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Epstein-Barr virus (EBV) is an important DNA tumor virus that is associated with approximately 10% of gastric carcinomas and 99% of nasopharyngeal cancers (NPC). DNA methylation and microRNAs (miRNAs) are the most studied epigenetic mechanisms that can prompt disease susceptibility. This study aimed to detect the effect of EBV on Wnt inhibitory factor 1 (WIF1), Nemo-like kinase (NLK), and adenomatous polyposis coli (APC) gene methylation, and expression in gastric carcinoma and NPC. The WIF1, NLK, and APC gene mRNA expression levels were measured by real-time quantitative RT-PCR in four EBV-positive cell lines and four EBV-negative cell lines. Bisulfite genomic sequencing or methylation-specific PCR was used to detect the methylation status of the WIF1, NLK, and APC promoters. All cell lines were treated with 5-azacytidine (5-aza-dC), miR-BART19-3p mimics or an inhibitor, and analyzed by flow cytometry and MTT cell proliferation assays. The WIF1, NLK, and APC promoters were hypermethylated in all eight cell lines. 5-Aza-dC displayed a growth inhibitory effect on cells . After transfection with miR-BART19-3p mimics, the expression of WIF1, and APC decreased, and the cellular proliferation rate increased. After transfection with the miR-BART19-3p inhibitor, the expression levels were higher, and the cell growth was inhibited. In the NPC and GC cell lines, the promoters of WIF1, NLK, and APC are highly methylated, and the expression of these three genes is regulated by miR-BART19-3p. The activity of the Wnt pathway in EBV-associated tumors may be enhanced by miR-BART19-3p., (© 2017 Wiley Periodicals, Inc.)

Both human papillomavirus (HPV) infection and the aberrant Ras associated domain family gene 1A (RASSF1A) promoter methylation status participate in the pathogenesis of cervical cancer. Some studies suggest that E6, and E7 are involved in the pathogenetic mechanisms of RASSF1A. We mainly explored a possible involvement of HPV16 oncogenes E6 or/and E7 in RASSF1A promoter methylation status and possible roles of RASSF1A gene methylation in cervical cancer. Bisulfite genomic sequencing (BGS) PCR combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation status of the RASSF1A gene promoter in HPV16/18-positive and HPV-negative cervical cancer cell lines; ectopically expressed HPV16 E6, E7 and E6/E7 cervical cancer cell lines; normal cervical and cervical cancer tissues. The mRNA and protein expression of RASSF1A was detected by RT-PCR and western blotting. Re-expression and downregulated promoter methylation status were detected in the ectopically expressed HPV16 E6 and E7 cervical cancer cell line HT-3. The methylation status and expression of RASSF1A could be downregulated or reactivated by 5-Aza-dc in HT-3 and C33A cells. Additionally, statistics showed significant hypermethylation of RASSF1A in cervical cancer samples compared to that in normal cervical samples (P<0.05). The false negative rate (FNR) was 6.25% by HC2 method, when reconfirmed by HPV detection combining the MY09/11, GP5+/6+ and SPF1/2 methods. The ectopic expression of HPV16 E6 and/or E7 may be involved in aberrant methylation and expression of the RASSF1A gene. RASSF1A gene expression could be regulated by its promoter methylation status. Additionally, the false negativity of the HPV detection may contribute to the uncertain relationship between HPV infection and aberrant RASSF1A promoter methylation.

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., (Copyright © 2016 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

Promoter CpG methylation is a fundamental regulatory process of gene expression. TET proteins are active CpG demethylases converting 5-methylcytosine to 5-hydroxymethylcytosine, with loss of 5 hmC as an epigenetic hallmark of cancers, indicating critical roles of TET proteins in epigenetic tumorigenesis. Through analysis of tumor methylomes, we discovered TET1 as a methylated target, and further confirmed its frequent downregulation/methylation in cell lines and primary tumors of multiple carcinomas and lymphomas, including nasopharyngeal, esophageal, gastric, colorectal, renal, breast and cervical carcinomas, as well as non-Hodgkin, Hodgkin and nasal natural killer/T-cell lymphomas, although all three TET family genes are ubiquitously expressed in normal tissues. Ectopic expression of TET1 catalytic domain suppressed colony formation and induced apoptosis of tumor cells of multiple tissue types, supporting its role as a broad bona fide tumor suppressor. Furthermore, TET1 catalytic domain possessed demethylase activity in cancer cells, being able to inhibit the CpG methylation of tumor suppressor gene (TSG) promoters and reactivate their expression, such as SLIT2, ZNF382 and HOXA9. As only infrequent mutations of TET1 have been reported, compared to TET2, epigenetic silencing therefore appears to be the dominant mechanism for TET1 inactivation in cancers, which also forms a feedback loop of CpG methylation during tumorigenesis.

Background: Cervical cancer (CC) is a leading cause of mortality in females, especially in developing countries. The two viral oncoproteins E6 and E7 mediate the oncogenic activities of high-risk human papillomavirus (hrHPV), and hrHPV, especially HPV16 or/and HPV18 (HPV16/18) play critical roles in CC through different pathways. STK31 gene of which the expression has been proven to be regulated by the methylation status of its promoter, is one of the novel cancer/testis (CT) genes and plays important roles in human cancers. Reasearches have indicated that viral infection is correlated to the methylation statuses of some genes. Herein, we detected methylation status of the STK31 gene in cervical tumors and explored its interaction with HPV16 or/and HPV18 (HPV16/18) infection., Methods: Bisulfite genomic sequencing PCR (BGS) combined with TA clone, methylation-specific PCR (MSP) were used to analyze methylation statuses of the STK31 gene promoter/exon 1 region in HPV16/18-positive, HPV-negative CC cell lines; ectopically expressed HPV16 E6, -E7, and -E6/E7 CC cells; normal cervical tissues and cervical tumor tissues of different stages. The mRNA and protein expressions of STK31 were detected by RT-PCR and western blotting., Results: The STK31 gene promoter/exon 1 was hypomethylated in the HPV16/18-positive cell lines HeLa, SiHa and CaSki, and the mRNA and protein expression were detected. In contrast, the STK31 gene exhibited hypermethylation and silenced expression in the HPV-negative CC cells C33A and HT-3. Compared with the primary HPV-negative CC cell lines, the STK31 methylation was downregulated, and STK31 expression was induced in the HPV16E7/E67 transfected cells. The methylation statuses and expressions of STK31 were verified in the cervical tumor samples at different stages. Additionally, chemotherapy treatment may influence STK31 expression by regulating its methylation status., Conclusions: STK31 may be a novel cellular target gene for the HPV16 oncogeneE7. The HPV16 oncogene E7 may affect STK31 expression through a methylation-mediated mechanism. The aberrant methylation of the STK31 promoter/exon 1 region may be a precursor of human cervical carcinogenesis and a potential DNA aberrant methylation biomarker of conditions ranging from precancerous disease to invasive cancer.

Closely located at the tumor suppressor locus 16q22.1, CKLF-like MARVEL transmembrane domain-containing member 3 and 4 (CMTM3 and CMTM4) encode two CMTM family proteins, which link chemokines and the transmembrane-4 superfamily. In contrast to the broad expression of both CMTM3 and CMTM4 in normal human adult tissues, only CMTM3 is silenced or down-regulated in common carcinoma (gastric, breast, nasopharyngeal, esophageal, and colon) cell lines and primary tumors. CMTM3 methylation was not detected in normal epithelial cell lines and tissues, with weak methylation present in only 5 of 35 (14%) gastric cancer adjacent normal tissues. Furthermore, immunohistochemistry showed that CMTM3 protein was absent in 12 of 35 (34%) gastric and 1 of 2 colorectal tumors, which was well correlated with its methylation status. The silencing of CMTM3 is due to aberrant promoter CpG methylation that could be reversed by pharmacologic demethylation. Ectopic expression of CMTM3 strongly suppressed the colony formation of carcinoma cell lines. In addition, CMTM3 inhibited tumor cell growth and induced apoptosis with caspase-3 activation. Thus, CMTM3 exerts tumor-suppressive functions in tumor cells, with frequent epigenetic inactivation by promoter CpG methylation in common carcinomas.

BACKGROUND: Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a common malignant tumor associated with EBV infection. Insulin-like growth factor 2 (IGF2) is an imprinted gene and a key protein that regulates growth, especially during normal fetal development. Loss of imprinting (LOI), is a common epigenetic anomaly in a variety of human cancers. However, the promoter methylation, imprinting status and function of IGF2 gene in GC are unclear. OBJECTIVE: To explore the role of IGF2 in the occurrence and development of gastric cancer. METHODS: The biological function of IGF2 in gastric cancer was investigated by Transwell, wound healing, CCK-8 and flow cytometry assays. IGF2 imprinting status and gene promoter methylation in gastric cancer tissues were detected by PCR-RFLP and BGS. RESULTS: The results showed that the expression of IGF2 was higher in GC tissues than adjacent tissues. IGF2 gene promoter methylation and LOI were significantly higher in EBVaGC tissues than in EBV-negative gastric cancer (EBVnGC) tissues. The high expression of IGF2 in gastric cancer can promote the migration and proliferation of gastric cancer cells. CONCLUSION: Our data suggest that IGF2 is involved in the occurrence and development of gastric cancer. Targeting IGF2 may be a potential therapeutic target for gastric cancer. [ABSTRACT FROM AUTHOR]

EBV is an important human tumor virus and is closely related to the occurrence of a variety of tumors, involving 10% of gastric cancer. In EBV-associated gastric carcinoma (EBVaGC), EBV expresses restrict viral genes including EBV nuclear antigen 1, EBV encoded small RNAs, Bam HI-A rightward transcripts, latent membrane protein 2A and miRNAs. The role of EBV in gastric carcinogenesis has received increasing attention and is considered to be another pathogenic factor in addition to Helicobacter pylori. A typical characteristic of EBVaGC is the extensive methylation of viral and host genome. Combined with other epigenetic mechanisms, EBV infection acts as an epigenetic driver of EBVaGC oncogenesis. In this review we discuss recent findings of EBV effect on host epigenetic alterations in EBVaGC and its role in oncogenic process. [ABSTRACT FROM AUTHOR]

Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinomas that promoter hypermethylation of tumor-related genes is extremely frequent to be found. Aquaporin 3 (AQP3) is a small membrane transport protein that plays a crucial role in cancer progression and metastasis. However, there is no experimental study on the expression of AQP3 in EBVaGC and the regulation mechanism of EBV on AQP3. In this study, the loss of AQP3 was contributed by the hypermethylation status of AQP3 promoter in EBVaGC which was caused by elevated expression of DNMT3a. In addition, stable and transient transfection system in SGC7901 showed that viral latent membrane protein 2A (LMP2A) activated phosphorylated ERK and up-regulated DNMT3a. Taken together, LMP2A induced the phosphorylation of ERK, which activated DNMT3a transcription and caused AQP3 expression loss through CpG island methylation of AQP3 promoter in EBVaGC. [ABSTRACT FROM AUTHOR]