• Title/Summary/Keyword: knock down

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Knockdown of GCF2/LRRFIP1 by RNAi Causes Cell Growth Inhibition and Increased Apoptosis in Human Hepatoma HepG2 Cells

  • Li, Jing-Ping;Cao, Nai-Xia;Jiang, Ri-Ting;He, Shao-Jian;Huang, Tian-Ming;Wu, Bo;Chen, De-Feng;Ma, Ping;Chen, Li;Zhou, Su-Fang;Xie, Xiao-Xun;Luo, Guo-Rong
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.6
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    • pp.2753-2758
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    • 2014
  • Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

Gene Expression Profiling by Ginsenoside Rb1 in Keratinocyte HaCaT Cells (피부각질세포 HaCaT에서 진세노사이드 Rb1에 의한 유전자 발현 양상)

  • Lee, Dong Woo;Kim, Jung Min;Bang, In Seok
    • Journal of Life Science
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    • v.29 no.5
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    • pp.514-523
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    • 2019
  • We investigated the gene expression patterns and the mechanisms of action of the apoptotic response by microarray analysis of human keratinocyte HaCaT cells treated with ginsenoside Rb1, a saponin of Panax ginseng C. A. Meyer. Genes related to apoptosis, the G2/M transition of the mitotic cell cycle, cell division, mitotic nuclear division, and intracellular protein transport were 2-fold up-regulated in HaCaT cells treated with the ginsenoside Rb1, whereas genes related to DNA repair, regeneration fission, and extracellular matrix organization were 2-fold down-regulated. Apoptosis signaling may be mediated by FAS and PLA2G4A, and pathway analysis indicated that STAT3 might be an upstream regulator of these genes. The activity of FAS and PLA2G4A was verified by qPCR, which showed that FAS was increased about 2-fold in HaCaT cells treated with $10{\mu}g/ml$ of ginsenoside Rb1 for 24 hr, PLA2G4A was increased about twice after 6 hours, and gene expression was increased more than 2-fold after 24 hr. Knockdown of STAT3 with siRNA decreased FAS expression and increased PLA2G4A expression but only FAS was passed from the upstream regulator STAT3. These results indicate that STAT3, which is an upstream regulator, induces apoptosis via FAS during treatment with ginsenoside Rb1.

SOCS1 counteracts ROS-mediated survival signals and promotes apoptosis by modulating cell cycle to increase radiosensitivity of colorectal cancer cells

  • Ryu, Ji-Yoon;Oh, Jiyoung;Kim, Su-Min;Kim, Won-Gi;Jeong, Hana;Ahn, Shin-Ae;Kim, Seol-Hee;Jang, Ji-Young;Yoo, Byong Chul;Kim, Chul Woo;Lee, Choong-Eun
    • BMB Reports
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    • v.55 no.4
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    • pp.198-203
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    • 2022
  • As negative regulators of cytokine signaling pathways, suppressors of cytokine signaling (SOCS) proteins have been reported to possess both pro-tumor and anti-tumor functions. Our recent studies have demonstrated suppressive effects of SOCS1 on epithelial to mesenchymal signaling in colorectal cancer cells in response to fractionated ionizing radiation or oxidative stress. The objective of the present study was to determine the radiosensitizing action of SOCS1 as an anti-tumor mechanism in colorectal cancer cell model. In HCT116 cells exposed to ionizing radiation, SOCS1 over-expression shifted cell cycle arrest from G2/M to G1 and promoted radiation-induced apoptosis in a p53-dependent manner with down-regulation of cyclin B and up-regulation of p21. On the other hand, SOCS1 knock-down resulted in a reduced apoptosis with a decrease in G1 arrest. The regulatory action of SOCS1 on the radiation response was mediated by inhibition of radiation-induced Jak3/STAT3 and Erk activities, thereby blocking G1 to S transition. Radiation-induced early ROS signal was responsible for the activation of Jak3/Erk/STAT3 that led to cell survival response. Our data collectively indicate that SOCS1 can promote radiosensitivity of colorectal cancer cells by counteracting ROS-mediated survival signal, thereby blocking cell cycle progression from G1 to S. The resulting increase in G1 arrest with p53 activation then contributes to the promotion of apoptotic response upon radiation. Thus, induction of SOCS1 expression may increase therapeutic efficacy of radiation in tumors with low SOCS1 levels.

Characterization of a PyrR-deficient Mutant of Bacillus subtilis by a Proteomic Approach (프로테옴 분석에 의한 Bacillus subtilis PyrR 돌연변이체의 특성)

  • Seul, Keyung-Jo;Cho, Hyun-Soo;Ghim, Sa-Youl
    • Microbiology and Biotechnology Letters
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    • v.39 no.1
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    • pp.9-19
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    • 2011
  • The Bacillus subtilis pyrimidine biosynthetic (pyr) operon encodes all of the enzymes for the de novo biosynthesis of Uridine monophosphate (UMP) and additional cistrones encoding a uracil permease and the regulatory protein PyrR. The PyrR is a bifunctional protein with pyr mRNA-binding regulatory funtion and uracil phosphoribosyltransferase activity. To study the global regulation by the pyrR deletion, the proteome comparison between Bacillus subtilis DB104 and Bacillus subtilis DB104 ${\Delta}$pyrR in the minimal medium without pyrimidines was employed. Proteome analysis of the cytosolic proteins from both strains by 2D-gel electrophoresis showed the variations in levels of protein expression. On the silver stained 2D-gel with an isoelectric point (pI) between 4 and 10, about 1,300 spots were detected and 172 spots showed quantitative variations in which 42 high quantitatively variant proteins were identified. The results showed that production of the pyrimidine biosynthetic enzymes (PyrAA, PyrAB, PyrB, PyrC, PyrD, and PyrF) were significantly increased in B. subtilis DB104 ${\Delta}$pyrR. Besides, proteins associated carbohydrate metabolism, elongation protein synthesis, metabolism of cofactors and vitamins, motility, tRNA synthetase, catalase, ATP-binding protein, and cell division protein FtsZ were overproduced in the PyrR-deficient mutant. Based on analytic results, the PyrR might be involved a number of other metabolisms or various phenomena in the bacterial cell besides the pyrimidine biosynthesis.

Anti-inflammatory Effect of Achyranthoside E Dimethyl Ester in LPS-stimulated RAW 264.7 Cells (LPS로 인한 RAW 264.7 세포의 염증반응에 미치는 achyranthoside E dimethyl ester의 효과)

  • Bang, Soo Young;Kim, Ji-Hee;Moon, Hyung-In;Kim, Young Hee
    • Journal of Life Science
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    • v.23 no.6
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    • pp.736-742
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    • 2013
  • Achyranthoside E dimethyl ester (AEDE) is an oleanolic acid glycoside from Achyranthes japonica. In this study, we investigated the effects of AEDE on nitric oxide (NO) production and underlying molecular mechanisms in lipopolysaccharide (LPS)-stimulated macrophages. AEDE inhibited LPS-induced NO secretion as well as inducible NO synthase (iNOS) expression, without affecting cell viability. Further study demonstrated that AEDE induced heme oxygenase-1 (HO-1) gene expression. In addition, the inhibitory effects of AEDE on iNOS expression were abrogated by small interfering RNA-mediated knock-down of HO-1. Moreover, AEDE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. AEDE-induced expression of HO-1 was inhibited by inhibitors of phosphatidylinositol 3-kinase (PI-3K) and extracellular signal regulated kinase (ERK1/2). AEDE phosphorylated Akt and ERK1/2 as well. Therefore, these results suggest that AEDE suppresses the production of pro-inflammatory mediator such as NO by inducing HO-1 expression via PI-3K/Akt/ERK-Nrf2 signaling. These findings provide the scientific rationale for anti-inflammatory therapeutic use of AEDE.

Site Selection using Port and Industry Clusters (제조산업의 항만클러스터 입지선정 모형에 관한 연구 - 수도권을 중심으로 -)

  • Gang, Sang-Gon;An, Seung-Beom;Lee, Chung-Hyo
    • Journal of Korea Port Economic Association
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    • v.24 no.4
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    • pp.237-255
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    • 2008
  • This paper aims to clarify if clustering effects among industries exist and if port-industry clustering effects exist. A knock-down approach was used in a survey and 16 industries were categorized. We defined which industry is more competitive in industry clusters and port-industry clusters. Another survey to experts was carried out to identify which industry is more appropriate to one of the three ports in Sudokwon (Seoul Metropolitan Areas): Incheon port, Pyungtaik port and Dangjin port. Five manufacturing industries are selected considering port-industry clustering relationships in this area and Analytic Hierarch Process was used for a pairwise comparison. Locational, social and economic factors are selected for 1st level. A result shows that Incheon port is more competitive in petroleum manufacturing, primary metal manufacturing and rubber and plastic manufacturing and Pyeontaik port is more competitive in metal assembly manufacturing and automobile and trailer manufacturing. However, sensitivity analysis shows a turnover of ranking in some industries. As there exist slight differences among three ports, cooperation is necessary when the government and Port Authorities make plans.

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Effects of Achyranthoside C Dimethyl Ester on Heme Oxygenase-1 Expression and NO Production (Heme Oxygenase-1 발현과 NO 생성에 미치는 Achyranthoside C Dimethyl Ester의 효과)

  • Bang, Soo Young;Song, Ji Su;Moon, Hyung-In;Kim, YoungHee
    • Journal of Life Science
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    • v.25 no.9
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    • pp.976-983
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    • 2015
  • Achyranthoside C dimethyl ester (ACDE) is an oleanolic acid glycoside from Achyranthes japonica which has been used in traditional medicine for the treatment of edema and arthritis. In this study, we investigated the anti-inflammatory effects of ACDE in RAW264.7 macrophages. ACDE significantly induced heme oxygenase-1 (HO-1) gene expression in RAW264.7 cells, while ACDE improved LPS-induced toxicity of cells. And ACDE induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. Further study demonstrated that ACDE-induced expression of HO-1 was inhibited by inhibitors of phosphatidylinositol 3-kinase (PI-3K) (LY294002), c-Jun kinase (JNK) (SP600125), extracellular signal regulated kinase (ERK) (PD98059) and p38 kinase (SB203580). Moreover, ACDE phosphorylated Akt, JNK, ERK, and p38 MAPK. In addition, ACDE inhibited LPS-induced NO secretion as well as inducible NO synthase (iNOS) expression in a dose-dependent manner. The inhibitory effects of ACDE on iNOS expression were abrogated by small interfering RNA (siRNA)-mediated knock-down of HO-1. Therefore, these results suggest that ACDE suppresses the production of pro-inflammatory mediator such as NO by inducing HO-1 expression via PI-3K/Akt/MAPK-Nrf2 signaling pathway. These findings could help us to understand the active principle included in the roots of A. japonica and the molecular mechanisms underlying anti-inflammatory action of ACDE.

Gardenia jasminoides Exerts Anti-inflammatory Activity via Akt and p38-dependent Heme Oxygenase-1 Upregulation in Microglial Cells (소교세포에서 heme oxygenase-1 발현 유도를 통한 치자(Gardenia jasminoides)의 항염증 효과)

  • Song, Ji Su;Shin, Ji Eun;Kim, Ji-Hee;Kim, YoungHee
    • Journal of Life Science
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    • v.27 no.1
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    • pp.8-14
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    • 2017
  • Died Gardenia jasminoides fruit is used as a dye in the food and clothes industries in Asia. The present study investigated the anti-inflammatory effects of aqueous extract of G. jasminoides fruits (GJ) in BV-2 microglial cells. GJ inhibited lipopolysaccharide-induced nitric oxide (NO) secretion, inducible nitric oxide synthase (iNOS) expression, and reactive oxygen species production, without affecting cell viability. Furthermore, GJ increased the expression of heme oxygenase-1 (HO-1) in a dose-dependent manner. Moreover, the inhibitory effect of GJ on iNOS expression was abrogated by small interfering RNA-mediated knock-down of HO-1. In addition, GJ induced nuclear translocation of nuclear factor E2-related factor 2 (Nrf2), a transcription factor that regulates HO-1 expression. GJ-mediated expression of HO-1 was suppressed by LY294002, a phosphoinositide 3-kinase (PI-3K) inhibitor, and SB203580, a p38 kinase inhibitor, but not by the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or c-Jun N-terminal kinase (JNK) inhibitor SP600125. GJ also enhanced the phosphorylation of Akt and p38. These results suggest that GJ suppresses the production of NO, a pro-inflammatory mediator, by inducing HO-1 expression via PI-3K/Akt/p38 signaling. These findings illustrate a novel molecular mechanism by which extract from G. jasminoides fruits inhibits neuroinflammation.

Characterization of α-Gal Epitope in Cells and Tissues from Homozygous α-1,3-Galactosyltransferase Knockout Pigs

  • Hwang, In-Sul;Kwon, Dae-Jin;Kwak, Tae-Uk;Oh, Keon Bong;Ock, Sun-A;Chung, Hak-Jae;Im, Gi-Sun;Hwang, Seongsoo
    • Reproductive and Developmental Biology
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    • v.39 no.4
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    • pp.127-132
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    • 2015
  • To overcome the hyperacute immune rejection during pig-to-non-human primates xenotranasplantation, we have produced and bred ${\alpha}$-1,3-galactosyltransferase knock-out ($GalT^{-/-}$) pigs. In this study, the somatic cells and tissues from the $GalT^{-/-}$ pigs were characterized by an analysis of the expression of Gal${\alpha}$-1,3-Gal (${\alpha}-Gal$) epitope. Briefly, ear fibroblast cell lines of 19 homozygous $GalT^{-/-}$ pigs were established and cryopreserved. The expression of ${\alpha}-Gal$ epitope in the cells was measured by fluorescence activated cell sorter (FACS) analysis using BS-I-B4 lectin. Also, the homozygous ($GalT^{-/-}$) cells and tissues samples were immunostained with BS-I-B4 lectin for analysis of ${\alpha}-Gal$ epitope expression. The results showed that the expression of ${\alpha}-Gal$ epitope in $GalT^{-/-}$ cells (0.2 %) were significantly (p<0.05) down-regulated to the range of cynomolgus monkey fibroblast (0.2 %) cells compared to heterozygous ($GalT^{-/+}$) (9.3 %) and wild type ($GalT^{+/+}$) (93.7 %) fibroblast cells. In the immunostaining results, while the expression of ${\alpha}-Gal$ epitope was detected a partly in $GalT^{-/+}$ cells and mostly in $GalT^{+/+}$ cells, it was almost not detected in the $GalT^{-/-}$ cells. Also, immunostaining results from various tissues of the $GalT^{-/-}$ pig showed that the expression of ${\alpha}-Gal$ epitope was not detectable, whereas various tissues from $GalT^{+/+}$ pig showed a strong expression of ${\alpha}-Gal$ epitope. Our results demonstrated that ${\alpha}-Gal$ epitope expressions from $GalT^{-/-}$ pigs were successfully knocked out to prevent hyperacute immune rejection for further study of xenotransplantation.

Insecticidal Activities of Polymers and Surfactants Against Sweet Potato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) (담배가루이에 대한 폴리머와 전착제의 살충효과)

  • Yoon, Chang-Mann;Cho, Sun-Ran;Moon, Sang-Rae;Shin, Youn-Ho;Kim, Gil-Hah
    • The Korean Journal of Pesticide Science
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    • v.15 no.2
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    • pp.177-187
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    • 2011
  • This study was performed to evaluate the polymers and surfactants as the potential control agents of sweet potato whitefly Bemisia tabaci, which is causing problems in ornamental garden and greenhouse. Polymers have an insecticidal activity to knock down and to be lethal to small winged insects by its viscosity. Among five polymers tested at 0.2% concentration, polinol P-24 showed the highest insecticidal activity as 59.4% against B. tabaci adult in cylindrical chamber, and followed by polinol P-20 (insecticidal activity, 57.1%). When treated at 0.1 % or 0.3% concentrations, Polinol P-24 also showed the highest insecticidal activity with 43.3% and 54.5%, respectively. Among eight surfactants tested, insecticidal activity was the highest in 0.0005% NP10 treatment (70.0%), and followed by 0.001% NP7 (67.4%). The synergistic effect between polinol P-24 and eight surfactants was evaluated. After bioassays, the 0.2% polinol P-24 plus 0.005% NP10 was selected as a candidate control agent for controlling of B. tabaci adults. Polinol P-24/NP10 was showed the highest control efficacy against B. tabaci adults applied three times at three day-intervals in square rearing cage. In the greenhouse, the mixture treatment showed good control value over 70% seven days after treatment.