BACKGROUND: Disabled-2 (Dab2) is known as a tumor suppressor as well as a Wnt pathway inhibitor. We previously reported that Dab2 was down-regulated due to gene promoter hypermethylation in lung cancer. Here, we aim to study if X-ray irradiation can induce de-methylation of the Dab2 gene and subsequently up-regulate its expression, and also to attempt to suppress the malignant biological behavior of and enhance the radiosensitivity in lung cancer cells with hypermethylation of the Dab2 gene.
METHODS: Immunostaining was performed to investigate the relationship between Dab2 expression and lung cancer clinicopathological characteristics. Bisulfite sequencing PCR (BSP) was used to evaluate the methylation status of lung cancer cells with or without X-ray treatment. Real-time PCR and western Blot were performed to investigate the expression of Dab2, Wnt pathway factors, DNMTs and methyl CpG binding protein 2 (MeCP2). Colony Formation, matrigel invasion and xenograft experiment were performed to evaluate the malignant biological behavior of lung cancer cells with irradiation.
RESULTS: The result of immunostaining of Dab2 in lung cancer tissues showed that decreased Dab2 expression was positively correlated with poor differentiation, lymph node metastasis, advanced TNM stage and poor prognosis. X-ray treatment significantly up-regulated Dab2 expression and inhibited Wnt factors in LK2 cells (with hypermethylation of the Dab2 gene promoter, P < 0.05), but not in SPC-A-1 cells (with hypomethylation of the Dab2 gene promoter); however, the effect could be reversed by Dab2 or Axin knockdown (P < 0.05). Decreased expression of DNMT1, DNMT3b and MeCP2 could be detected in both LK2 and SPC-A-1 cells compared to non-irradiated cells (P < 0.05). Both in vitro studies and in vivo xenograft tumor growth demonstrated that X-ray could significantly inhibit the proliferation and invasion of LK2 but not SPC-A-1 cells (P < 0.05).
CONCLUSION: In general, X-ray-induced up-regulation of Dab2 and inhibition of the Wnt pathway may be mediated by de-methylation of a hypermethylated Dab2 gene promoter. X-ray treatment significantly inhibits proliferation and invasion of lung cancer cells with hypermethylation of the Dab2 gene promoter, but is less effective in lung cancer cells with hypomethylation of the Dab2 gene promoter. These results indicate that the methylation status of the Dab2 gene promoter might be a potential predictor of the radiosensitivity of lung cancer cells.

BACKGROUND: Colorectal cancer (CRC) is one of the most common digestive malignant tumors, and DMTN is a transcriptionally differentially expressed gene that was identified using CRC mRNA sequencing data from The Cancer Genome Atlas (TCGA). Our preliminary work suggested that the expression of DMTN was downregulated in CRC, and the Rac1 signaling pathway was significantly enriched in CRC tissues with low DMTN expression. However, the specific functions and underlying molecular mechanisms of DMTN in the progression of CRC and the upstream factors regulating the downregulation of the gene remain unclear.
METHODS: DMTN expression was analyzed in CRC tissues, and the relationship between DMTN expression and the clinicopathological parameters was analyzed. In vitro and in vivo experimental models were used to detect the effects of DMTN dysregulation on invasion and metastasis of CRC cells. GSEA assay was performed to explore the mechanism of DMTN in invasion and metastasis of CRC. Westernblot, Co-IP and GST-Pull-Down assay were used to detect the interaction between DMTN and ARHGEF2, as well as the activation of the RAC1 signaling. Bisulfite genomic sequence (BSP) assay was used to test the degree of methylation of DMTN gene promoter in CRC tissues.
RESULTS: We found that the expression of DMTN was significantly decreased in CRC tissues, and the downregulation of DMTN was associated with advanced progression and poor survival and was regarded as an independent predictive factor of CRC patient prognosis. The overexpression of DMTN inhibited, while the knockdown of DMTN promoted, invasion and metastasis in CRC cells. Moreover, hypermethylation and the deletion of DMTN relieved binding to the ARHGEF2 protein, activated the Rac1 signaling pathway, regulated actin cytoskeletal rearrangements, and promoted the invasion and metastasis of CRC cells.
CONCLUSION: Our study demonstrated that the downregulation of DMTN promoted the metastasis of colorectal cancer cells by regulating the actin cytoskeleton through RAC1 signaling activation, potentially providing a new therapeutic target to enable cancer precision medicine for CRC patients.

BACKGROUND/AIMS: Choriocarcinoma (CC) is a highly aggressive gestational trophoblastic neoplasia; however, the underlying molecular mechanisms of its invasiveness and metastasis remain poorly understood. Human secreted frizzled-related protein 2 (SFRP2) could function as a tumor promoter or suppressor in different tumors, yet the role it plays in CC's invasion and metastasis is thoroughly unclear. The current study was aimed to explore the function and underlying mechanism of SFRP2 in CC.
METHODS: The expression of SFRP2 in CC tissues was examined via immunohistochemistry. The methylation level and expression of SFRP2 in CC cell lines, JEG-3 and JAR were examined via bisulfite sequencing PCR (BSP), western blotting and quantitative RT-PCR. The biological role of increasing expressed SFRP2 through its promoter demethylation with 5-Aza-2'-deoxycytidine (5-Aza) was examined by a series of in vitro functional studies. Furthermore, lentivirus transfection technology was adopted to investigate the biological roles of SFRP2 knockdown in JEG-3 and JAR cells in vitro and in vivo. Moreover, its downstream signaling pathway was investigated.
RESULTS: SFRP2 was downregulated in CC tissues, and its expression was inversely related to its promoter hypermethylation frequency in JEG-3 and JAR cells. Increased SFRP2 through its promoter demethylation inhibited cell migration, invasion and colony formation in JEG-3 and JAR cells, whereas decreased SFRP2 reversed the epithelial-mesenchymal transition (EMT) process and stemness in JEG-3 and JAR cells both in vitro and vivo. Mechanistically, SFRP2 regulated the EMT and stemness of CC cell lines via canonical Wnt/β-catenin signaling, validated by the usage of a Wnt activator and inhibitor.
CONCLUSION: The current study indicates that downregulated SFRP2 has potent tumor-promotive effects in CC through the modulation of cancer stemness and the EMT phenotype via activation of Wnt/β-catenin signaling in vitro and in vivo.

A disintegrin and metalloproteinase domain 33 (ADAM33) gene is a transmembrane glycoprotein that mediates changes in cell adhesion and plays an important role in cancer progression. Since bisphenol A (BPA) and phthalates are epigenetically toxic, the purpose of this study was to examine whether BPA and phthalate metabolites, including monoethyl phthalate (MEP), mono‑n‑butyl phthalate (MBP), mono‑isobutyl phthalate (MIBP), mono(2‑ethylhexyl) phthalate (MEHP), mono(2‑ethyl‑5‑hydroxyhexyl) phthalate (MEHHP), mono(2‑ethyl‑5‑carboxypentyl) phthalate (MECPP), and mono(2‑ethyl‑5‑oxohexyl) phthalate (MEOHP), have an epigenetic impact on ADAM33 and the incidence of breast cancer. CpG islands of breast cancer microarray datasets obtained from the Gene Expression Omnibus (GEO) were used to assess the ADAM33 methylation profile. We designed a case‑control study including 44 cases and 22 age‑matched controls to detect the methylation status of intron 1 in ADAM33 from peripheral blood mononuclear cells (PBMCs) in blood, using BSP, nested PCR, and bisulfite sequencing, and measured the in vivo gene expression of ADAM33 and the urinary concentrations of endocrine‑disrupting chemicals (EDCs), using real‑time PCR, high‑performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC‑MS). Only one dataset, GSE32393, reached significance (P=0.016). ADAM33 expression and methylation frequencies at CpG site 3 in intron 1 were higher in the control group. We found a positive association between intron 1 methylation level and ADAM33 expression as well as urinary concentrations of MEHHP, MECPP, MEOHP and Σ4MEHP (the sum of MEHP, MECPP, MEHHP, and MEOHP) in the cases. This study suggests that metabolites of phthalate such as MEHHP, MECPP, MEOHP and Σ4MEHP may increase the intron 1 methylation level to elevate ADAM33 gene expression and have a protective effect on reducing the risk of breast cancer.

Herein we evaluated the genotoxic effects of BnSP-6, a Lys-49 phospholipase A

OBJECTIVE: Primordial odontogenic tumour (POT) is a rare benign mixed epithelial and mesenchymal odontogenic tumour. POT is composed of dental papilla-like tissue covered with cuboidal to columnar epithelium that resembles to inner and outer enamel epithelium of the enamel organ without dental hard tissue formation. The aim of this study was to examine pathogenesis of POT based on tumourigenesis and odontogenesis.
SUBJECTS AND METHODS: Six cases of POT were submitted for study. DNA analysis and transcriptome analysis were performed by next-generation sequencing. Expression of amelogenin, ameloblastin and dentin sialophosphoprotein (DSPP) was examined by immunohistochemistry.
RESULTS: There were no gene mutations detected in any of analysed 151 cancer- and 42 odontogenesis-associated genes. Enamel protein-coding genes of Amelx, Ambn and Enam, and dentin protein-coding genes of Col1a1, Dspp, Nes and Dmp1 were expressed, whereas expression of dentinogenesis-associated genes of Bglap, Ibsp and Nfic was negative or very weak suggesting inhibition of dentin formation in POT after odontoblast differentiation. Immunoreactivity of amelogenin, ameloblastin and DSPP was detected in POT.
CONCLUSIONS: Pathogenesis of POT is considered to be genetically different from other odontogenic tumours. It is suggested that inhibition of enamel and dentin formation in POT is due to defects in dentin formation process.

PURPOSE: Doxorubicin is one of the most active agents in the first-line therapy for metastatic breast cancer, but its utility is partially limited by the frequent emergence of doxorubicin resistance. In this study, we aimed to investigate the role of ATP-binding cassette sub-family B, member 4 (ABCB4) in acquired doxorubicin resistance in breast cancer cells, as well as its potential mechanism.
METHODS: In doxorubicin-sensitive and -resistant breast cancer cell lines MCF-7 and MDA-MB-231, the expression levels of ABCB4 were detected using real-time quantitative PCR and Western blot analysis, the DNA methylation and histone acetylation status of ABCB4 gene were investigated by bisulfite-sequencing PCR (BSP) and chromatin immunoprecipitation (ChIP) assays, and the doxorubicin sensitivity and intracellular doxorubicin accumulation were observed using cell cytotoxicity assay and flow cytometry. In Madin-Darby Canine Kidney (MDCKII) cells, In vitro transport assay was used to assess the ABCB4-mediated transport of doxorubicin.
RESULTS: ABCB4 was overexpressed in doxorubicin-resistant breast cancer cells compared to their doxorubicin-sensitive counterparts, which was associated with reduced DNA methylation as well as increased histone acetylation at the ABCB4 promoter. ABCB4 could actively pump doxorubicin out of the cells, and knockdown of ABCB4 increased doxorubicin sensitivity and intracellular accumulation in doxorubicin-resistant breast cancer cells.
CONCLUSIONS: Our results indicate that ABCB4 is overexpressed in breast cancer cells with acquired doxorubicin resistance, which could be attributed, at least partially, to the epigenetic modifications of ABCB4 gene. ABCB4 mediates the efflux transport of doxorubicin, and contributes to the acquired resistance of doxorubicin in breast cancer cells.

Hepatocellular carcinoma (HCC) is one of the most lethal and prevalent cancers worldwide and has recently become the second most common cause of cancer-related deaths in men of developing countries. Guanine nucleotide-binding protein (G protein) has been reported to be associated with the early process of HCC. In our previous study, GNAO1, one of members of G protein, was found to be down-regulated in HCC. Thus, the present study aimed to throw light upon the mechanism of the abnormal expression of GNAO1 in HCC. First, qPCR results from two HCC cell lines (SMMC-7721 and QGY-7703) confirmed the down-expression of GNAO1, followed by the validation of the methylation status of the promoter region by bisulfite sequence PCR (BSP). Moreover, 5-Aza-2'-deoxycytidine (DAC) with Trichostatin A (TSA) treatment made it much clear that GNAO1 transcription was inhibited by promoter hypermethylation, contributing to its low expression. It was further revealed that the silencing effect was regulated by methyltransferase 1 (DNMT1), and was further enhanced by transforming growth factor β (TGF-β). In addition, the up-regulation of GNAO1 with the help of recombinant plasmid was also found to accelerate cell apoptosis, confirmed by flow cytometry and western blotting analysis. All these results above indicated that the promoter hypermethylation of GNAO1 might play an important role in HCC, suggesting that it might be used as a promising biomarker for HCC diagnosis and targeted therapy.

BACKGROUND: Recent studies have shown that interferon-γ (IFN-γ)-induced galectin-9 expression in Kupffer cells plays an essential role in modulatingthe microenvironment of hepatitis-associated hepatocellular carcinoma (HCC). However, whether or not IFN-γ induces galectin-9 expression in HCC cells, its biological role and regulatory mechanism in HCC development and progression are poorly defined.
METHODS: Quantitative PCR and western blotting analysis were used to detect galectin-9 and EZH2 levels in HCC cell lines stimulated with IFN-γ. Bioinformatics analysis and luciferase reporter assay were utilized to confirmthe binding ofmiR-22 to the 3' untranslated region (3'-UTR) of galectin-9. The methylation status of miR-22 promoter was analyzed by MSP (Methylation specific PCR) and BSP (bisulfite sequencing PCR), while chromatin immunoprecipitation (ChIP) assay identify the occupation status of EZH2 and H3K27me3 at the promoter. Furthermore, the effect of ectopic expression of galectin-9 and miR-22 on cell proliferation, migration, invasion and cell apoptosis was assessed by using CCK-8, transwell assays and flow cytometric analysis, respectively.
RESULTS: IFN-γ induces up-regulation of galectin-9 and EZH2 in HCC cell lines. Galectin-9 is a target of miR-22 and EZH2 facilitates galectin-9 expression by tri-methylation of H3K27 on miR-22 promoter but not hyper-methylation status of DNA. MiR-22 overexpression suppressed HCC cell growth, invasion, and metastasis both in vitro and in vivo. Interestingly, galectin-9 also exhibited antitumor effects, and restoring galectin-9 expression in miR-22 overexpressing cells strengthened its antitumor effects.
CONCLUSIONS: These findings indicated that EZH2 facilitates galectin-9 expression by epigenetically repressing miR-22 and that galectin-9, which is known as an immunosuppressant, also functions as a tumor suppressor in HCC.

Neurofibromatosis type 1 (NF1) is a common genetic disorder caused by mutations in the NF1 gene. Recalcitrant bone healing following fracture (i.e. pseudarthrosis) is one of the most problematic skeletal complications associated with NF1. The etiology of this condition is still unclear; thus, pharmacological options for clinical management are limited. Multiple studies have shown the reduced osteogenic potential of Nf1-deficient osteoprogenitors. A recent transcriptome profiling investigation revealed that EREG and EGFR, encoding epiregulin and its receptor Epidermal Growth Factor Receptor 1, respectively, were among the top over-expressed genes in cells of the NF1 pseudarthrosis site. Because EGFR stimulation is known to inhibit osteogenic differentiation, we hypothesized that increased EREG and EGFR expression in NF1-deficient skeletal progenitors may contribute to their reduced osteogenic differentiation potential. In this study, we first confirmed via single-cell mRNA sequencing that EREG over-expression was associated with NF1 second hit somatic mutations in human bone cells, whereas Transforming Growth Factor beta 1 (TGFβ1) expression was unchanged. Second, using ex-vivo recombined Nf1-deficient mouse bone marrow stromal cells (mBMSCs), we show that this molecular signature is conserved between mice and humans, and that epiregulin generated by these cells is overexpressed and active, whereas soluble TGFβ1 expression and activity are not affected. However, blocking either epiregulin function or EGFR signaling by EGFR1 or pan EGFR inhibition (using AG-1478 and Poziotinib respectively) did not correct the differentiation defect of Nf1-deficient mBMSCs, as measured by the expression of Alpl, Ibsp and alkaline phosphatase activity. These results suggest that clinically available drugs aimed at inhibiting EGFR signaling are unlikely to have a significant benefit for the management of bone non-union in children with NF1 PA.

BACKGROUND: The potential mechanisms regarding how methylation of microRNA(miRNA) CpG Island could regulate cancer cell chemo-resistance remains unclear. This study aims to explore the epigenetic dysregulation mechanism of miRNA-493 and the ability to modulate lung cancer cell chemotherapy resistance.
METHODS: Real-time quantitative PCR (qRT-PCR) and In situ hybridization (ISH) were used to analyze the expression of miR-493 in lung cancer cell lines and tumor tissue, respectively. Bisulfite sequencing PCR (BSP) was used to exam the promoter CpG Island of miR-493. The effect of miR-493 on chemosensitivity was evaluated by cell viability assays, apoptosis assays and in vivo experiment. The DNA damage was measured by γ-H2AX immunofluorescence. Luciferase reporter assay was used to assess the target genes of miR-493. Expression of target proteins and downstream molecules were analyzed by Western blot.
RESULTS: miR-493 is silenced in resistant lung cancer cell due to the aberrant DNA methylation. Enforced expression of miR-493 in lung cancer cells promotes chemotherapy sensitivity to cisplatin through impairing the DNA damage repair and increasing the cells apoptosis in vitro and in vivo. Furthermore, we identify that TCRP1 is a direct functional target of miR-493. Ectopic expression of TCRP1 attenuated increased apoptosis in miR-493-overexpressing lung cancer cells upon cisplatin treatment. Meanwhile, miR-493 level is negatively correlated with TCRP1 expression in lung cancer patients and TCRP1 expression were correlated with poor survival.
CONCLUSIONS: Our results highlight that hyper-methylation of miR-493CpG island might play important roles in the development of lung cancer chemo-resistance by targeting TCRP1, which might be used as a potential therapeutic target in preventing the chemo-resistance of lung cancer.

Piezoelectric films of poly(vinylidenedifluoride-trifluoroethylene) (P(VDF-TrFE)) and of P(VDF-TrFE)/boron nitride nanotubes (BNNTs) were prepared by cast-annealing and used for SaOS-2 osteoblast-like cell culture. Films were characterized in terms of surface and bulk features, and composite films demonstrated enhanced piezoresponse compared to plain polymeric films (d

In breast cancer (BC), silencing of miRNA genes due to miRNA gene promoter methylation are the important mechanisms directly contributing to tumorigenesis and tumor progression. miRNA-495 (miR-495) has been reported to be a tumor suppressor gene in various cancers, but its role and regulation in BC remains unclear. In the present study, the level of miR-495 was inversely correlated with the expression of STAT-3 in BC tissues and cell lines. miR-495 can directly target 3'-UTR of STAT-3 mRNA and thereby decrease the expression of STAT-3 in MCF-7 and HCC1973 cells by Targetscan and Dual-luciferase assay. We further analyzed miR-495 promoter methylation by sodium bisulfite sequencing method (BSP), and found DNA methyltransferase inhibitor, 5-AzaC concomitantly upregulated expression of miR-495 and downregulated its target gene STAT-3 and its downstream target VEGF. Furthermore, we further observed that 5-AzaC treatment, miR-495 mimics and STAT-3 knockdown significantly inhibited cell function in breast cancer by Transwell assay, EdU flow cytometry, Annexin V-FITC/PI combined with flow cytometry and Hoechst staining. Taken together, our data are first to demonstrate that the miR-495 is silenced due to promoter methylation in breast cancer. DNA methyltransferase inhibitor 5-AzaC could reverse miR‑495 (suppressor gene) and STAT-3 (oncogene). The anticancer properties of 5-AzaC were preliminarily confirmed in breast cancer.

Chronic exposure to aflatoxin B

BACKGROUND: Aberrant DNA methylation patterns play a major role in tumorigenesis and the effects of nutrients, especially folate in the diet, on methylation changes is of great importance in colorectal cancer (CRC). Folate deficiency would disrupt methylation patterns; however, its exact effects on DNA methylation patterns in CRC are unclear. This study was performed to gain insight into the methylation changes induced by folate deficiency and the putative role of methylation pattern diversities of related genes in the clinical outcome of CRC.
METHODS: The NimbleGen MeDIP chip (Methylated DNA Immunoprecipitation chip) assay was used in high-resolution mapping of DNA methylation patterns in the normal human colon mucosal epithelial cell line, NCM460 cultured with or without folate. Aberrant CpG island methylation patterns in the promoter of genes were identified by chip assay and then were confirmed in paired colorectal tissues and corresponding non-malignant tissues obtained from patients by bisulfate sequencing PCR (BSP). Of the total, the expression of cystathionine-beta-synthase (CBS) involved in methyl metabolism and its important substrate, homocysteine, were all detected by realtime RT-PCR and immunostaining. We also analyzed the data of its hypermethylation level statistically correlated with pathological parameters and the clinical outcome in malignant tissues.
RESULTS: The chip assay showed that there are 17 genes with hyper or hypomethylation in CpG islands of promoter on chromosome 21, and 8 of them seemed to be associated with tumorigenesis. Among the total, a hypermethylation patterns existed in the promoter of CBS in CRC (p < 0.001), and the hypermethylation is related with the down-regulation of CBS and the accumulation of homocysteine in vitro and vivo (p < 0.001). Univariate analysis showed CBS hypermethylation level is correlated with age (p < 0.001), pT stage (p = 0.008), pN stage (p = 0.038), liver metastasis (p = 0.017), pTNM stage (p = 0.032), Dukes' stage (p = 0.022), recurrence (p = 0.041), five-year survival (p = 0.034), recurrence-free probability (p = 0.011), and overall survival (p = 0.018). Multivariate analysis showed that CBS hypermethylation level significantly correlated with recurrence rate (p = 0.039) and overall survival (p = 0.012) independent of pT stage, pN stage, and liver metastasis.
CONCLUSIONS: Folate deficiency could induce aberrant DNA methylation patterns and gene expressions in CRC. CBS plays a critical role in tumorigenesis and could serve as a prognostic marker for tumor progression.

BACKGROUND: γ-Glutamyl hydrolase (GGH) regulates intracellular folates and antifolates such as methotrexate (MTX) for proper nucleotide biosynthesis and antifolate-induced cytotoxicity, respectively. In addition to genetic polymorphism and karyotypic abnormalities, methylation of CpG island 1 (CpG1) in the promoter region is found to modulate GGH activity by reducing GGH mRNA expression in acute lymphoblastic leukemia (ALL) cells. We aim to investigate methylation status of two CpG islands (CpG1 and CpG2) in the GGH promoter region in pediatric patients with ALL and acute myelogenous leukemia (AML).
METHODS: 70B-ALL, 29 AML, 10 ITP (idiopathic thrombocytopenic purpura) and 40 healthy children are recruited in the present study. MS-HRM (methylation-sensitive high-resolution melting) and bisulfite sequencing PCR (BSP) are used to detect methylation change and its level in CpG1 and CpG2 in the GGH promoter region. GGH mRNA expression is quantified by real-time PCR. Correlation between CpG island methylation and GGH mRNA expression is assessed by statistical software.
RESULTS: Methylations of CpG1 are detected in leukemia cells samples obtained from 30.9% (21/68) of patients with ALL and 20.7% (6/29) of patients with AML. These methylations are not detected in the controls. Methylations of CpG2 are detected in leukemia cell samples obtained from 44.1% (30/68) of the ALL patients and 37.9% (11/29) of the AML patients. These percentages are significantly higher than that observed in the control cell samples: 6.0% (3/50) (Fisher's exact test, P = 0.000). The abundance of CpG1 methylation in all leukemia cell samples is classified as Grade I (methylation level is less than 10%) and the abundance of CpG2 methylation in leukemia cell samples is classified in separate grades. Our results indicate that methylation of CpG1 or hypermethylation (the methylation level is greater than 10%) of CpG2 could significantly reduce GGH mRNA expression in leukemia cells from the ALL and AML patients (ALL-CpG1: t = 4.632, P = 0.000; ALL-CpG2: t = 3.250, P = 0.006; AML-CpG1: t = -2.254, P = 0.037; AML-CpG2: t = 1.328, P = 0.202).
CONCLUSION: Either methylation of CpG1 or hypermethylation of CpG2 in GGH promoter region can significantly reduce GGH mRNA expression in pediatric patients with acute leukemia, which can improve the response to treatment.

BACKGROUND: Inactivation of tumor suppressor genes by promoter hypermethylation plays a key role in the tumorgenesis. It is necessary to uncover the detailed pattern of whole genome-wide abnormal DNA methylation during the development of gastric cancer (GC).
METHOD: We performed a genome-wide methylation detection using 12 paired of GC tissues and their corresponding normal tissues. Methylation-specific PCR (MSP) and bisulphite sequencing (BSP) were used to measure methylation status of specific CpG site. Based on the bioinformatic analysis, the cell phenotypes and mouse model experiments were constructed to detect effect of the target gene. Using the Kaplan-Meier survival curve, the clinical value of KCNMA1 was assessed in GC patients.
RESULTS: The CpG site cg24113782 located at the promoter of KCNMA1 showed the most significant difference, contributing to the commonly silenced KCNMA1in gastric cancer cells and primary GC tissues. The promoter methylation of KCNMA1 was detected in 68.7% (77/112) of tumor tissues, compared with 16.2% (18/112) of normal tissues (P < 0.001). The survival curve indicated that KCNMA1 hypermethylation was significantly associated with the shortened survival in GC patients (P = 0.036). KCNMA1 significantly inhibited biological malignant behavior of gastric cancer cell by inducing cell apoptosis in vitro, and suppressed xenograft tumor growth in subcutaneous mouse models (both P < 0.001). Furthermore, the anti-tumor effect of KCNMA1was mediated through suppressing the expression of PTK2.
CONCLUSION: KCNMA1 is a critical tumor suppressor in gastric carcinogenesis and its hypermethylation is an independent prognostic factor in patients with gastric cancer.

BACKGROUND: DNA methylation modification has been proved to influence the phenotype of polycystic ovary syndrome (PCOS). Genome-wide association studies (GWAS) demonstrate that yes-associated protein (YAP1) genetic sites are associated with PCOS. The study aims to detect the methylation status of YAP1 promoter in ovary granulosa cells (GCs) of PCOS patients and explore novel therapeutic targets for PCOS.
METHODS: Randomized controlled trial was applied and a total of 72 women were included in the study, including 36 cases of PCOS patients and 36 cases of health controls. Ovary GCs were extracted from in vitro fertilization embryo transfer. Methylation status of YAP1 promoter was detected by bisulfite sequencing PCR (BSP). Protein and mRNA expression of YAP1 were measured by western blotting and real-time quantitate PCR.
RESULTS: Overall methylation level of YAP1 promoter region from PCOS group was significantly lower than that from control group. CpG sites analysis revealed that 12 sites (-443, -431, -403, -371, -331, -120, -49, -5, +1, +9, +15, +22) were significantly hypomethylated in women with PCOS (P < 0.05). A significant upregulation of YAP1 mRNA and protein expression levels was observed. Testosterone concentration could alleviate the methylation status and demonstrate obvious dose-dependent relation.
CONCLUSION: Our research achievements manifest that hypomethylation of YAP1 promoter promotes the YAP1 expression, which plays a key role in the pathogenesis and accelerate PCOS.

Epigenetic inactivation of GPX3 has been identified in various cancers including leukemia. Moreover, aberrant DNA methylation was also found as a dominant mechanism of disease progression in myelodysplastic syndrome (MDS). This study intended to explore GPX3 promoter methylation and its clinical relevance in 110 patients with MDS. GPX3 methylation was examined by real-time quantitative methylation-specific PCR (RQ-MSP) and bisulfite sequencing PCR (BSP). GPX3 methylation was identified in 15% (17/110) MDS patients, and significantly higher than controls, and lower than acute myeloid leukemia (AML) patients (P = 0.024 and 0.041). GPX3 methylated patients had older age and higher frequency of DNMT3A mutation (P = 0.015 and 0.066). Cases with GPX3 methylation showed significantly shorter overall survival (OS) time than those with GPX3 unmethylation analyzed with Kaplan-Meier analysis (P = 0.012). Moreover, Cox regression analysis revealed that GPX3 methylation might act as an independent prognostic indicator in MDS (HR = 1.847, P = 0.072). GPX3 methylation density was significantly increased during the progression from MDS to secondary acute myeloid leukemia (sAML) in three follow-up paired patients. Our study concludes that GPX3 methylation in bone marrow is associated with adverse prognosis and leukemia transformation in MDS.

Early reports suggest that nuclear factor IA (NFIA) is important in the pathogenesis of glioma. Our previous study demonstrated that the long non‑coding RNA (lncRNA), RP5‑833A20.1, suppressed the expression of NFIA in THP‑1 macrophage-derived foam cells. However, the effect and possible mechanism of RP5‑833A20.1 on glioma remains to be fully elucidated, and whether the NFIA-dependent pathway is involved in its progression has not been investigated. In the present study, the mechanisms by which RP5‑833A20.1 regulates the expression of NFIA in glioma were investigated. The expression levels of RP5‑833A20.1 and NFIA were determined in U251 cells and clinical samples using reverse transcription‑quantitative polymerase chain reaction (PCR) analysis. The effects of RP5‑833A20.1 on cell proliferation, invasion, cell cycle and apoptosis were evaluated using in vitro assays. The potential changes in protein expression were investigated using western blot analysis. The methylation status of the CpG island in the NFIA promoter was determined using bisulfite PCR (BSP) sequencing. It was found that the expression of RP5‑833A20.1 was downregulated, whereas the expression of NFIA was upregulated in glioma tissues, compared with corresponding adjacent nontumor tissues from 20 patients with glioma. The overexpression of RP5‑833A20.1 inhibited proliferation and cell cycle progression, and induced apoptosis in the U251 cells. The mRNA and protein levels of NFIA were markedly inhibited by overexpression of RP5‑833A20.1 in the U251 cells. The overexpression of RP5‑833A20.1 increased the expression of microRNA‑382‑5p in the U251 cells. The BSP assay revealed that the overexpression of RP5‑833A20.1 enhanced the methylation level of the NFIA promoter. These results demonstrated that RP5‑833A20.1 inhibited tumor cell proliferation, induced apoptosis and inhibited cell‑cycle progression by suppressing the expression of NFIA in U251 cells. Collectively, these results indicated RP5‑833A20.1 as a novel therapeutic target for glioma.

Bromodomains (BRDs) have emerged as compelling targets for cancer therapy. The development of selective and potent BET (bromo and extra-terminal) inhibitors and their significant activity in diverse tumor models have rapidly translated into clinical studies and have motivated drug development efforts targeting non-BET BRDs. However, the complex multidomain/subunit architecture of BRD protein complexes complicates predictions of the consequences of their pharmacological targeting. To address this issue, we developed a promiscuous BRD inhibitor [bromosporine (BSP)] that broadly targets BRDs (including BETs) with nanomolar affinity, creating a tool for the identification of cellular processes and diseases where BRDs have a regulatory function. As a proof of principle, we studied the effects of BSP on leukemic cell lines known to be sensitive to BET inhibition and found, as expected, strong antiproliferative activity. Comparison of the modulation of transcriptional profiles by BSP after a short exposure to the inhibitor resulted in a BET inhibitor signature but no significant additional changes in transcription that could account for inhibition of other BRDs. Thus, nonselective targeting of BRDs identified BETs, but not other BRDs, as master regulators of context-dependent primary transcription response.

Wnt signaling is implicated in bone formation and activated in breast cancer cells promoting primary and metastatic tumor growth. A compelling question is whether osteogenic miRNAs that increase Wnt activity for bone formation are aberrantly expressed in breast tumor cells to support metastatic bone disease. Here we report that miR-218-5p is highly expressed in bone metastases from breast cancer patients, but is not detected in normal mammary epithelial cells. Furthermore, inhibition of miR-218-5p impaired the growth of bone metastatic MDA-MB-231 cells in the bone microenvironment in vivo. These findings indicate a positive role for miR-218-5p in bone metastasis. Bioinformatic and biochemical analyses revealed a positive correlation between aberrant miR-218-5p expression and activation of Wnt signaling in breast cancer cells. Mechanistically, miR-218-5p targets the Wnt inhibitors Sclerostin (SOST) and sFRP-2, which highly enhances Wnt signaling. In contrast, delivery of antimiR-218-5p decreased Wnt activity and the expression of metastasis-related genes, including bone sialoprotein (BSP/IBSP), osteopontin (OPN/SPP1) and CXCR-4, implicating a Wnt/miR-218-5p regulatory network in bone metastatic breast cancer. Furthermore, miR-218-5p also mediates the Wnt-dependent up-regulation of PTHrP, a key cytokine promoting cancer-induced osteolysis. Antagonizing miR-218-5p reduced the expression of PTHrP and Rankl, inhibited osteoclast differentiation in vitro and in vivo, and prevented the development of osteolytic lesions in a preclinical metastasis model. We conclude that pathological elevation of miR-218-5p in breast cancer cells activates Wnt signaling to enhance metastatic properties of breast cancer cells and cancer-induced osteolytic disease, suggesting that miR-218-5p could be an attractive therapeutic target for preventing disease progression.

BACKGROUND: In addition to treating acute promyelocytic leukemia, arsenic trioxide (ATO) suppresses other solid tumors, including chondrosarcoma. However, the effects of ATO on metastasis in chondrosarcoma cells, and the underlying molecular mechanisms remain unclear.
METHODS: The effects of ATO on the migratory and invasive capacities of chondrosarcoma cells were investigated by Wound healing, Transwell and EMT assays. The expression of miR-125b in human chondrosarcoma tissues and cell lines was detected by real-time PCR analysis. Bisulfite sequencing analysis (BSP) was used to detect the effects of ATO on the expression of miR-125b. The gain-of-function and loss-of-function experiments were performed on chondrosarcoma cell lines to investigate the effects of miR-125b on chondrosarcoma invasion, and to determine whether signal transducer and activator of transcription 3(Stat3) mediates these effects. Dual-luciferase reporter assay was used to identify whether Stat3 is a direct target of miR-125b.
RESULTS: MiR-125b was significantly downregulated in human metastatic chondrosarcoma tissues and cell lines but not in non-metastatic chondrosarcoma tissues. ATO up-regulates the expression of miR-125b by the demethylation of DNA. ATO induces MET and attenuates the invasive capacities of chondrosarcoma cells through miR-125b. Stat3 was verified as a direct target of miR-125b, which is involved in ATO regulating EMT-associated traits.
CONCLUSIONS: These findings, for the first time, provides evidence that the miR-125b-mediated inhibition of Stat3 is involved in the ATO-induced attenuation of metastasis in chondrosarcoma cells.

SATB2 (special AT-rich binding protein-2), a transcription factor and chromatin modulator, regulates the expression of genes required for maintaining pluripotency and self-renewal. The molecular mechanisms by which human pancreatic normal ductal epithelial cells are transformed to cancer cells are not well understood. The main goal of the paper is to examine the molecular mechanisms by which SATB2 regulates transformation of human pancreatic normal ductal epithelial (HPNE) cells, and assess whether transformed HPNE cells gained the phenotypes of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in pancreatic CSCs, primary tissues and cell lines, but not in HPNE cells. SATB2 induces cellular transformation, stemness and epithelial to mesenchymal transition in HPNE cells, and inhibition of its expression suppresses these activities. Overexpression of SATB2 in HPNE cells resulted in induction of stem cell markers (CD44, CD24 and CD133), and transcription factors (Oct4, Sox2 and Nanog). SATB2 can directly bind to promoters of Bcl-2, Bsp, Nanog, c-Myc, XIAP, Klf4 and Hoxa2, suggesting the role of SATB2 in pluripotency, cell survival and proliferation. SATB2-overexpressing HPNE cells (HPNE/SATB2) formed tumors in Balb C nude mice, whereas HPNE/Empty vector cells did not form any tumor. Since SATB2 is highly expressed in human pancreatic cancer tissues and cell lines, but not in HPNE cells and normal pancreatic tissue, it can drive pancreatic cancer growth and metastasis. Our findings suggest that SATB2 can induce dedifferentiation by inducing stemness and may have a role in pancreatic carcinogenesis, and can be used as a diagnostic biomarker.

BACKGROUND: The association of hepatitis B virus (HBV) preS1 and preS2 deletions with progressive liver diseases are not fully understood.
OBJECTIVE: The study aimed to investigate characteristics of HBV preS deletion in HBV-infected patients with different illness categories.
STUDY DESIGN: Total of 539 HBV-infected patients were enrolled in the study, including 146 with chronic hepatitis B (CHB), 111 with HBV-related liver cirrhosis (LC), 146 with HBV-related acute-on-chronic liver failure (ACLF), and 136 with HBV-related hepatocellular carcinoma (HCC). PreS deletion was determined by sequencing. Replicons containing representative preS1 and preS2 deletion mutants and wild-type were respectively constructed and transfected into HepG2 cells for phenotypic analysis.
RESULTS: The detection rates of overall preS deletion were 15.8%, 26.1%, 24.0%, and 34.6% in CHB, LC, ACLF, and HCC patients, respectively. PreS1 deletion was most frequently detected in LC patients while preS2 deletion was most frequently detected in HCC patients, both frequencies were significantly higher than that in CHB patients (17.1% vs. 4.8%, P<0.01; 19.1% vs. 4.8%, P<0.01). The deletion patterns across preS gene were different among the 4 illness categories. Compared with wild-type strain, the preS1 deletion mutant had defected preS1 expression, significantly decreased viral mRNA level and SP II promoter activity; while preS2 deletion mutant had defected preS2 expression, and significantly decreased viral mRNA level.
CONCLUSIONS: HBV preS deletion was associated with advancement of liver diseases not only presented in preS deletion incidence, but also in the deletion pattern. Patients with preS2 deletion might have a higher risk to develop HCC.

BACKGROUND: Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC).
METHODS: The expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies.
RESULTS: We found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3β and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo.
CONCLUSIONS: Our findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

BACKGROUND: Promoter CpG methylation of Epstein-Barr virus (EBV) genome plays an essential role in maintaining viral latency. Latent membrane protein (LMP) 1, 2A and 2B of EBV exert multiple oncogenic properties by activating multiple signal pathways and modulating the expression of various oncogenes.
AIMS: To study the methylation and expression of LMP1, 2A and LMP2B in EBV-positive cell lines and EBV-associated tumors.
METHODS: The methylation profiles of LMP1p, LMP2Ap and LMP2Bp were evaluated by methylation-specific PCR (MSP) and bisulfite sequencing PCR (BSP), as well as their expression by quantitative real-time (qRT)-PCR in 41 EBV-associated carcinomas (EBVaGCs) and 5 EBV-positive cell lines.
RESULTS: All LMP promoters were methylated at different degrees in EBV-positive cell lines and hypermethylated in EBV-associated gastric carcinomas, while unmethylated LMP2Ap alleles were detected in B95-8 cell line. Following 5-aza-2'-deoxycytidine (5-aza) treatment, the LMP1 expression was restored along with concomitant promoter demethylation; changes of LMP2A and LMP2B expression were different in different cells.
CONCLUSION: Methylation of LMP1, 2A and 2B promoters mediates the silencing of LMP1, 2A and 2B in EBV-associated carcinomas and cell lines in varying degrees, and could be reactivated by demethylation agent and thus may contribute to the therapy of EBVaGCs.

Aberrant expression of the miR-129 family has been found in several types of cancer, yet its expression and potential biologic role in breast cancer remain largely unknown. In the present study, we found that miR-129-2 was consistently downregulated in the breast cancer specimens and cell lines. Overexpression of miR-129-2-3p markedly suppressed breast cancer cell proliferation and induced its apoptosis. In addition, a luciferase reporter assay revealed that miR-129-2-3p suppressed BCL2L2 expression. Furthermore, BCL2L2 was able to reverse miR-129-2-3p-mediated cell apoptosis, indicating that BCL2L2 plays a crucial role in mediating the tumor-suppressive role of miR-129-2-3p. Moreover, bisulfite DNA sequencing PCR (BSP) analysis identified that promoter hypermethylation was responsible for the downregulation of miR-129-2 in breast cancer. Collectively, our findings indicate that miR-129-2 is downregulated in breast cancer cells by promoter hypermethylation. Moreover, downregulation of miR-129-2 results in BCL2L2 overexpression and disease progression in breast cancer patients.

This study aims to investigate the biological function of microRNA-200b and BMI1, predicted target of microRNA-200b in human hepatocellular carcinoma (HCC). MicroRNA-200b and BMI1 expression in HCC tissues were evaluated by qPCR. A luciferase reporter assay was used to validate BMI1 as a direct target of microRNA-200b. The effect of microRNA-200b on HCC progression was studied in vitro and in vivo. Methylation specific PCR (MSP) and bisulfite sequencing PCR (BSP) were used to detect the methylation status of the microRNA-200b promoter. Significant downregulation of microRNA-200b was observed in 83.3% of HCC tissues. By contrast, BMI1 was significantly overexpressed in 66.7% of HCC tissues. The results of the luciferase assay confirmed BMI1 as a direct target gene of microRNA-200b. Forced expression of microRNA-200b in HCC cells dramatically repressed proliferation, colony formation, cell cycle progression, and invasion. Moreover, microRNA-200b synergized with 5-fluorouracil to induce apoptosis in vitro and suppressed tumorigenicity in vivo. In addition, MSP analysis and BSP revealed that CpG sites in the promoter region of microRNA-200b were extensively methylated in HCC, with concomitant downregulation of microRNA-200b expression. Furthermore, microRNA-200b was activated in HCC cells after treatment with 5-azacytidine, whereas BMI1 expression was clearly downregulated. Our results indicate that microRNA-200b is partially silenced by DNA hypermethylation and that it can repress tumor progression by directly targeting BMI1 in HCC.

5-Aza-2'-deoxycytidine (5-Aza-CdR) is currently acknowledged as a demethylation drug, and causes a certain degree of demethylation in a variety of cancer cells, including pancreatic cancer cells. Emodin, a traditional Chinese medicine (TCM), is an effective monomer extracted from rhubarb and has been reported to exhibit antitumor activity in different manners in pancreatic cancer. In the present study, we examined whether emodin caused demethylation and increased the demethylation of three tumor-suppressor genes P16, RASSF1A and ppENK with a high degree of methylation in pancreatic cancer when combined with 5-Aza-CdR. Our research showed that emodin inhibited the growth of pancreatic cancer Panc-1 cells in a dose- and time-dependent manner. Dot-blot results showed that emodin combined with 5-Aza-CdR significantly suppressed the expression of genome 5mC in PANC-1 cells. In order to verify the effect of methylation, methylation-specific PCR (MSP) and bisulfite genomic sequencing PCR (BSP) combined with TA were selected for the cloning and sequencing. Results of MSP and BSP confirmed that emodin caused faint demethylation, and 5-Aza-CdR had a certain degree of demethylation. When emodin was combined with 5-Aza-CdR, the demethylation was more significant. At the same time, fluorescent quantitative PCR and western blot analysis results confirmed that when emodin was combined with 5-Aza-CdR, the expression levels of P16, RASSF1A and ppENK were increased more significantly compared to either treatment alone. In contrast, the expression levels of DNA methyltransferase 1 (DNMT1) and DNMT3a were more significantly reduced with the combination treatment than the control or either agent alone, further proving that emodin in combination with 5-Aza-CdR enhanced the demethylation effect of 5-Aza-CdR by reducing the expression of methyltransferases. In conclusion, the present study confirmed that emodin in combination with 5-Aza-CdR enhanced the demethylation by 5-Aza-CdR of tumor-suppressor genes p16, RASSF1A and ppENK by reducing the expression of methyltransferases DNMT1 and DNMT3a.