• 생명과학회지
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• 제28권7호
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• pp.765-771
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• 2018

$PPAR{\gamma}$는 지방세포의 분화를 조절하는 핵심적인 전사 인자로서 이를 조절하는 후성유전학적 조절 기전이 비만억제 연구에서 중요하게 주목 받고 있다. 선행연구에서 CACUL1이 $PPAR{\gamma}$의 전사 활성 및 지방세포의 분화를 억제하는 corepressor로서 작용함을 밝힌 바 있으며 본 연구에서는 CACUL1의 새로운 결합 단백질로 발굴된 protein arginine methyltransferase 5 (PRMT5)의 $PPAR{\gamma}$ 조절 기능을 분석하였다. PRMT5가 CACUL1과 결합함을 immunoprecipitation assay in vivo와 GST-pull down assay in vitro를 통하여 확인하였다. Luciferase reporter assay 결과로 두 단백질이 상호 협력하여 $PPAR{\gamma}$의 전사 활성을 억제함을 확인하였다. PRMT5가 안정적으로 과발현 또는 knockdown되는 3T3-L1 세포주를 제작하여 지방세포 분화에 미치는 영향을 분석한 결과, PRMT5가 3T3-L1세포의 지방세포 분화를 억제함을 증명하였다. 같은 맥락으로 PRMT5는 $PPAR{\gamma}$의 타겟 유전자인 Lpl과 aP2의 발현을 억제하는 것을 RT-qPCR로 확인하였다. 이상의 연구 결과로 PRMT5이 CACUL1과 결합하여 $PPAR{\gamma}$의 전사 활성을 방해, 나아가 지방세포의 분화를 억제하는 기존에 알려지지 않은 분자적 기전을 처음으로 밝혔다. 따라서, PRMT5 효소 활성의 조절은 비만 억제를 위한 약물 개발에 단서를 제공할 것이다.

• Journal of Gastric Cancer
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• 제13권4호
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• pp.232-241
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• 2013

Purpose: Gastrokine 1 plays an important role in gastric mucosal defense. Additionally, the Gastrokine 1-miR-185-DNMT1 axis has been shown to suppress gastric carcinogenesis through regulation of epigenetic alteration. Here, we investigated the effects of Gastrokine 1 on DNA methylation and gastritis. Materials and Methods: Expression of Gastrokine 1, DNMT1, EZH2, and c-Myc proteins, and the presence of Helicobacter pylori CagA protein were determined in 55 non-neoplastic gastric mucosal tissue samples by western blot analysis. The CpG island methylation phenotype was also examined using six markers (p16, hMLH1, CDH1, MINT1, MINT2 and MINT31) by methylation-specific polymerase chain reaction. Histological gastritis was assessed according to the updated Sydney classification system. Results: Reduced Gastrokine 1 expression was found in 20 of the 55 (36.4%) gastric mucosal tissue samples and was closely associated with miR-185 expression. The Gastrokine 1 expression level was inversely correlated with that of DNMT1, EZH2, and c-Myc, and closely associated with the degree of gastritis. The H. pylori CagA protein was detected in 26 of the 55 (47.3%) gastric mucosal tissues and was positively associated with the expression of DNMT1, EZH2, and c-Myc. In addition, 30 (54.5%) and 23 (41.9%) of the gastric mucosal tissues could be classified as CpG island methylation phenotype-low and CpG island methylation phenotype-high, respectively. Reduced expression of Gastrokine 1 and miR-185, and increased expression of DNMT1, EZH2, and c-Myc were detected in the CpG island methylation phenotype-high gastric mucosa. Conclusions: Gastrokine 1 has a crucial role in gastric inflammation and DNA methylation in gastric mucosa.

• 한국가금학회지
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• 제41권3호
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• pp.217-225
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• 2014

텔로미어는 진핵 세포의 염색체 말단으로, 직렬 반복 DNA 염기 서열과 shelterin 단백질 복합체로 구성되어 있다. 텔로미어의 기능은 염색체를 보호하는 것으로 체세포의 텔로미어 길이는 세포 분열시 DNA 복제 결실로 인해 연령이 증가함에 따라 감소하는 경향이 있다. 그러나 유전적, 후생유전학적 및 환경적 수준에서 여러 가지 요인이 텔로미어 길이에 영향을 미친다. 따라서 본 연구에서는 닭의 텔로미어 길이의 유전력을 추정하고, 이들의 유전전이 양상을 살펴보고자 하였다. 텔로미어 길이는 백혈구를 이용하여 양적 형광접합보인법(Q-FISH)과 양적 중합효소 연쇄반응법(qRT-PCR)으로 분석하였다. 분석 결과, 텔로미어 길이의 유전력은 자손과 부모 회귀 분석에 의해 출생 시 0.9로 추정되었고, 10 주령 및 30주령 때 부 분산 분석에 의해 0.03과 0.04로 추정되었다. 부와 자손 간(r=0.348) 및 모와 자손 간(r=0.380) 텔로미어 길이는 모두 유의한 정의 상관 관계를 보였다. 따라서 닭 텔로미어의 유전 전이 양상은 부모 양쪽 모두로부터 비슷하게 자식에 영향을 미치는 것으로 판단된다. 더불어 암수 자손에 미치는 영향 또한 유사한 것으로 나타났다. 이러한 결과는 부모의 텔로미어 길이의 각인이 성염색체의 유전자가 아닌 상염색체의 유전자에 의해 조절되는 것을 의미한다. 또한, 산모 연령에 따른 출생 자손의 텔로미어 길이는 차이가 없는 것으로 나타났다. 따라서 모체의 연령이 출생 자손의 텔로미어 길이에 영향을 미치지 않는데, 이는 수정란의 초기 배아 단계에서 세포적 reprogramming이 이루어지기 때문으로 사료된다.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
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• pp.27-27
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• 2003

A diversified and concentrative approach of methylation player can be one of the most powerful studies in the understanding of global epigenetic modifications. Previous studies have suggested that DNA methylation contributes to transcriptional silencing through the several DNA methylation-mediated repression systems by hypermethylation, including methyltransferases (DNMTs), DNA methyltransferase association protein 1 (DMAPl), methyl-CpG binding domain (MBD), and histone deacetylases (HDACs). Assembly of these regulatory protein complexes act sequentially, reciprocally, and interdependently on the newly composed DNA strand through S phase. Therefore, these protein complexes have a role in coupling DNA replication to the designed turn-off system in genome. In this study, we attempted to address the role of DNA methylation by the functional analysis of the methyltransferase molecule, we described the involvement of DMAP1 and DNMTs in cell divistion and the effect of their loss. We also described distinct patterns that DMAP1 and DNMTs are spatially reorganized and displaced from condensing chromosomes as cells progress through mitosis in HeLa cell, COS7, and HIH3T3 cell cycle progressions. DNMT1, DNMT3b, and DMAP1 do not stably contact the genetic material during chromosome compaction and repressive expression. These finding show that the loss of activities of DNMTs and DMAP1 occure stage specifically during the cell cycle, may contribute to the integral balance of global DNA methylation. This is consistent with previous studies resulted in decreased histone acetyltransferases and HDACs, and differs from studies resulted in increased histone methyltransferases. Our results suggest that DNA methylation by DNMTs and DMAP1 during mitosis acts to antagonize hypermethylation by which this mark is epigenetical mitotic-specific methylation.

• Asian-Australasian Journal of Animal Sciences
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• 제33권2호
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• pp.203-211
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• 2020

Objective: Staphylococcus aureus (S. aureus) is one of the major microorganisms responsible for subclinical mastitis in dairy cattle. The present study was designed with the aim to explore the DNA methylation patterns using the Fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) techniques in a S. aureus-infected mouse model. Methods: A total of 12 out-bred Institute of Cancer Research female mice ranging from 12 to 13 weeks-old were selected to construct a mastitis model. F-MSAP analysis was carried out to detect fluctuations of DNA methylation between control group and S. aureus mastitis group. Results: Visible changes were observed in white cell counts in milk, percentage of granulocytes, percentage of lymphocytes, CD⁴⁺/CD⁸⁺ ratio (CD⁴⁺/CD⁸⁺), and histopathology of mice pre- and post-challenge with S. aureus. These findings showed the suitability of the S. aureus-infected mouse model. A total of 369 fragments was amplified from udder tissue samples from the two groups (S. aureus-infected mastitis group and control group) using eight pairs of selective primers. Results indicated that the methylation level of mastitis mouse group was higher than that in the control group. In addition, NCK-associated protein 5 (Nckap5) and transposon MTD were identified to be differentially methylated through secondary polymerase chain reaction and sequencing in the mastitis group. These observations might play an important role in the development of S. aureus mastitis. Conclusion: Collectively, our study suggests that the methylation modification in Nckap5 and transposon MTD might be considered as epigenetic markers in resistance to S. aureus-infected mastitis and provided a new insight into S. aureus mastitis research in dairy industry and public health.

• Asian Pacific Journal of Cancer Prevention
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• 제15권23호
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• pp.10255-10262
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• 2015

High risk human papillomavirus (HR-HPV) E2 proteins play roles in transcriptional regulation and are commonly functionally disrupted when the HPV genome integrates into host chromosomes. Some 15-40% of cancer cases, however, contain an intact E2 gene or episomal HPV. In these cases, polymorphism of the E2 gene might be involved. This study aimed to determine polymorphisms of the E2 gene in episomal HPV16 detected in high grade squamous intraepithelial lesions and squamous cell carcinomas and altered functions compared to the E2 prototype. The E2 gene was amplified and sequenced. Two expression vectors containing E2 gene polymorphisms were constructed and transfected in SiHa and C33A cells, then E6 gene as well as Il-10 and TNF-${\alpha}$ expression was determined by quantitative RT-PCR. Expression vectors and reporter vectors containing the HPV16 long control region (LCR) were co-transfected and transcriptional activity was determined. The results showed that a total of 32 nucleotides and 23 amino acids were changed in all 20 cases of study, found in the transactivation (TA) domain, hinge (H) region and DNA binding (DB) domain with 14, 5 and 13 nucleotide positions. They mostly caused amino acid change. The expressing vectors containing different E2 gene polymorphisms showed E6 mRNA suppression, TNF-${\alpha}$ mRNA suppression and IL-10 induction but no statistically significant differences when compared to the E2 prototype. Moreover, promoter activity in HPV16 LCR was not affected by E2 protein with different gene polymorphisms, in contrast to nucleotide variations in LCR that showed an effect on transcription activity. These results demonstrated that E2 gene polymorphisms of episomal HPV16 did not affect transcriptional regulation and suggested that nucleotide variation as well as epigenetic modification of the LCR might play a role in inducing malignant transformation of cells containing episomal HPV16.

• Asian Pacific Journal of Cancer Prevention
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• 제15권1호
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• pp.75-84
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• 2014

Aberrant DNA methylation of tumor suppressor genes has been reported in all major types of leukemia with potential involvement in the inactivation of regulatory cell cycle and apoptosis genes. However, most of the previous reports did not show the extent of concurrent methylation of multiple genes in the four leukemia types. Here, we analyzed six key genes (p14, p15, p16, p53, DAPK and TMS1) for DNA methylation using methylation specific PCR to analyze peripheral blood of 78 leukemia patients (24 CML, 25 CLL, 12 AML, and 17 ALL) and 24 healthy volunteers. In CML, methylation was detected for p15 (11%), p16 (9%), p53 (23%) and DAPK (23%), in CLL, p14 (25%), p15 (19%), p16 (12%), p53 (17%) and DAPK (36%), in AML, p14 (8%), p15 (45%), p53 (9%) and DAPK (17%) and in ALL, p15 (14%), p16 (8%), and p53 (8%). This study highlighted an essential role of DAPK methylation in chronic leukemia in contrast to p15 methylation in the acute cases, whereas TMS1 hypermethylation was absent in all cases. Furthermore, hypermethylation of multiple genes per patient was observed, with obvious selectiveness in the 9p21 chromosomal region genes (p14, p15 and p16). Interestingly, methylation of p15 increased the risk of methylation in p53, and vice versa, by five folds (p=0.03) indicating possible synergistic epigenetic disruption of different phases of the cell cycle or between the cell cycle and apoptosis. The investigation of multiple relationships between methylated genes might shed light on tumor specific inactivation of the cell cycle and apoptotic pathways.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7529-7536
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• 2013

Background and Aims: MicroRNA-21 (miR-21) is reported to be overexpressed and to contribute to proliferation, apoptosis and gemcitabine resistance in pancreatic ductal adenocarcinomas (PDACs). The aims of this study were to explore regulation of miR-21 expression by epigenetic change and its impact on chemoresistance and malignant properties of of pancreatic cancer. Materials and methods: We retrospectively collected 41 cases of advanced pancreatic cancer patients who were sensitive or resistant to gemcitabine and assessed levels of serum circulating miR-21 for correlation with cytotoxic activity. Histone acetylation in the miR-21 promoter was also studied in gemcitabine-sensitive and gemcitabine-resistant PDAC cells. Gemcitabine-resistant HPAC and PANC-1 cells were transfected with pre-miR-21 precursors (pre-miR-21) and antisense oligonucleotides (anti-miR-21), and were treated with TSA. Finally, invasion and metastasis assays were performed and alteration in mir-21, PTEN, AKT and pAKT level was evaluated in these cells. Results: Serum miR-21 levels were increased in gemcitabine-resistant PDAC patients compared with gemcitabine-sensitive subjects. The miR-21 levels were increased in 6 PDAC cells treated with gemcitabine significantly, associated with 50% inhibitory concentrations ($IC_{50}s$). Histone acetylation levels at miR-21 promoter were increased in PDAC cells after treatment with gemcitabine. Enhanced invasion and metastasis, increased miR-21 expression, decreased PTEN, elevated pAKT level were demonstrated in gemcitabine-resistant HPAC and PANC-1 cells. Pre-miR-21 transfection or TSA treatment further increased invasion and metastasis ability, decreased PTEN, and elevated pAKT levels in these two lines. In contrast, anti-miR-21 transfection could reverse invasion and metastasis, and PTEN and pAKT expressions induced by gemcitabine. Conclusions: MiR-21 upregulation induced by histone acetylation in the promoter zone is associated with chemoresistance to gemcitabine and enhanced malignant potential in pancreatic cancer cells.

• Maxillofacial Plastic and Reconstructive Surgery
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• 제19권4호
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• pp.414-434
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• 1997

Though many genetic and epigenetic alterations have been identified in hamster oral carcinogenesis model, there is no information about the possible role of transforming growth factor related with oral cancer. The purpose of this paper was to find the expression patterns of transforming growth factor alpha and beta during the stages of complete oral carcinogenesis model in hamster. 0.5% 9, 10-dimethyl-1, 2-benzanthracene(DMBA) in mineral oil was topically applied to the buccal pouch of 75 hamster three times a week during the experimental periods. The experimental animals were subdivided into two groups of control and experiment. Only the mineral oil was applied to the control group. 0.5% DMBA in mineral oil was applied to the experimental groups of 6, 8, 10, 12, 14, 16, 18 and 20 weeks. The expression of the $TGF-{\alpha}$ and $TGF-{\beta}$ protein were evaluated by the distribution and intensity of positive cells during the carcinogenesis using the immunohistochemical study. The following results were obtained ; 1. The buccal pouch epithelium of hamster was histologically changed to the dysplasia at 6, 8, 10 weeks, carcinoma in situ at 12 weeks, and squamous cell carcinoma at 14 weeks. 2. The expression of the $TGF-{\alpha}$ was restricted to the parabasal and basal layers of the normal and dysplastic mucosa, but those positive cells were extended to the spinous layers of the epithelium in the carcinoma. 3. The degree of $TGF-{\alpha}$ expression was markedly decreased in the carcinoma at 16, 18, 20. The strong positive staining in the center of cancer islands and weak positive staining in periphery of tumor were seen at the stage of squamous cell carcinoma. 4. The positive index of the $TGF-{\alpha}$ had a tendency to increase with DMBA- applied time. There was a statistically significant difference between 12, 18, 20 experimental group and control group (p<0.05). 5. The expression of the $TGF-{\beta}$ was shown at the cytoplasm of all control and experimental groups, and the parabasal and basal layers of the normal and dyslastic mucosa, but it was shown at the basal layers of the epithelium in the carcinoma. 6. $TGF-{\beta}$ was expressed diffusely at 16, 18, 20 experimental group. The strong positive staining in the center of cancer islands and positive staining in periphery of tumor were seen at the stage of squamous cell carcinoma. From the above findings, the expression of $TGF-{\alpha}$ and ${\beta}$ in oral carcinogenesis model seems to have two formal stages, the first being an overexpression step as reaction to uncontrolled growth and the second being one in which external protein accumulate in the surrounding stroma and intracytoplasm. Overexpression of $TGF-{\alpha}$ and ${\beta}$ may have important cooperative roles for the promotion of cancer and factor of prognosis.

• Asian Pacific Journal of Cancer Prevention
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• 제16권2호
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• pp.541-549
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• 2015

Triple negative breast cancer (TNBC) is an aggressive subtype of breast cancer (BC) with higher metastatic rate and both local and systemic recurrence compared to non-TNBC. The generation of reactive oxygen species (ROS) secondary to oxidative stress is associated with DNA damage, chromosomal degradation and alterations of both hypermethylation and hypomethylation of DNA. This study concerns differential methylation of promoter regions in specific groups of genes in TNBC and non-TNBC Saudi females in an effort to understand whether epigenetic events might be involved in breast carcinogenesis, and whether they might be used as markers for Saudi BCs. Methylation of glutathione S-transferase P1 (GSTP1), T-cadherin (CDH13), Paired box protein 5 (PAX5), death associated protein kinase (DAPK), twist-related protein (TWIST), DNA-binding protein inhibitor (ID4), High In Normal-1 (HIN-1), cyclin-dependent kinase inhibitor 2A (p16), cyclin D2 and retinoic acid receptor-${\beta}$ ($RAR{\beta}1$) genes was analyzed by methylation specific polymerase chain reaction (MSP) in 200 archival formalin-fixed paraffin embedded BC tissues divided into 3 groups; benign breast tissues (20), TNBC (80) and non-TNBC (100). The relationships between methylation status, and clinical and pathological characteristics of patients and tumors were assessed. Higher frequencies of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 hypermethylation were found in TNBC than in non-TNBC. Hypermethylation of GSTP1, CDH13, ID4, DAPK, HIN-1 and PAX5 increased with tumor grade increasing. Other statistically significant correlations were identified with studied genes. Data from this study suggest that increased hypermethylation of GSTP1, ID4, TWIST, DAPK, PAX5 and HIN-1 genes in TNBC than in non-TNBC can act as useful biomarker for BCs in the Saudi population. The higher frequency of specific hypermethylated genes paralleling tumor grade, size and lymph node involvement suggests contributions to breast cancer initiation and progression.