non-GCB, 4 cases (10.0%)], and mutations resulting in Y196 substitution, which is known to be a phosphorylation site critical for signal transduction, were confirmed in 13/15 cases (86.7%). Mutations in the coiled-coil (C-C) domain of CARD11 were detected in 4 cases [2.9%; GCB, 1 case (2.7%) and non-GCB, 1 case (2.5%)]. TIR (Toll/IL1 receptor) mutations in MYD88 were detected in 17 cases [12.3%; GCB, 3 cases (8.1%) and non-GCB, 6 cases (15%)], and Y265P substitution was confirmed in 14/17 cases (82%). Y641 substitution in EZH2 was detected in 9 cases [6.5%; GCB, 6 cases (16.2%) and non-GCB, 2 cases (5.0%)]. The frequency of EZH2 mutation was almost equivalent with previous reports, but the mutation frequencies of CD79B, CARD11, and MYD88 were much lower. Genetic mutations were confirmed in both CD79B and MYD88 in 6 cases (4.3%), while the frequency of CD79B and MYD88 mutations individually were 40% and 35.3%. Overall survival was compared between patients with or without each genetic mutation. Patients with CD79B and MYD88 mutations tended to have relatively poor outcomes, but the differences were not statistically significant (p=0.12 and 0.40, respectively). Patients with the EZH2 mutation showed relatively better outcomes, but the difference was not statistically significant (p=0.57). The overall survival rate of subjects with the CARD11 C-C mutation was significantly lower (p<0.0001), with the median overall survival of mutation(+) and (−) of 308.5 and 2950 days, respectively. Clinical records indicate that all cases with mutations were diagnosed as DLBCL at stages I (1 case), II (1 case), and IV (2 cases) and died due to disease progression within 1 year after the first diagnosis. Although we could not demonstrate statistically significant differences in overall survival for presence of CD79B, MYD88, and EZH2 mutations in this investigation, the tendencies observed here warrant further investigation with larger numbers of patients. Since prognosis was significantly poor in patients with the CARD11 C-C mutation, a more detailed extraction of their clinical symptoms is needed to confirm which phenomenon are related to the CARD11 C-C mutation. Disclosures:Naoe:Zenyaku-Kogyo: Research Funding; Novartis Pharma.: Honoraria, Speakers Bureau; Chugai Pharma.: Research Funding; Dainipponn-Sumitomo Pharma.: Research Funding; Kyowa-Hakko Kirin.: Research Funding; Otsuka Pharma.: Research Funding.