• Biomolecules & Therapeutics
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• 제28권5호
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• pp.405-413
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• 2020

Fibroblast-like synoviocytes (FLS) play a crucial role in initiating rheumatoid arthritis. B-cell activating factor (BAFF) plays a role in FLS survival as well as in B cell maturation and maintenance. Here, we investigated whether tumor necrosis factor (TNF)-α-induced BAFF expression controls FLS migration and whether BAFF expression in FLS could be regulated by KR33426 which is the inhibitor of BAFF binding to BAFF receptors (BAFF-R) by using MH7A synovial cells transfected with the SV40 T antigen. More TNF-α-treated cells migrated compared to the control. TNF-α increased BAFF expression in FLS, significantly. FLS migration was inhibited by the transfection with BAFF-siRNA. KR33426 also inhibited BAFF expression increased by TNF-α treatment in FLS as judged by western blotting, PCR, and transcriptional activity assay. Kinases including JNK, p38 and Erk were activated by TNF-α treatment. While JNK and p38 were inhibited by KR33426 treatment, no changes in Erk were observed. Transcription factors including p65, c-Fos, CREB and SP1 were enhanced by TNF-α treatment. Among them, c-Fos was inhibited by KR33426 treatment. Small interference(si)-RNA of c-fos decreased BAFF transcriptional activity. FLS migration induced by TNF-α was inhibited by the transfection with BAFF-siRNA. KR33426 increased Twist, Snail, Cadherin-11 and N-Cadherin. In contrast, KR33426 decreased E-cadherin and TNF-α-enhanced CCL2. Taken together, our results demonstrate that synovial cell migration via CCL2 expression could be regulated by BAFF expression which is decreased by KR33426 and c-Fos-siRNA. It suggests for the first time that the role of BAFF-siRNA on FLS migration might be matched in the effect of KR33426 on BAFF expression.

• Journal of Preventive Medicine and Public Health
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• 제45권4호
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• pp.251-258
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• 2012

Objectives: The purpose of this paper was to elucidate the potential methylation levels of adjacent normal and cancer tissues by comparing them with normal colorectal tissues, and to describe the correlations between the methylation and clinical parameters in Korean colorectal cancer (CRC) patients. Methods: Hypermethylation profiles of nine genes (RASSF1, APC, $p16^{INK4a}$, Twist1, E-cadherin, TIMP3, Smad4, COX2, and ABCB1) were examined with 100 sets of cancer tissues and 14 normal colorectal tissues. We determined the hypermethylation at a given level by a percent of methylation ratio value of 10 using quantitative methylation real-time polymerase chain reaction. Results: Nine genes' hypermethylation levels in Korean CRC patient tissues were increased more higher than normal colorectal tissues. However, the amounts of $p16^{INK4a}$ and E-cadherin gene hypermethylation in normal and CRC tissues were not significantly different nor did TIMP3 gene hypermethylation in adjacent normal and cancer tissues differ significantly. The hypermethylation of TIMP3, Ecadherin, ABCB1, and COX2 genes among other genes were abundantly found in normal colorectal tissues. The hypermethylation of nine genes' methylation in cancer tissues was not significantly associated with any clinical parameters. In Cohen's kappa test, it was moderately observed that RASSF1 was related with E-cadherin, and Smad4 with ABCB1 and COX2. Conclusions: This study provides evidence for different hypermethylation patterns of cancer-associated genes in normal and CRC tissues, which may serve as useful information on CRC cancer progression.

• The Korean Journal of Physiology and Pharmacology
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• 제26권6호
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• pp.479-499
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• 2022

The lack of a clonal renin-secreting cell line has greatly hindered the investigation of the regulatory mechanisms of renin secretion at the cellular, biochemical, and molecular levels. In the present study, we investigated whether it was possible to induce phenotypic switching of the renin-expressing clonal cell line As4.1 from constitutive inactive renin secretion to regulated active renin secretion. When grown to postconfluence for at least two days in media containing fetal bovine serum or insulin-like growth factor-1, the formation of cell-cell contacts via N-cadherin triggered downstream cellular signaling cascades and activated smooth muscle-specific genes, culminating in phenotypic switching to a regulated active renin secretion phenotype, including responding to the key stimuli of active renin secretion. With the use of phenotype-switched As4.1 cells, we provide the first evidence that active renin secretion via exocytosis is regulated by phosphorylation/dephosphorylation of the 20 kDa myosin light chain. The molecular mechanism of phenotypic switching in As4.1 cells described here could serve as a working model for full phenotypic modulation of other secretory cell lines with incomplete phenotypes.

• BMB Reports
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• 제56권2호
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• pp.120-125
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• 2023

Karyopherin-α3 (KPNA3), a karyopherin-α isoform, is intimately associated with metastatic progression via epithelial-mesenchymal transition (EMT). However, the molecular mechanism underlying how KPNA3 acts as an EMT inducer remains to be elucidated. In this report, we identified that KPNA3 was significantly upregulated in cancer cells, particularly in triple-negative breast cancer, and its knockdown resulted in the suppression of cell proliferation and metastasis. The comprehensive transcriptome analysis from KPNA3 knockdown cells indicated that KPNA3 is involved in the regulation of numerous EMT-related genes, including the downregulation of GATA3 and E-cadherin and the up-regulation of HAS2. Moreover, it was found that KPNA3 EMT-mediated metastasis can be achieved by TGF-β or AKT signaling pathways; this suggests that the novel independent signaling pathways KPNA3-TGF-β-GATA3-HAS2/E-cadherin and KPNA3-AKT-HAS2/E-cadherin are involved in the EMT-mediated progress of TNBC MDA-MB-231 cells. These findings provide new insights into the divergent EMT inducibility of KPNA3 according to cell and cancer type.

• Clinical and Experimental Pediatrics
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• 제52권8호
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• pp.944-952
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• 2009

목 적 : Epithelial to mesenchymal transition (EMT)은 태생기에 있어 필수 불가결한 발달과정일 뿐 아니라, 신 섬유화에 있어서도 중요한 역할을 하며, nestin은 고전적인 줄기세포 표지자로 신 세뇨관 간질 손상에 있어 새로운 표지자로 밝혀지고 있다. 신생 백서 신장에서 Angiotensin (Ang) II가 EMT에 미치는 영향을 알아보고자, 안지오텐신 전환 효소 억제제를 투여한 신생 백서의 신장에서 EMT 표지자 및 nestin의 발현 양상을 조사하였다. 방 법 : 7일 동안 신생 백서에게 enalapril (30 mg/kg/d) 또는 vehicle을 투여하였으며, ${\alpha}-smooth$ muscle actin (SMA), E-cadherin, vimentin 및 nestin에 대한 면역 조직 화학 염색을 시행하였다. 결 과 : enalapril 투여군에서 대조군에 비해 신 피질 및 수질 모두에서 ${\alpha}-SMA$ 발현이 증가하였으며, 이는 확장된 세뇨관 상피 세포에서 뚜렷하였다(P<0.05). E-cadherin 발현은 enalapril 투여군의 신 피질 및 수질의 세뇨관 상피 세포에서 확연히 감소하였다(P<0.05). vimentin 및 nestin 발현은 신 피질에서는 양군간의 차이가 없었으나, 신 수질에서는 enalapril 투여군에서 세뇨관 간질 세포에서 발현이 의미있게 증가하였다(P<0.05). 결 론 : 신생 백서 신장에서 Ang II 억제는 ${\alpha}-SMA$ 발현을 증가시키고, E-cadherin 발현을 감소시킴으로써 발달하는 신장의 EMT를 증가시켰다. Enalapril 투여는 또한 신 수질에서 vimentin과 nestin의 발현을 증가시켰으며, 이는 신생 백서 신장에서의 Ang II 억제로 인한 EMT 과정 중 신 수질의 변화가 더욱 뚜렷한 것을 시사하며, Ang II 억제가 EMT 표지자들의 발현을 다르게 변화시키는 것으로 사료된다.

• Tuberculosis and Respiratory Diseases
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• 제79권3호
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• pp.143-152
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• 2016

Background: Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal lung disease characterized by the accumulation of excessive fibroblasts and myofibroblasts in the extracellular matrix. The transforming growth factor ${\beta}1$ (TGF-${\beta}1$)-induced epithelial-to-mesenchymal transition (EMT) is thought to be a possible source of fibroblasts/myofibroblasts in IPF lungs. We have previously reported that apolipoprotein A1 (ApoA1) has anti-fibrotic activity in experimental lung fibrosis. In this study, we determine whether ApoA1 modulates TGF-${\beta}1$-induced EMT in experimental lung fibrosis and clarify its mechanism of action. Methods: The A549 alveolar epithelial cell line was treated with TGF-${\beta}1$ with or without ApoA1. Morphological changes and expression of EMT-related markers, including E-cadherin, N-cadherin, and ${\alpha}$-smooth muscle actin were evaluated. Expressions of Smad and non-Smad mediators and TGF-${\beta}1$ receptor type 1 ($T{\beta}RI$) and type 2 ($T{\beta}RII$) were measured. The silica-induced lung fibrosis model was established using ApoA1 overexpressing transgenic mice. Results: TGF-${\beta}1$-treated A549 cells were changed to the mesenchymal morphology with less E-cadherin and more N-cadherin expression. The addition of ApoA1 inhibited the TGF-${\beta}1$-induced change of the EMT phenotype. ApoA1 inhibited the TGF-${\beta}1$-induced increase in the phosphorylation of Smad2 and 3 as well as that of ERK and p38 mitogen-activated protein kinase mediators. In addition, ApoA1 reduced the TGF-${\beta}1$-induced increase in $T{\beta}RI$ and $T{\beta}RII$ expression. In a mouse model of silica-induced lung fibrosis, ApoA1 overexpression reduced the silica-mediated effects, which were increased N-cadherin and decreased E-cadherin expression in the alveolar epithelium. Conclusion: Our data demonstrate that ApoA1 inhibits TGF-${\beta}1$-induced EMT in experimental lung fibrosis.

• Journal of Gastric Cancer
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• 제6권4호
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• pp.227-236
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• 2006

목적: 유전자 메틸화는 유전자의 서열에 영향을 주지 않으면서 유전자의 발현을 억제하고 세포분열 후 그대로 보존되는 후성적 변화이다. 위암조직과 정상위조직에서 hMLH1, p16, p14, COX-2, MGMT, E-cadherin 유전자와 MINT (MINT1, 2, 12, 25, 31)의 메틸화 상태를 검사하여 위암의 발생 과정에서의 작용과 CIMP 및 Helicobacter pylori균 감염을 포함한 임상병리학적인자와의 연관성을 알아보고자 하였다. 대상 및 방법: 위암과 정상위 신선 동결 조직 각각 36예를 대상으로 MSP (methylation-specific PCR)방법을 이용하여 메틸화 상태를 분석하였고 CIMP의 분석은 MINT1, MINT2, MINT12, MINT25, MINT31의 5개 marker를 대상으로 시행하였다. Helicobacter pylori균 감염여부는 Warthin-Starry silver 염색을 통하여 분류하였다. 결과: 위암 관련 유전자인 p14, p16, MGMT, COX-2, E-cadherin, hMLH1의 메틸화는 각각 14예(38.9%), 13예(36.1%), 8예(22.2%), 10예(27.8%), 21예(58.3%), 6예(16.7%)였다. MINT1과 MINT25의 메틸화는 위암조직에서 정상위조직에서보다 통계학적으로 유의하게 높게 관찰되었다. CIMP 양성률은 위암조직에서 44.4%로 높게 나타났으며 CIMP-H 위암은 환자의 연령과 종양크기와 연관이 있었다. CIMP 양성 위암은 p16 유전자의 메틸화와 연관이 있었고 p16 유전자의 메틸화는 조직학적으로 저분화, 미만형, 궤양형성하는 위암에서 낮게 나타났다. MINT1의 메틸화는 Helicobacter pylori균과 연관성이 있었다. 결론: 위암에서 hMLH1, p16, p14, COX-2, MGMT, E-cadherin, MINT (MINT1, 2, 12, 25, 31)의 불활성화에 DNA 메틸화가 작용함을 알 수 있었고, Helicobacter pylori균에 의한 위암발생에 MINT1의 메틸화가 연관이 있음을 알 수 있었다.

• 생명과학회지
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• 제29권8호
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• pp.904-915
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• 2019

전립선암은 전이성 종양 중의 하나로 치료를 위해 호르몬 요법이나 외과 적 거세 방법이 주로 수행되지만 많은 부작용을 나타내었다. 최근 많은 연구자들이 이러한 상황을 해결하기 위해 종양 미세 환경을 연구하고 있으며 그 중 면역 세포, 특히 대식세포는 종양 미세 환경의 중요한 구성요소이다. 정상적인 조건에서 대식세포는 여러 암세포에 대해 약한 종양 살균 활성을 갖으나 $interferon-{\gamma}$ 또는 lipopolysaccharide에 의해 활성화되면, 염증성 사이토카인 및 케모카인을 분비함으로써 암세포를 직접 또는 간접적으로 사멸 시키게 된다. 본 연구에서는, 마우스 대식세포인 RAW 264.7 세포에 Phellinus linteus 추출물을 처리하여 산화질소의 방출과 pro-inflammatory cytokine들을 real-time PCR과 ELISA 방법으로 분석하였다. RAW 264.7의 조정 배지는 48시간 동안 전립선 암세포처리하여 상피간엽세포전이 관련 유전자의 발현을 측정 하였다. 그 때에 mesenchymal 관련 유전자들인 N-cadherin, snail, twist, slug 및 cadherin 11이 감소했을 뿐만 아니라 epithelial 관련 유전자인 E-cadherin은 증가하였다. 또한 암 전이 및 신생 혈관 형성에 관여하는 vimentin, ccl2 및 vegfa가 감소되었는데, 이는 EMT가 암세포의 이동과 침범에 밀접한 관련이 있기 때문이다. 따라서 Phellinus linteu에 의해 자극된 RAW 264.7 세포의 조정 배지는 인간 전립선 암세포주인 PC-3 세포의 이동과 전이를 억제하고 EMT 경로를 조절한다는 것을 나타낸다.

• Tuberculosis and Respiratory Diseases
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• 제64권1호
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• pp.22-27
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• 2008

연구배경: 본 연구에서는 황색포도알균이 숙주세포 내로 침입하여 증식하는 정도를 관찰하고자 한다. 이때 세포 외 바탕 단백질의 변화가 수반될 것으로 가설을 설정하고, 이러한 변화에 영향을 미칠 것으로 생각되는 matrix metalloproteinase (MMP)의 발현과 역할에 대해 연구하고자 하였다. 방법: 황색포도알균은 $10^6{\sim}10^7CFU/ml$을 $10^5$개의 기관지상피세포인 BEAS-2B 세포에 2시간 동안 침입시킨다. 이후 세척으로 세포 밖에 있는 황색포도알균을 제거한 후, BEAS-2B 세포를 다양한 시간 동안(4, 6, 8, 12 시간) 배양한 후 황색포도알균의 집락수(CFU/ml)를 측정하였고, 단백질을 분리하여 세포 외 바탕단백질의 발현 정도와 MMP의 활성도를 측정하였다. 또한 MMP 억제제인 GM6001을 전처치한 후 황색포도알균을 세포에 침입시킨 후 세포 내에서의 균의 집락수 및 세포 외 바탕단백질의 변화를 관찰하였다. 결과: 황색포도알균의 집락을 측정한 결과 4시간과 12시간을 비교해 볼 때 MOI가 증가할수록, 감염시킨 시간이 길수록 숙주세포 내로 침입이 유의하게 증가하였다. BEAS-2B 세포에서 황색포도알균을 침입시킨 시간이 길수록, MOI가 증가할수록 MMP 2 및 MMP 9의 활성도와 dysadherin의 발현은 증가하였고, 이와는 대조적으로 E-cadherin의 발현은 감소하였다. MMP억제제인 GM6001을 전 처치 한 결과 황색포도알균의 세포 내 침입을 유의하게 감소시켰다. 결론: 황색포도알균이 기관지 상피세포 내로 침입할 때 dysadherin 및 E-cadherin 같은 세포 외 바탕 단백질의 변화를 동반하며, MMP 활성도가 균의 세포 내 침입에 관여하는 것으로 보인다.

• Asian Pacific Journal of Cancer Prevention
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• 제16권9호
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• pp.4065-4069
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• 2015

Background: Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. Materials and Methods: EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NCPANC-1, and si-PANC-1 cells, respectively. Results: After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. Conclusions: Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.