The mitogen-activated protein kinase phosphatase-1 (MKP-1) gene is a potential methylation biomarker for malignancy of breast cancer

The mitogen-activated protein kinase (MAPK) phosphatase- 1 (MKP-1) belongs to the MAPK cascades which are central to cell proliferation and apoptosis. The carcinogenic role of MKP-1 has been reported in many types of cancer but it has rarely been investigated in breast cancer. The present study was designed to evaluate the MKP-1 mRNA expression and its possible regulation by methylation of MKP-1 promoter in the model of several breast cancer cell lines and tissues as well as controls. Our data demonstrate MKP-1 mRNA expression significantly decreased in five breast cancer cell lines compared to breast controls (P<0.01). Using the methylation-specific PCR (MSP) analysis, the unmethylated reaction (U) is dominant in both normal cell lines and benign breast tumors (100% vs. 86.2%), whereas the methylated reaction (M) is dominant in both breast cancer cell lines and invasive breast tumors (100% vs. 57.2%). In terms of methylation ratio (M/M+U), methylation level in MKP-1 promoter is significantly higher in the invasive breast tumor tissues (n = 152) than in benign breast tumor tissues (n = 29) (P<0.0001). Assessing the methylation ratio of the promoter of MKP-1 gene to diagnose the breast malignancy (invasive vs. benign), the area under the receiver- operating characteristic (ROC) curve was 0.809 (95% CI: 0.711-0.906, P<0.001). The best performance for this prediction has a sensitivity of 76.32% and a specificity of 82.76% at the cutoff value of 0.38. Taken together, we firstly demonstrated that the promoter methylation of MKP-1 gene is a potential breast cancer biomarker for breast malignancy.     

Epigenetic silencing of UBXN8 contributes to leukemogenesis in t(8;21) acute myeloid leukemia

The formation of the RUNX1-RUNX1T1 fusion protein, resulting from the t(8;21) translocation, is considered to be one of the initiating events of t(8;21) acute myeloid leukemia (AML). However, the mechanisms of the oncogenic mechanism of RUNX1-RUNX1T1 remain unclear. In this study, we found that RUNX1-RUNX1T1 triggers the heterochromatic silencing of UBXN8 by recognizing the RUNX1-binding sites and recruiting chromatin-remodeling enzymes to the UBXN8 promoter region. Decitabine, a specific inhibitor of DNA methylation, upregulated the expression of UBXN8 in RUNX1-RUNX1T1⁺ AML cell lines. Overexpression of UBXN8 inhibited the proliferation and colony-forming ability of and promoted cell cycle arrest in t(8;21) AML cell lines. Enhancing UBXN8 levels can significantly inhibit tumor proliferation and promote the differentiation of RUNX1-RUNX1T1⁺ cells in vivo. In conclusion, our results indicated that epigenetic silencing of UBXN8 via methylation of its promoter region mediated by the RUNX1-RUNX1T1 fusion protein contributes to the leukemogenesis of t(8;21) AML and that UBXN8 targeting may be a potential therapeutic strategy for t(8;21) AML.Subject terms: Acute myeloid leukaemia, Tumour heterogeneity     

Genome-scale DNA methylation pattern profiling of human bone marrow mesenchymal stem cells in long-term culture

Human bone marrow mesenchymal stem cells (MSCs) expanded in vitro exhibit not only a tendency to lose their proliferative potential, homing ability and telomere length but also genetic or epigenetic modifications, resulting in senescence. We compared differential methylation patterns of genes and miRNAs between early-passage [passage 5 (P5)] and late-passage (P15) cells and estimated the relationship between senescence and DNA methylation patterns. When we examined hypermethylated genes (methylation peak ≥ 2) at P5 or P15, 2,739 genes, including those related to fructose and mannose metabolism and calcium signaling pathways, and 2,587 genes, including those related to DNA replication, cell cycle and the PPAR signaling pathway, were hypermethylated at P5 and P15, respectively. There was common hypermethylation of 1,205 genes at both P5 and P15. In addition, genes that were hypermethylated at P5 (CPEB1, GMPPA, CDKN1A, TBX2, SMAD9 and MCM2) showed lower mRNA expression than did those hypermethylated at P15, whereas genes that were hypermethylated at P15 (MAML2, FEN1 and CDK4) showed lower mRNA expression than did those that were hypermethylated at P5, demonstrating that hypermethylation at DNA promoter regions inhibited gene expression and that hypomethylation increased gene expression. In the case of hypermethylation on miRNA, 27 miRNAs were hypermethylated at P5, whereas 44 miRNAs were hypermethylated at P15. These results show that hypermethylation increases at genes related to DNA replication, cell cycle and adipogenic differentiation due to long-term culture, which may in part affect MSC senescence.     

The role of promoter methylation in Epstein-Barr virus (EBV) microRNA expression in EBV-infected B cell lines

Epstein-Barr virus (EBV) microRNAs (miRNAs) are expressed in EBV-associated tumors and cell lines, but the regulation mechanism of their expression is unclear yet. We investigated whether the expression of EBV miRNAs is epigenetically regulated in EBV-infected B cell lines. The expression of BART miRNAs was inversely related with the methylation level of the BART promoter at both steady-state and following 5-aza-2'-deoxycytidine treatment of the cells. The expression of BHRF1 miRNAs also became detectable with the demethylation of Cp/Wp in latency I EBV-infected cell lines. Furthermore, in vitro methylation of the BART and Cp promoters reduced the promoter-driven transactivation. In contrast, tricostatin A had little effect on the expression of EBV miRNA expression as well as on the BART and Cp/Wp promoters. Our results suggest that promoter methylation, but not histone acetylation, plays a role in regulation of the EBV miRNA expression in EBV-infected B cell lines.     

Inactivation patterns of p16/INK4A in oral squamous cell carcinomas

The p16/INK4A is one of the major target genes in carcinogenesis and its inactivation has frequently been reported in other types of tumors. The purpose of this study is to evaluate inactivation patterns of p16/INK4A in oral squamous cell carcinoma. Six different oral cancer cell lines, SCC-4, SCC-9, SCC-15, SCC-25, KB, and SNUDH- 379 were examined for inactivation of p16/INK4A genes. In the analysis of p16/INK4A gene inactivation, PCR amplification, direct sequencing, and methylation-specific PCR methods were adopted for evaluation of homozygous deletion, point mutation, and promoter hypermethylation, respectively. Homozygous deletion was detected in SCC-25 and SCC-9. SCC-15 showed hypermethylated promoter region within p16/INK4A gene. It is suggestive in the present study that inactivation patterns of p16/INK4A were mainly homozygous deletion, promoter methylation rather than point mutation in oral squamous cancer cell lines, so treatment modalities of oral squamous cell carcinoma should be focused on these types of inactivation.     

DERL3 functions as a tumor suppressor in gastric cancer

BackgroundGastric cancer is a common malignant tumor in the clinic with a high mortality rate, ranking the first among malignant tumors of the digestive system. Early gastric cancer exhibits no specific clinical symptoms and signs, and most of the patients were diagnosed as advanced gastric cancer. The prognosis is poor, and the 5-year overall survival rate is still lower than 30%, seriously threatening people’s life and health. However, the pathogenesis of gastric cancer is still unclear.MethodsThis study aimed to identify methylated differentially expressed genes in gastric cancer and to study the cellular functions and pathways that may be involved in its regulation, as well as the biological functions of key methylated differentially expressed genes. The gene expression data set and methylation data set of gastric cancer genes based on TCGA were analyzed to identify prognostic methylated genes.ResultsThis study showed that the methylation of the DERL3 promoter was correlated with the clinical analysis of tumors. Further studies were conducted on genes co-expressed with DERL3, whose functions and pathways to inhibit gastric cancer were adaptive immune response, T cell activation, immune response-regulating pathway, cell surface on molecules, and natural killer cell-mediated cytotoxicity. Finally, cell proliferation assay, cell scratch assay, and cell invasion assay confirmed that DERL3 as a tumor suppressor gene inhibited the malignant evolution of gastric cancer.ConclusionsThe analysis of key methylated differentially expressed genes helped elucidate the epigenetic regulation mechanism in the development of gastric cancer. DERL3, as a methylation biomarker, has a predictive and prognostic value in the accurate diagnosis and treatment of gastric cancer and provides potential targets for the precision treatment of gastric cancer.Trial RegistrationNot applicable.     

Advent of the cancer methylome

DNA hypermethylation of CpG islands plays an important role in gene regulation during cancer development. Many techniques have been developed to detect global DNA methylation in cancer cells compared to normal tissues. This knowledge helps us to better understand cancer progression and also aids in the development of new biomarker for early cancer detection. New prognostic tools for monitoring drug efficacy during cancer treatment can also be developed. In this review, we will examine the different techniques that have been used to study DNA methylation, as well as the emerging high resolution, high throughput techniques for identification of methylated regions to defining cancer related genes in the cancer methylome.     

DNA Methylation: Site-Specific Methylations Cause Gene Inactivation

In the molecular biology of eukaryotic organisms, the elucidation of mechanisms involved in the regulation of gene expression has assumed an important role. All cells of an organism carry the same genes, but differ in the patterns of genes they express. There is an increasing amount of evidence that cancer cells exhibit a pattern of gene expression which can be very different from that of normal cells. One of the molecular signals that has been recognized in the regulation of gene expression in eukaryotes is the modified nucleotide 5-methylcytosine (5-mC). Through experiments in well-characterized eukaryotic systems, evidence has been adduced that the introduction of 5-mC into highly specific sequences, particularly into the 5′ and promoter regions of a gene, can cause gene inactivation. Viral and other eukaryotic systems have helped in the recognition of this cause-and-effect relationship. Inactive genes are frequently hypermethylated in the promoter region; active genes are hypomethylated. However, these correlations are not always as simple and straightforward. The biochemical mechanisms by which site-specific DNA methylations cause gene inactivation have not yet been determined. It is plausible to postulate that promoter methylations could somehow affect the binding of cellular enzymes involved in recognizing the promoter of a gene. Structural alterations of DNA promoter sequences arising from DNA methylations could also be important. DNA methylation is likely to represent a long-term inactivation signal, since it is presently thought that patterns of DNA methylation can be changed only by DNA replication and specific inhibition of post-replicative maintenance methylation.     

Differential promoter methylation may be a key molecular mechanism in regulating BubR1 expression in cancer cells

The BubR1 mitotic-checkpoint protein monitors proper attachment of microtubules to kinetochores, and links regulation of chromosome-spindle attachment to mitotic-checkpoint signaling. Thus, disruption of BubR1 activity results in a loss of checkpoint control, chromosomal instability caused by a premature anaphase, and/or the early onset of tumorigenesis. The mechanisms by which deregulation and/or abnormalities of BubR1 expression operate, however, remain to be elucidated. In this study, we demonstrate that levels of BubR1 expression are significantly increased by demethylation. Bisulfite sequencing analysis revealed that the methylation status of two CpG sites in the essential BubR1 promoter appear to be associated with BubR1 expression levels. Associations of MBD2 and HDAC1 with the BubR1 promoter were significantly relieved by addition of 5-aza-2'-deoxycytidine, an irreversible DNA methyltransferase inhibitor. However, genomic DNA isolated from 31 patients with colorectal carcinomas exhibited a +84A/G polymorphic change in approximately 60% of patients, but this polymorphism had no effect on promoter activity. Our findings indicate that differential regulation of BubR1 expression is associated with changes in BubR1 promoter hypermethylation patterns, but not with promoter polymorphisms, thus providing a novel insight into the molecular regulation of BubR1 expression in human cancer cells.     

PTEN/MMAC1 enhances the growth inhibition by anticancer drugs with downregulation of IGF-II expression in gastric cancer cells

PTEN/MMAC1 is a tumor suppressor gene that is mutated in a variety of advanced and metastatic cancers. Its major function is likely to be the phosphatase activity that regulates the phosphotidylinositol (PI)3-kinase/Akt pathway. On the other hand, IGF system plays an important role in cell proliferation and cell survival via PI3-kinase/Akt and mitogen-activated protein kinase pathways in many cancer cells. To evaluate effect of PTEN on cell growth and IGF system in gastric cancer, human gastric adenocarcinoma cells (SNU-5 & -216) were transfected with human PTEN cDNA. Those PTEN- transfected gastric cancer cells had a lower proliferation rate than the pcDNA3-transfected cells. PTEN overexpression induced a profound decrease in the IGF-II and IGF-IR expression levels, and downregulation of IGF-II expression by PTEN was mediated through the regulation of the IGF-II promoter. In addition, a PI3-kinase inhibitor, LY294002, induced the downregulation of IGF-II expression. The PTEN-overexpressing SUN-5 and -216 cells were more sensitive to death induced by etoposide and adriamycin that induce DNA damage than the pcDNA3-transfected cells. These findings suggest that PTEN suppresses the cell growth through modulation of IGF system and sensitizing cancer cells to cell death by anticancer drugs.     

污染水产去甲基化表观遗传毒性EGFP评价方法

pEGFP-C3质粒经过体外人工甲基化处理后,被转染进入HepG2细胞以构建重组细胞株．以5-AZA为阳性去甲基化毒物与重组细胞共培养,通过亚硫酸氢钠测序法定量检测EGFP基因启动子区甲基化状态,通过实时定量PCR检测EGFP基因表达,借助流式细胞术和荧光摄片定量检测共培养细胞的绿色荧光强度,在DNA甲基化、EGFP基因mRNA表达、GFP蛋白等多个层次研究5-AZA染毒处理与其去甲基化能力和荧光表达改变的响应关系．对天津污染水产的去甲基化能力进行了实际样品测试．结果表明,5-AZA与重组细胞的DNA甲基化、基因表达、蛋白产物变化之间存在显著关联,具有较低的检出浓度和良好的重复性．天津污染海域的水产去甲基化能力较强．本文初步建立了一种污染物去甲基化表观遗传毒性评价方法．     

Screening of exon methylation biomarkers for colorectal cancer via LC‐MS/MS strategy

The identification of biomarkers would be of benefit for the diagnosis and treatment of colorectal cancer. DNA methylation in specific genomic regions, which had shown strongly association with disease genotypes, was an effective indicator to reveal the occurrence and development of cancers. To screen out methylation biomarkers for colorectal cancer (CRC), genomic DNA was isolated from colorectal cancerous and corresponding cancer‐adjacent tissues collected from 30 CRC patients and then bisulfite‐converted. The exon regions of 5 targeted genes (CNRIP1 , HIC1 , RUNX3 , p15 , and SFRP2 ) were amplified by using nested polymerase chain reaction with specific primers, and the amplicon was purified and hydrolyzed. The methylation levels of these specific regions were detected by liquid chromatography tandem mass spectrometry (LC‐MS/MS). The results showed that 5 targeted exon regions were successfully amplified and confirmed by sequencing. The methodological validations indicated that LC‐MS/MS was highly sensitive and accurate. The methylation levels of CNRIP1 and RUNX3 were remarkably high in CRC tissues with statistical difference when compared with corresponding cancer‐adjacent individuals, while that of HIC1 , p15 , and SFRP2 had no difference between 2 subjects. These findings supported CNRIP1 and RUNX3 as potential DNA methylation biomarkers for CRC diagnosis and treatment, and our LC‐MS/MS approach exhibited great advantages in the identification of regional DNA methylation biomarkers.     

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.