• Asian Pacific Journal of Cancer Prevention
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• v.15 no.6
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• pp.2485-2489
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• 2014

In the present study, we studied the hypermethylation of the human riboflavin transporter 2 (hRFT2) gene and regulation of protein expression in biopsies from resected tissues from Uighur cervical squamous cell carcinoma (CSCC) patients and their neighboring normal tissues. hRFT2 gene promoter region methylation sequences were mapped in cervical cancer cell line SiHa by bisulfite-sequencing PCR and quantitative detection of methylated DNA from 30 pairs of Uighur's CSCCs and adjacent normal tissues by MassARRAY (Sequenom, San Diego, CA, USA) and hRFT2 protein expression was analyzed by immunohistochemistry. In SiHa, we identified 2 CG sites methylated from all of 12CpG sites of the hRFT2 gene. Analysis of the data from quantitative analysis of single CpG site methylation by Sequenom MassARRAY platform showed that the methylation level between two CpG sites (CpG 2 and CpG 3) from CpG 1~12 showed significant differences between CSCC and neighboring normal tissues. However, the methylation level of whole target CpG fragments demonstrated no significant variation between CSCC ($0.476{\pm}0.020$) and neighboring normal tissues ($0.401{\pm}0.019$, p>0.05). There was a tendency for translocation the hRFT2 proteins from cytoplasm/membrane to nucleus in CSCC with increase in methylation of CpG 2 and CpG 3 in hRFT2gene promoter regions, which may relate to the genesis of CSCC. Our results suggested that epigenetic modifications are responsible for aberrant expression of the hRFT2 gene, and may help to understand mechanisms of cervical carcinogenesis.

• BMB Reports
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• v.43 no.6
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• pp.400-406
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• 2010

Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5'-aza-2'-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.9
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• pp.4071-4075
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• 2015

Background: Retinoblastoma protein-interacting zinc finger gene 1(RIZ1) functions as a tumor suppressor. Hypermethylation-mediated RIZ1 silencing has been reported in several cancers, but not in renal cell carcinoma (RCC) yet. Materials and Methods: We examined the RIZ1 expression and methylation in a panel of RCC cell lines and 50 primary tumors using semiquantitative/quantitative polymerase chain reaction (PCR), methylation specific PCR, and bisulfite sequencing genomic. We also explored the relationship between methylation status of RIZ1 and clinicopathological features in RCC patients. Results: RIZ1 expression was down-regulated or lost in OS-RC-2, 769-P, Caki-1, 786-O and A498 RCC cell lines. Restored expression of RIZ1 was detected after addition of 5-aza-2'-deoxycytidine with/without trichostatin A, suggesting that DNA methylation directly mediates its silencing. The RIZ1 expression was significantly reduced in RCCs compared to adjacent non-malignant renal samples (P<0.001). Aberrant methylation was detected in 15 of 50 (30%) RCCs and in 2 of 28 (7%) adjacent non-malignant renal samples (P=0.02). No statistically significant correlation between methylated and unmethylated cases with regard to age, gender, pathological stage and grade was observed. Conclusions: RIZ1 expression is down-regulated in human RCC, and this down-regulation is associated with methylation. RIZ1 methylation may play a role in renal carcinogenesis.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.7
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• pp.1037-1043
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• 2016

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

• Asian-Australasian Journal of Animal Sciences
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• v.32 no.2
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• pp.170-175
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• 2019

Objective: Uncoupling protein 3 gene (UCP3) is a candidate gene associated with the meat quality of pigs. The aim of this study was to explore the regulation mechanism of UCP3 expression and provide a theoretical basis for the research of the function of porcine UCP3 gene in meat quality. Methods: Bisulfite sequencing polymerase chain reaction (PCR) and quantitative real-time PCR (Q-PCR) were used to analyze the methylation of UCP3 5′-flanking region and UCP3 mRNA expression in the adipose tissue or skeletal muscle of three pig breeds at different ages (1, 90, 210-day-old Putian Black pig; 90-day-old Duroc; and 90-day-old Dupu). Results: Results showed that two cytosine-guanine dinucleotide (CpG) islands are present in the promoter region of porcine UCP3 gene. The second CpG island located in the core promoter region contained 9 CpG sites. The methylation level of CpG island 2 was lower in the adipose tissue and skeletal muscle of 90-day-old Putian Black pigs compared with 1-day-old and 210-day-old Putian Black pigs, and the difference also existed in the skeletal muscle among the three 90-day-old pig breeds. Furthermore, the obvious changing difference of UCP3 mRNA expression was observed in the skeletal muscle of different groups. However, the difference of methylation status and expression level of UCP3 gene was not significant in the adipose tissue. Conclusion: Our data indicate that UCP3 mRNA expression level was associated with the methylation status of UCP3 promoter in the skeletal muscle of pigs.

• Experimental and Molecular Medicine
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• v.50 no.12
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• pp.1.1-1.14
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• 2018

DNA methylation is a regulatory mechanism in epigenetics that is frequently altered during human carcinogenesis. To detect critical methylation events associated with gastric cancer (GC), we compared three DNA methylomes from gastric mucosa (GM), intestinal metaplasia (IM), and gastric tumor (GT) cells that were microscopically dissected from an intestinal-type early gastric cancer (EGC) using methylated DNA binding domain sequencing (MBD-seq) and reduced representation bisulfite sequencing (RRBS) analysis. In this study, we focused on differentially methylated promoters (DMPs) that could be directly associated with gene expression. We detected 2,761 and 677 DMPs between the GT and GM by MBD-seq and RRBS, respectively, and for a total of 3,035 DMPs. Then, 514 (17%) of all DMPs were detected in the IM genome, which is a precancer of GC, supporting that some DMPs might represent an early event in gastric carcinogenesis. A pathway analysis of all DMPs demonstrated that 59 G protein-coupled receptor (GPCR) genes linked to the hypermethylated DMPs were significantly enriched in a neuroactive ligand-receptor interaction pathway. Furthermore, among the 59 GPCRs, six GI hormone receptor genes (NPY1R, PPYR1, PTGDR, PTGER2, PTGER3, and SSTR2) that play an inhibitory role in the secretion of gastrin or gastric acid were selected and validated as potential biomarkers for the diagnosis or prognosis of GC patients in two cohorts. These data suggest that the loss of function of gastrointestinal (GI) hormone receptors by promoter methylation may lead to gastric carcinogenesis because gastrin and gastric acid have been known to play a role in cell differentiation and carcinogenesis in the GI tract.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.22 no.2
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• pp.164-173
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• 2000

The p16 protein is a cyclin dependent kinase inhibitor that inhibits cell cycle progression from $G_1$ phase to S phase in cell cycle. Many p16 gene mutations have been noted in many cancer-cell lines and in some primary cancers, and alterations of p16 gene function by DNA methylation have been noticed in various kinds of cancer tissues and cell-lines. There have been a large body of literature has accumulated indicating that abnormal patterns of DNA methylation (both hypomethylation and hypermethylation) occur in a wide variety of human neoplasma and that these aberrations of DNA methylation may play an important epigenetic role in the development and progression of neoplasia. DNA methylation is a part of the inheritable epigenetic system that influences expression or silencing of genes necessary for normal differentiation and proliferation. Gene activity may be silenced by methylation of up steream regulatory regions. Reactivation is associated with demethylation. Although evidence or a high incidence of p16 alterations in a variety of cell lines and primary tumors has been reported, that has been contested by other investigators. The precise mechanisms by which abnormal methylation might contribute to carcinogenesis are still not fully elucidated, but conceivably could involve the modulation of oncogene and other important regulatory gene expression, in addition to creating areas of genetic instability, thus predisposing to mutational events causing neoplasia. There have been many variable results of studies of head and neck squamous cell carcinoma(HNSCC). This investigation was studied on 13 primary HNSCC for p16 gene status by protein expression in immunohistochemistry, and DNA genetic/epigenetic analyzed to determine the incidence, the mechanisms, and the potential biological significance of its Inactivation. As methylation detection method of p16 gene, the methylation specific PCR(MSP) is sensitive and specific for methylation of any block of CpG sites in a CpG islands using bisulfite-modified DNA. The genomic DNA is modified by treatment with sodium bisulfate, which converts all unmethylated cytosines to uracil(thymidine). The primers designed for MSP were chosen for regions containing frequent cytosines (to distinguish unmodified from modified DNA), and CpG pairs near the 5' end of the primers (to provide maximal discrimination in the PCR between methylated and unmethylated DNA). The two strands of DNA are no longer complementary after bisulfite treatment, primers can be designed for either modified strand. In this study, 13 paraffin embedded block tissues were used, so the fragment of DNA to be amplified was intentionally small, to allow the assessment of methylation pattern in a limited region and to facilitate the application of this technique to samlples. In this 13 primary HNSCC tissues, there was no methylation of p16 promoter gene (detected by MSP and automatic sequencing). The p16 protein-specific immunohistochemical staining was performed on 13 paraffin embedded primary HNSCC tissue samples. Twelve cases among the 13 showed altered expression of p16 proteins (negative expression). In this study, The author suggested that low expression of p16 protein may play an important role in human HNSCC, and this study suggested that many kinds of genetic mechanisms including DNA methylation may play the role in carcinogenesis.

• Reproductive and Developmental Biology
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• v.31 no.2
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• pp.83-90
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• 2007

Insulin-like growth factor II (IGF2) and H19 genes are mutually imprinted genes which may be responsible for abnormalities in the cloned fetuses and offspring. This study was performed to identify putative differentially methylated regions (DMRs) of porcine H19 locus and to explore its genomic imprinting in in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) embryos. Based on mice genomic data, we identified DMRs on H19 and found porcine H19 DMRs that included three CTCF binding sites. Methylation patterns in IVF and SCNT embryos at the 2-, 4-, $8{\sim}16$-cells and blastocyst stages were analyzed by BS (Bisulfite Sequencing)-PCR. The CpGs in CTCF1 was significantly unmethylated in the 2-cell stage IVF embryos. However, the 4- (29.1%) and $8{\sim}16$-cell (68.2%) and blastocyst (48.2%) stages showed higher methylation levels (p<0.01). On the other hand, SCNT embryos were unmethylayted ($0{\sim}2%$) at all stages of development. The CpGs in CTCF2 showed almost unmethylation levels at the 2-,4- and $8{\sim}16$-cell and blastocyst stages of development in both IVF ($0{\sim}14.1%$) and SCNT ($0{\sim}6.4%$) embryos. At all stages of development, CTCF3 was unmethylated in IVF ($0{\sim}17.3%$) and SCNT ($0{\sim}1.2%$) embryos except at the blastocyst stage (54.5%) of IVF embryos. In conclusion, porcine SCNT embryos showed an aberrant methylation pattern comprised to IVF embryos. Therefore, we suggest that the aberrant methylation pattern of H19 loci may be a reason for increased abnormal fetus after embryo transfer of porcine SCNT embryos.

• Reproductive and Developmental Biology
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• v.34 no.3
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• pp.181-184
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• 2010

Osmolarity of culture media is one of the most important factors affecting in vitro development. This study was conducted to investigate the DNA methylation status of Pre-1 and satellite sequence in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaG or 0.05M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. Previous our studies have reported that pNT embryos cultured in both hypertonic media showed significantly higher blastocyst formation rate than that of control. The DNA methylation status of the satellite sequences in blastocyst was characterized using bisulfite-sequencing technology. The satellite region had a similar methylation pattern of in vivo blastocyst among two culture groups excepting the control group. Each level of methylation is that the satellite DNA moderately methylated (43.10% of PZM-3; 56.12% of NaCl; 55.06% of sucrose; 60.00% of in vivo embryos). As a result of the sequence of PRE-1, CpG methylation pattern was similar to three groups, including in vivo group. In case of the satellite DNA region, the osmolarity conditions were affected CpG DNA methylation status while PRE-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. These results indicate that the modification of osmolarity in a culture media may influence to spatially change of DNA methylation of repetitive sequence for pNT embryo development.

• Reproductive and Developmental Biology
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• v.35 no.1
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• pp.93-97
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• 2011

Previously, we reported that the osmolarity conditions in the satellite region were affected CpG DNA methylation status while Pre-1 sequence was not affected CpG DNA methylation in pNT blastocyst stage. This study was conducted to investigate the DNA methylation status of repeat sequences in pig nuclear transfer (pNT) embryos produced under different osmolarity culture conditions. Control group of pNT embryos was cultured in PZM-3 for six days. Other two treatment groups of pNT embryos were cultured in modified PZM-3 with 138 mM NaCl or 0.05 M sucrose (mPZM-3, 320 mOsmol) for two days, and then cultured in PZM-3 (270 mOsmol) for four days. The DNA methylation status of the Pre-1 sequences in blastocysts was characterized using a bisulfite-sequencing method. Intriguingly, in the present study, we found the unique DNA methylation at several non-CpG sequences at the Pre-1 sequences in all groups. The non-CpG methylation was hypermethylated in all three groups, including in vivo group (86.90% of PZM-3; 83.87% of NaCl; 84.82% of sucrose; 90.94% of in vivo embryos). To determine whether certain non-CpG methylated sites were preferentially methylated, we also investigated the methylation degree of CpA, CpT and CpC. Excepting in vivo group, preference of methylation was CpT>CpC>CpA in all three groups investigated. These results indicate that DNA methylation of Pre-1 sequences was hypermethylated in CpG as well as non-CpG site, regardless modification of osmolarity in a culture media.