Bisulfite Pyrosequencing Assays Research Articles

Deregulated hsa‐miR‐23a‐3p and hsa‐mir‐148a‐3p influence key processes in classic Hodgkin lymphoma (cHL) pathogenesis

Introduction: We previously performed global miRNA expression profiling of cHL vs. non-Hodgkin lymphoma (NHL) cell lines and sorted germinal centre B-cells (GCB). In the group of 79 deregulated miRNAs (p < 0.05) we became particularly interested in the upregulated miR-23a-3p, a potential oncomiR, predicted to regulate the expression of TNFAIP3 and the downregulated miR-148a-3p, a potential tumor suppressor miRNA, predicted to regulate the expression of IL15. Therefore, we aimed to decipher the role of miR-23a-3p and miR-148a-3p in cHL pathogenesis. Methods: Expression analyses were performed with TaqMan Advanced miRNA Assays versus two miRNA controls. Laser capture microdissection was used for primary HRS collection. 1,000 (miRNA expression) and 2 × 200 (DNA methylation) HRS cells were collected per case. Bisulfite pyrosequencing assay was designed using the PyroMark Software and sequencing performed using the PyroMark Q24 sequencer. MiRNA-mRNAs interactions were validated with Dual-Glo Luciferase Assay using respective miRNA mimics. For overexpression pre-miRNAs were cloned into the pCDH-CMV-MCS-EF1α-GreenPuro vector and transductions were performed in triplicate. Cell proliferation was measured using CCK-8 in four replications in three independent reactions by the GloMax®96 Microplate reader. Results: We confirmed the overexpression of miR-23a-3p (p < 0.001) and downregulation of miR-148a-3p (p < 0.014) in microdissected HRS cells (n = 10) vs. GCB cells (n = 10). Moreover, we observed inverse correlation of miR-148a-3p expression with DNA methylation level of an adjacent CpG island (r = −0.72, p < 0.01; cHL n = 7, NHL n = 10). Elevated DNA methylation of this CpG island was detected also in 2/6 cHL primary cases. Next, we validated interactions between miR-23a-3p and miR-148a-3p and their target genes. A reduction by 34% (p < 0.001) and 40% (p < 0.01) of the luciferase signal was observed in consequence of the interaction of miR-23a-3p mimic—TNFAIP3 3’UTR and miR-148a-3p mimic—IL15 3’UTR, respectively. Moreover, the abundance of TNFAIP3 protein was reduced after overexpression of the miR-23a (coexpressed with miR-27a) in L428 (p = 0.0018) and GCB6-16 (p = 0.0083) cell lines, compared to empty vector transductions. Whereas overexpression of miR-148a in three cHL cell lines caused a 32% decrease (p < 0.05) in proliferation in the KM-H2 cell line compared to empty vector transductions. The research was funded by: This research was funded by the National Science Center, Poland grants: 2014/14/M/NZ2/00529 to M.G. and the 2019/32/T/NZ5/00441 Etiuda scholarship to JP. SH is supported by the German Research Foundation (DFG, HA6145/3-1). MG, ADz-K and NR received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 952304. Keywords: Hodgkin lymphoma, Tumor Biology and Heterogeneity No conflicts of interests pertinent to the abstract.

CpG site-specific methylation as epi-biomarkers for the prediction of health risk in PAHs-exposed populations

Environmental insults can lead to alteration in DNA methylation of specific genes. To address the role of altered DNA methylation in prediction of polycyclic aromatic hydrocarbons (PAHs) exposure-induced genetic damage, we recruited two populations, including diesel engine exhausts (low-level) and coke oven emissions (high-level) exposed subjects. The positive correlation was observed between the internal exposure marker (1-hydroxypyrene) and the extents of DNA damage (P < 0.05). The methylation of representative genes, including TRIM36, RASSF1a, and MGMT in peripheral blood lymphocytes was quantitatively examined by bisulfite-pyrosequencing assay. The DNA methylation of these three genes in response to PAHs exposure were changed in a CpG-site-specific manner. The identified hot CpG site-specific methylation of three genes exhibited higher predictive power for DNA damage than the respective single genes in both populations. Furthermore, the dose-response relationship analysis revealed a nonlinear U-shape curve of TRIM36 or RASSF1a methylation in combined population, which led to determination of the threshold of health risk. Furthermore, we established a prediction model for genetic damage based on the unidirectional-alteration MGMT methylation levels. In conclusion, this study provides new insight into the application of multiple epi-biomarkers for health risk assessment upon PAHs exposure.

Curcumin induces re‑expression of BRCA1 and suppression of γ synuclein by modulating DNA promoter methylation in breast cancer cell lines.

Restoration of normal DNA promoter methylation and expression states of cancer-related genes may be an option for the prevention as well as the treatment of several types of cancer. Constitutional promoter methylation of BRCA1 DNA repair associated (BRCA1) gene is linked with a high risk of developing breast and ovarian cancer. Furthermore, hypomethylation of the proto-oncogene γ synuclein (SNCG) is associated with the metastasis of breast and ovarian cancer and reduced disease-free survival (DFS). In the present study, we evaluated the potential of curcumin to re-express hypermethylated BRCA1 and to suppress hypomethylated SNCG in triple-negative breast cancer (TNBC) cell line HCC-38, the estrogen receptor-negative/progesterone receptor-negative (ER−/PR−) cell line UACC-3199, and the ER+/PR+ cell line T47D. The cells were treated with 5 and 10 µM curcumin for 6 days and with 5-aza-2′-deoxycytidine (5′-aza-CdR) for 48 h. Methylation-specific PCR and bisulfite pyrosequencing assays were used to assess DNA promoter methylation while gene expression levels were analyzed using quantitative real-time PCR and immunoblotting. We found that curcumin treatment restored BRCA1 gene expression by reducing the DNA promoter methylation level in HCC-38 and UACC-3199 cells and that it suppressed the expression of SNCG by inducing DNA promoter methylation in T47D cells. Notably, 5′-aza-CdR restored BRCA1 gene expression only in UACC-3199, and not in HCC-38 cells. Curcumin-induced hypomethylation of the BRCA1 promoter appears to be realized through the upregulation of the ten-eleven translocation 1 (TET1) gene, whereas curcumin-induced hypermethylation of SNCG may be realized through the upregulation of the DNA methyltransferase 3 (DNMT3) and the downregulation of TET1. Notably, miR-29b was found to be reversely expressed compared to TET1 in curcumin- and 5′-aza-CdR-treated cells, suggesting its involvement in the regulation of TET1. Overall, our results indicate that curcumin has an intrinsic dual function on DNA promoter methylation. We believe that curcumin may be considered a promising therapeutic option for treating TNBC patients in addition to preventing breast and ovarian cancer, particularly in cancer-free females harboring methylated BRCA1.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P&lt;0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

FLT1 hypermethylation is involved in polycyclic aromatic hydrocarbons-induced cell transformation

Coke oven emissions (COEs) are common particle pollutants in occupational environment and the major constituents of COEs are polycyclic aromatic hydrocarbons (PAHs). Previously, we identified aberrant methylation of the fms related tyrosine kinase 1 (FLT1) gene over the course of benzo(a)pyrene (BaP)-induced cell transformation via genome-wide methylation array. To quantify FLT1 methylation, we established a bisulfite pyrosequencing assay and examined the FLT1 hypermethylation in several human cancers. The results revealed that 70.0% (21/30 pairs) of lung cancers harbored hypermethylated FLT1 and concomitant suppression of gene expression compared to the adjacent tissues. This implies that FLT1 hypermethylation might play a role in malignant cell transformation. In addition, FLT1 hypermethylation and gene suppression appeared in primary human lymphocytes in a dose-response manner following COEs treatment. To explore whether FLT1 methylation is correlated with COEs exposure and DNA damage, we recruited 144 male subjects who had been exposed to high levels of COEs and 84 male control subjects. Notably, the FLT1 methylation in peripheral blood lymphocytes (PBLCs) of the COEs-exposed group (19.8 ± 3.2%) was enhanced by 17.9% compared to that of the control group (16.8 ± 2.8%) (P < 0.001). The FLT1 methylation status was positively correlated with urinary 1-hydroxypyrene (1-OHP) levels, an internal exposure marker of PAHs (β = 0.029, 95% CI = 0.010–0.048, P = 0.003) and positively correlated with DNA damage (βOTM = 0.024, 95% CI = 0.007–0.040, P = 0.005; βTail DNA = 0.035, 95% CI = 0.0017–0.054, P < 0.001) indicated by comet assay. Taken together, these findings indicate that FLT1 might be a tumor suppressor, and its hypermethylation might contribute to PAHs-induced carcinogenicity.

SOX11 hypermethylation as a tumor biomarker in endometrial cancer

We previously reported that SOX4 is overexpressed in endometrial cancer and that it partially contributes to hypermethylation of miR-129-2 and miR-203. The current study seeks to identify methylation and expression levels of the SOX gene family in endometrial carcinomas. Methylation levels of the 16 SOX gene family members were measured by combining bisulfite restriction analysis (COBRA), MassARRAY, and pyrosequencing assays of cell lines and endometrial cancer samples. Gene expression was determined by RT-qPCR. The methylation level of the SOX11 locus was correlated with clinicopathologic factors in primary endometrial tumors and in TCGA endometrial cohort. It was also examined in DNA of serum and endometrial specimens from a longitudinal cohort of early stage endometrial cancer patients. COBRA assays indicated that hypermethylation of SOX1, SOX2, SOX11, SOX14, SOX15, SOX17, and SOX18 was present in endometrial cancer cell lines and not in the normal control. SOX11 expression was reactivated only by a DNA methylation inhibitor. Moreover, aberrant DNA methylation of SOX11 was detected in the majority of endometrioid endometrial carcinomas (n=114) and none of the 22 adjacent normal endometrial samples (P<0.0001). The methylation status of SOX11 associated significantly with microsatellite instability and MLH1 methylation in endometrial tumors (P<0.0001), and this finding was validated in TCGA endometrial cohort. Furthermore, SOX11 was not hypermethylated in serum DNA from early stage endometrial cancer patients. This study found that hypermethylation of SOX11 is common in endometrial carcinomas and strongly associates with microsatellite instability and MLH1 methylation.

Methylation of BRCA1 and MGMT genes in white blood cells are transmitted from mothers to daughters

BackgroundConstitutive methylation of tumor suppressor genes are associated with increased cancer risk. However, to date, the question of epimutational transmission of these genes remains unresolved. Here, we studied the potential transmission of BRCA1 and MGMT promoter methylations in mother-newborn pairs.MethodsA total of 1014 female subjects (cancer-free women, n = 268; delivering women, n = 295; newborn females, n = 302; breast cancer patients, n = 67; ovarian cancer patients, n = 82) were screened for methylation status in white blood cells (WBC) using methylation-specific PCR and bisulfite pyrosequencing assays. In addition, BRCA1 gene expression levels were analyzed by quantitative real-time PCR.ResultsWe found similar methylation frequencies in newborn and adults for both BRCA1 (9.9 and 9.3%) and MGMT (12.3 and 13.1%). Of the 290 mother-newborn pairs analyzed for promoter methylation, 20 mothers were found to be positive for BRCA1 and 29 for MGMT. Four mother-newborn pairs were positive for methylated BRCA1 (20%) and nine pairs were positive for methylated MGMT (31%). Intriguingly, the delivering women had 26% lower BRCA1 and MGMT methylation frequencies than those of the cancer-free female subjects. BRCA1 was downregulated in both cancer-free woman carriers and breast cancer patients but not in newborn carriers. There was a statistically significant association between the MGMT promoter methylation and late-onset breast cancers.ConclusionsOur study demonstrates that BRCA1and MGMT epimutations are present from the early life of the carriers. We show the transmission of BRCA1 and MGMT epimutations from mother to daughter. Our data also point at the possible demethylation of BRCA1and MGMT during pregnancy.

Pyrosequencing analysis of methylation levels of clock genes in leukocytes from Parkinson’s disease patients

DNA methylation of neuronal PAS domain protein 2 (NPAS2) and cryptochrome circadian clock 1 (CRY1) promoters may be associated with Parkinson’s disease (PD). However, there is no simple and cost-effective method to quantify DNA methylation in these regions. Additionally, it is not clear whether DNA methylation of NPAS2 and CRY1 promoters is altered in peripheral blood of PD patients, especially newly diagnosed drug-naïve PD patients, and thus can be used as a PD biomarker. In the present study, we utilized bisulfite pyrosequencing assays to examine DNA methylation levels of six CpG sites in the NPAS2 promoter and five CpG sites in the CRY1 promoter. We compared DNA methylation levels at these sites in leukocytes from 80 medicated PD patients, 30 drug-naïve PD patients, and 80 healthy controls. Our results indicate that NPAS2 hypomethylation occurs at the early stage of PD and is a moderate biomarker for distinguishing PD patients from healthy subjects.

Association of etheno-DNA adduct and DNA methylation level among workers exposed to diesel engine exhaust

Objective: To investigate the association between etheno-DNA adduct and the promoter of DNA methylation levels of cyclin dependent kinase inhibitor 2A (P16), Ras association domain family 1 (RASSF1A) and O-6-methylguanine-DNA methyltransferase (MGMT) in workers with occupational exposure to diesel engine exhaust (DEE). Methods: We recruited 124 diesel engine testing workers as DEE exposure group and 112 water pump operator in the same area as control group in Henan province in 2012 using cluster sampling. The demographic data were obtained by questionnaire survey; urine after work and venous blood samples were collected from each subject. The urinary etheno-DNA adducts were detected using UPLC-MS/MS, including 1,N6-etheno-2'-deoxyadenosine (εdA) and 3,N4-etheno-2'-deoxycytidine(εdC). The DNA methylation levels of P16, RASSF1A, and MGMT were evaluated using bisulfite-pyrosequencing assay. The percentage of methylation was expressed as the 5-methylcytosine (5mC) over the sum of cytosines (%5mC). Spearman correlation and multiple linear regression were applied to analyze the association between etheno-DNA adducts and DNA methylation of P16, RASSF1A, and MGMT. Results: The median (P(25)-P(75)) of urinary εdA level was 230.00 (98.04-470.91) pmol/g creatinine in DEE exposure group, and 102.10 (49.95-194.48) creatinine in control group. The level of εdA was higher in DEE exposure group than control group (P<0.001). DNA methylation levels of P16, RASSF1A and MGMT were 2.04±0.41, 2.19 (1.94-2.51), 2.22 (1.94-2.46)%5mC in exposure group, and 2.19±0.40, 2.41 (2.11-2.67), 2.44 (2.15-2.91)%5mC in control group. DNA methylation levels were lower in exposure group (P values were 0.005, 0.002 and 0.001, respectively). Spearman correlation analysis showed that DNA methylation levels of P16, RASSF1A, and MGMT were negative associated with urinary εdA level (r values were -0.155, -0.137, and -0.198, respectively, P<0.05). No significant correlation was observed between the εdC level and any measured DNA methylation levels (P>0.05) . Multiple linear regression confirmed the negative correlation between εdA and DNA methylation levels of P16, RASSF1A, and MGMT in non-smoking group (β (95%CI) was -0.068 (-0.132--0.003), -0.082 (-0.159--0.004) and -0.048 (-0.090--0.007), P values were 0.039, 0.039 and 0.024, respectively). Moreover, εdC was negative associated with DNA methylation level of MGMT in non-smoking group (β (95%CI) was -0.094 (-0.179--0.008), P=0.032). Conclusion: DEE exposure could induce the increased of εdA and decreased of DNA methylation levels of P16, RASSF1A and MGMT.

TRIM36 hypermethylation is involved in polycyclic aromatic hydrocarbons-induced cell transformation

Long term exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with the increasing risk of lung cancer. To identify differentially hypermethylated genes associated with PAHs-induced carcinogenicity, we performed genome-wide DNA methylation analysis in 20 μM benzo(a)pyrene (BaP)-transformed human bronchial epithelial (HBE) cells at different stages of cell transformation. Several methylated genes (CNGA4, FLT1, GAREM1, SFMBT2, TRIM36) were differentially hypermethylated and their mRNA was suppressed in cells at both pre-transformed and transformed stages. Similar results were observed in HBE cells transformed by 20 μg/mL coke oven emissions (COEs) mixture collected from a coking manufacturing facility. In particular, hypermethylation of TRIM36 and suppression of TRIM36 expression were gradually enhanced over the time of COEs treatment. We developed bisulfite pyrosequencing assay and assessed TRIM36 methylation quantitatively. We found that hypermethylation of TRIM36 and reduced gene expression was prevalent in several types of human cancers. TRIM36 hypermethylation appeared in 90.0% (23/30) of Non-Small Cell Lung Cancer (NSCLCs) tissues compared to their paired adjacent tissues with an average increase of 1.32 fold. Furthermore, an increased methylation rate (5.90% v.s 7.38%) and reduced levels of TRIM36 mRNA were found in peripheral lymphocytes (PBLCs) of 151 COEs-exposed workers. In all subjects, TRIM36 hypermethylation was positively correlated with the level of urinary 1-hydroxypyrene (P < 0.001), an internal exposure marker of PAHs, and the DNA damage (P = 0.013). These findings suggest that aberrant hypermethylation of TRIM36 might be involved in the acquisition of malignant phenotype and could be served as a biomarker for risk assessment of PAHs exposure.

Detection of Turner syndrome using X-chromosome inactivation specific differentially methylated CpG sites: A pilot study

BackgroundEarly diagnosis of Turner syndrome (TS) may improve preventive measures and treatment. X-chromosome inactivation specific differentially methylated CpG sites (XIDMSs) that are high methylated in inactive X chromosomes (Xi) and unmethylated in active X chromosomes (Xa) may be potential makers for TS detection. MethodsThe candidate XIDMSs were screened from 9 male and 12 female DNA samples with normal karyotypes using the Illumina 450k array and validated by bisulfite sequencing PCR and pyrosequencing assay. X chromosome dosage was calculated according to the methylation level of multiple XIDMSs. ResultsOverall, 108 candidate XIDMSs were screened by the 450k array. Validations indicated that XIDMSs gathered and formed the X-chromosome inactivation specific differentially methylated regions (XIDMRs). Using 3 XIDMRs at SAT1, UXT and UTP14A loci, 36 TS, 22 normal female and 6 male samples were analyzed. Methylation levels of the 20 XIDMSs in the XIDMRs could distinguish between TS and normal female DNA samples, the X chromosome dosage was consistent with karyotyping data. Analyzing samples of 2 triple X syndrome and 3 Klinefelter syndrome patients suggested that this method could be used to detect X chromosome aneuploids other than TS. ConclusionsXIDMSs are widely spread along the X chromosome and might be effective markers for detection of TS and other X chromosome aneuploids.

RIP140 down-regulation alleviates acute lung injury via the inhibition of LPS-induced PPARγ promoter methylation

Seriously inflammatory response of the lungs can induce acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) which are serious public health threats due to their high patient morbidity and mortality. While RIP140 is known to modulate proinflammatory cytokine production during an inflammatory response, its role in ALI/ARDS is unclear. In this study, we examined RIP140 and PPARγ protein expression in RAW 264.7 cells and lung tissue following LPS-induced ALI. RIP140 shRNA adenoviral knockdown significantly elevated PPARγ expression, inhibited TNF-α, IL-1β, and IL-6 production in vivo and in vitro. Conversely, treatment with a PPARγ antagonist (GW9662) reversed these outcomes. Furthermore, co-IP showed that endogenous and exogenous RIP140 interacted with DNMT3b in RAW 264.7 cells. Bisulfite conversion, pyrosequencing and activity assays demonstrated that PPARγ promoter methylation levels were increased and that PPARγ transcriptional activity was inhibited following LPS treatment in macrophages. Nevertheless, RIP140 knockdown reduced PPARγ promoter methylation levels and restored its transcriptional activity. These results indicate that RIP140 knockdown can inhibit the production of inflammation mediators and remit ALI via the repression of DNMT3b mediated PPARγ promoter methylation.

Associations between DNA methylation in DNA damage response-related genes and cytokinesis-block micronucleus cytome index in diesel engine exhaust-exposed workers.

Recently, diesel engine exhaust (DEE) was reclassified as a known carcinogen to humans. DNA methylation alterations in DNA damage response (DDR)-related genes have the potential to affect DEE exposure-related cancer risk. However, the evidence regarding the association between DEE exposure and methylation alterations in DDR-related genes is limited. In 117 DEE-exposed workers and 112 non-DEE-exposed workers, we measured urinary concentrations of six mono-hydroxylated polycyclic aromatic hydrocarbons (OH-PAHs). We also determined the methylation levels of three DDR-related genes (p16, RASSF1A, and MGMT) and LINE-1 by bisulfite-pyrosequencing assay. We found that DEE-exposed workers exhibited significantly lower mean promoter methylation levels of p16, RASSF1A, and MGMT than non-DEE-exposed workers (all p<0.001). In all study subjects and non-smoking workers, increasing quartiles of urinary summed OH-PAHs was associated with hypomethylation of p16, RASSF1A, and MGMT (all p<0.05). In non-smoking workers, methylation in p16, RASSF1A, and MGMT decreased by 0.36% [95% confidential interval (CI): -0.60, -0.11%], 0.46% (95% CI: -0.79, -0.14%), and 0.55% (95% CI: -0.95, -0.15%), respectively, in association with highest versus lowest quartile of urinary summed OH-PAHs. In addition, p16, RASSF1A, MGMT, and LINE-1 methylation levels showed negative correlations with cytokinesis-block micronucleus cytome index which was previously measured in the same workers (all p<0.05). In conclusion, our results clearly indicated that DEE exposure and increased genetic damage were associated with hypomethylation of p16, RASSF1A, and MGMT. Future studies with larger sample size are needed to confirm these associations.

The Role of Dietary Extra Virgin Olive Oil and Corn Oil on the Alteration of Epigenetic Patterns in the Rat DMBA-Induced Breast Cancer Model.

Disruption of epigenetic patterns is a major change occurring in all types of cancers. Such alterations are characterized by global DNA hypomethylation, gene-promoter hypermethylation and aberrant histone modifications, and may be modified by environment. Nutritional factors, and especially dietary lipids, have a role in the etiology of breast cancer. Thus, we aimed to analyze the influence of different high fat diets on DNA methylation and histone modifications in the rat dimethylbenz(a)anthracene (DMBA)-induced breast cancer model. Female Sprague-Dawley rats were fed a low-fat, a high corn-oil or a high extra-virgin olive oil (EVOO) diet from weaning or from induction with DMBA. In mammary glands and tumors we analyzed global and gene specific (RASSF1A, TIMP3) DNA methylation by LUMA and bisulfite pyrosequencing assays, respectively. We also determined gene expression and enzymatic activity of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) and evaluated changes in histone modifications (H3K4me2, H3K27me3, H4K20me3 and H4K16ac) by western-blot. Our results showed variations along time in the global DNA methylation of the mammary gland displaying decreases at puberty and with aging. The olive oil-enriched diet, on the one hand, increased the levels of global DNA methylation in mammary gland and tumor, and on the other, changed histone modifications patterns. The corn oil-enriched diet increased DNA methyltransferase activity in both tissues, resulting in an increase in the promoter methylation of the tumor suppressor genes RASSF1A and TIMP3. These results suggest a differential effect of the high fat diets on epigenetic patterns with a relevant role in the neoplastic transformation, which could be one of the mechanisms of their differential promoter effect, clearly stimulating for the high corn-oil diet and with a weaker influence for the high EVOO diet, on breast cancer progression.

Abstract 3132: Suppressor microRNA-145 is epigenetically regulated by promoter hypermethylation in esophageal squamous cell carcinoma

Abstract Objectives: DNA methylation change is common epigenetic change in cancer. The promoter CpG island hypermethylation is one of the most common mechanisms by which tumor suppressor miRNAs are inactivated during tumorigenesis. However, microRNA (miRNA) regulations by epigenetic alteration, especially CpG island hypermethylation, have been not yet substantially understood in esophageal squamous cell carcinoma (ESCC). Therefore, we performed the screening of miRNAs which should be up-regulated after 5-aza-20-deoxycytidine (5-Aza) treatment in ESCC cell lines, then from up-regulated miRNAs, we focused on a suppressor miR-145-5p. Since miRNA-145-5p attracts an increasing attention as a tumor suppressor miRNA in ESCC, we evaluated miRNA-145 promoter methylation level and miRNA-145-5p expression level in ESCC Methods: The screening of miRNAs was analyzed using the Human miFinder 384HC miScript miRNA PCR Array. DNA methylation level was evaluated by using bisulfite-pyrosequencing assay. miRNA expression level was evaluated by using TaqMan quantitative real-time reverse transcription-polymerase chain reaction. miRNA-145-5p expression and its promoter methylation levels were quantified in 15 ESCC and matched 15 normal adjacent esophageal mucosa frozen tissues and in 105 formalin-fixed paraffin-embedded ESCC tissues between September 2009 and June 2012 at Kumamoto University Hospital (Kumamoto, Japan). Results Twenty miRNAs including miR-145-5p were up-regulated more than 3 fold in both TE-1 and KYSE30 after 5-Aza treatment by using miRNA PCR array analysis. After 5-Aza treatment, miRNA-145 promoter methylation level decreased and the expression level of miRNA-145-5p was significantly enhanced in 8 of 10 ESCC cell lines. The miRNA-145-5p expression levels in the cancer tissues (mean ± SD; 6.37 ± 1.47) was significantly lower than in matched normal adjacent esophageal epithelial mucosa (235.6 ± 61.9) (p = 0.0024). Moreover, the miRNA-145 promoter in the cancer tissues (41.4 ± 3.05) was significantly hyper methylated than in matched normal adjacent epithelial mucosa (mean; 57.1 ± 3.36) (p = 0.0042 by the paired t test). In 105 ESCC samples, the “high miRNA-145-5p expression level group” and “low miRNA-145-5p expression level group” experienced similar overall survival (Kaplan-Meier analysis; log rank p = 0.58) and disease-free survival (Kaplan-Meier analysis; log rank p = 0.80) Conclusions: Our results suggest that hypermethylation of the miRNA-145 promoter region might regulate miRNA-145 expression in ESCC. This is the first report on the epigenetic regulation of miRNA-145 in ESCC. However, further work is needed to assess our results and to make them more definite. Citation Format: kazuto harada, Yoshifumi Baba, Keisuke Kosumi, Ryuma Tokunaga, Daisuke Izumi, Mayuko Ouchi, Kennichi Nakamura, Yuki Kiyozumi, Junji Kurashige, Yukiharu Hiyoshi, Shiro Iwagami, Yuji Miyamoato, Yasuo Sakamoto, Naoya Yoshida, Masayuki Watanabe, Hideo Baba. Suppressor microRNA-145 is epigenetically regulated by promoter hypermethylation in esophageal squamous cell carcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3132. doi:10.1158/1538-7445.AM2015-3132

A Strategy for Accurate Quantification of 5-Methylcytosine and 5-Hydroxymethylcytosine at CpG Sites Within Gene Promoter.

5-Methylcytosine (5mC) can be converted to 5-hydroxymethylcytosine (5hmC) in mammalian DNA by the ten-eleven translocation (TET) enzymes. Traditional bisulfite-based DNA methylation analysis techniques have been widely used in the detection of 5mC. However, they can not discriminate 5hmC from 5mC, leading to overestimate 5mC levels. We here introduce a strategy, combination of selective oxidation and bisulfite pyrosequencing (BS-Pyroseq), for quantification of both 5mC and 5hmC at CpG sites within the promoters of CDH1, DAPK, RARβ and RUNX3 genes in a panel of cell lines and clinical samples. As expected, oxidative bisulfite pyrosequencing (oxBS-Pyroseq) assay decreased overall or site-specific methylation levels of three of these genes in most cell lines as compared with BS-Pyroseq assay. Similarly, decreased overall or site-specific methylation levels of DAPK, RARβ and RUNX3 genes in laryngeal, gastric and thyroid cancer and their matched normal tissues, respectively, were also found by a comparison between these two techniques, particularly in cancerous tissues. In addition, by using this combined strategy and hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) assay, we demonstrated that TET1 up-regulated DAPK expression through promoter demethylation. Collectively, this strategy is easy to establish and accurately discriminates and quantifies 5mC and 5hmC at CpG sites within selected gene promoters.

DNA Hypermethylation of SHISA3 in Colorectal Cancer: An Independent Predictor of Poor Prognosis.

Shisa3 is a novel tumor suppressor identified in lung cancer. However, its antitumor activity in other human cancers and the mechanism of gene inactivation remain unknown. SHISA3 expression was measured by reverse transcription-PCR (RT-PCR) and quantitative RT-PCR (RT-qPCR). DNA methylation was determined by bisulfite sequencing and pyrosequencing. Down-regulation of SHISA3 expression was observed in all of 11 colorectal cancer (CRC) cell lines and was further confirmed in 34 (65.4 %) of 52 colorectal carcinomas by RT-qPCR. Four of six CRC cell lines could restore SHISA3 expression after treatment with 5-aza-2'-deoxycytidine. Tumor-specific methylation of five CpG sites in the first intron of SHISA3 was identified by bisulfite sequencing, and their methylation levels were quantified in 127 pairs of primary CRC tissues by bisulfite pyrosequencing. The methylation levels of SHISA3 in tumors were noticeably higher than that in their matched normal mucosae. In addition, SHISA3 hypermethylation was significantly associated with an increased risk of disease recurrence in patients with stage II and III disease (P = 0.007) and was an independent predictor of poor overall survival [hazard ratio (HR) 2.9, 95 % confidence interval (CI) 1.5-5.8; P = 0.002] and disease-free survival (HR 4.0, 95 % CI 1.6-10.2; P = 0.003) of CRC patients. SHISA3 gene is epigenetically inactivated in a substantial fraction of CRC, and its hypermethylation is of prognostic significance in predicting clinical outcome. The quantitative bisulfite pyrosequencing assay established could be a cost-effective tool for providing a potential biomarker of adverse prognosis in CRC.

A B-cell epigenetic signature defines three biologic subgroups of chronic lymphocytic leukemia with clinical impact.

Prospective identification of patients with chronic lymphocytic leukemia (CLL) destined to progress would greatly facilitate their clinical management. Recently, whole-genome DNA methylation analyses identified three clinicobiologic CLL subgroups with an epigenetic signature related to different normal B-cell counterparts. Here, we developed a clinically applicable method to identify these subgroups and to study their clinical relevance. Using a support vector machine approach, we built a prediction model using five epigenetic biomarkers that was able to classify CLL patients accurately into the three subgroups, namely naive B-cell-like, intermediate and memory B-cell-like CLL. DNA methylation was quantified by highly reproducible bisulfite pyrosequencing assays in two independent CLL series. In the initial series (n=211), the three subgroups showed differential levels of IGHV (immunoglobulin heavy-chain locus) mutation (P<0.001) and VH usage (P<0.03), as well as different clinical features and outcome in terms of time to first treatment (TTT) and overall survival (P<0.001). A multivariate Cox model showed that epigenetic classification was the strongest predictor of TTT (P<0.001) along with Binet stage (P<0.001). These findings were corroborated in a validation series (n=97). In this study, we developed a simple and robust method using epigenetic biomarkers to categorize CLLs into three subgroups with different clinicobiologic features and outcome.

Pyrosequencing analysis of SNCA methylation levels in leukocytes from Parkinson's disease patients

DNA methylation of a CpG island located in intron 1 of the α-synuclein gene (SNCA) has been reported to play an essential role in the regulation of α-synuclein transcription, and probably has a close association with Parkinson's disease (PD). However, there is no simple and cost effective method to quantify DNA methylation in this region. Additionally, whether this CpG island is hypomethylated in peripheral blood in PD and can be used as PD biomarker is still under debate. In the present study, we developed a set of bisulfite pyrosequencing assays which can be used to examine the DNA methylation level of 13 CpG sites in intron 1 of SNCA. We compared DNA methylation levels at these sites in leukocytes from 50 PD patients and 50 healthy controls. Our results indicated that there were no significant differences in DNA methylation between PD patients and controls.

NOS1 Methylation and Carotid Artery Intima-Media Thickness in Children

Nitric oxide (NO) plays an important role in cardiovascular health by maintaining and regulating vascular tone and blood flow. Epigenetic regulation of NO synthase (NOS), the genes responsible for NO production, may affect cardiovascular disease, including the development of atherosclerosis in children. We measured percentage DNA methylation using bisulfite conversion and pyrosequencing assays on DNA from buccal cells provided by 377 participants of the Children's Health Study on whom carotid artery intima-media thickness (CIMT) measurements were also collected. We examined a total of 16 CpG loci located within NOS1, NOS2A, NOS3, ARG1, and ARG2 genes responsible for NO production. CIMT was measured using high-resolution B-mode carotid ultrasound. The association between percentage DNA methylation in ARG and NOS genes with CIMT was evaluated using linear regression adjusted for sex, ethnicity, body mass index, age at CIMT, town of residence, and experimental plate for pyrosequencing reactions. Differences in the association by ethnicity and ancestral group were also evaluated. For a 1% increase in average DNA methylation of NOS1, CIMT increased by 1.2 μm (P=0.02). This association was greater in Hispanic children of Native American descent (β=2.3; P=0.004) than in non-Hispanic whites (β=0.3; P=0.71) or Hispanic whites (β=1.0; P=0.35). DNA methylation of NOS1 has a plausible role in atherogenesis through regulation of NO production, although ancestry may alter the magnitude of this association.