• Title/Summary/Keyword: reporter assay

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TC1 (C8orf4) is involved in ERK1/2 pathway-regulated G1- to S-phase transition

  • Wang, Yi-Dong;Bian, Guo-Hui;Lv, Xiao-Yan;Zheng, Rong;Sun, Huan;Zhang, Zheng;Chen, Ye;Li, Qin-Wei;Xiao, Yan;Yang, Qiu-Tan;Ai, Jian-Zhong;Wei, Yu-Quan;Zhou, Qin
    • BMB Reports
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    • v.41 no.10
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    • pp.733-738
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    • 2008
  • Although previous studies have implicated a role for TC1 (C8orf4) in cancer cell proliferation, the molecular mechanism of its action is still largely unclear. In this study, we showed, for the first time, that the mRNA levels of TC1 were upregulated by mitogens (FBS/thrombin) and at least partially, through the ERK1/2 signaling pathway. Interestingly, the over-expression of TC1 promoted the $G_1$- to S-phase transition of the cell cycle, which was delayed by the deficiency of ERK1/2 signaling in fibroblast cells. Furthermore, the luciferase reporter assay indicated that the over-expression of TC1 significantly increased Cyclin D1 promoter-driven luciferase activity. Taken together, our findings revealed that TC1 was involved in the mitogen-activated ERK1/2 signaling pathway and positively regulated $G_1$- to S-phase transition of the cell cycle. Our results may provide a novel mechanism of the role of TC1 in the regulation of cell proliferation.

Dehydrocostus lactone inhibits NFATc1 via regulation of IKK, JNK, and Nrf2, thereby attenuating osteoclastogenesis

  • Lee, Hye In;Lee, Gong-Rak;Lee, Jiae;Kim, Narae;Kwon, Minjeong;Kim, Hyun Jin;Kim, Nam Young;Park, Jin Ha;Jeong, Woojin
    • BMB Reports
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    • v.53 no.4
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    • pp.218-222
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    • 2020
  • Excessive and hyperactive osteoclast activity causes bone diseases such as osteoporosis and periodontitis. Thus, the regulation of osteoclast differentiation has clinical implications. We recently reported that dehydrocostus lactone (DL) inhibits osteoclast differentiation by regulating a nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), but the underlying mechanism remains to be elucidated. Here we demonstrated that DL inhibits NFATc1 by regulating nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and nuclear factor-erythroid 2-related factor 2 (Nrf2). DL attenuated IκBα phosphorylation and p65 nuclear translocation as well as decreased the expression of NF-κB target genes and c-Fos. It also inhibited c-Jun N-terminal kinase (JNK) but not p38 or extracellular signal-regulated kinase. The reporter assay revealed that DL inhibits NF-κB and AP-1 activation. In addition, DL reduced reactive oxygen species either by scavenging them or by activating Nrf2. The DL inhibition of NFATc1 expression and osteoclast differentiation was less effective in Nrf2-deficient cells. Collectively, these results suggest that DL regulates NFATc1 by inhibiting NF-κB and AP-1 via down-regulation of IκB kinase and JNK as well as by activating Nrf2, and thereby attenuates osteoclast differentiation.

Inhibitory Effects of Coptis japonica Alkaloids on the LPS-Induced Activation of BV2 Microglial Cells

  • Jeon, Se-Jin;Kwon, Kyung-Ja;Shin, Sun-Mi;Lee, Sung-Hoon;Rhee, So-Young;Han, Seol-Heui;Lee, Jong-Min;Kim, Han-Young;Cheong, Jae-Hoon;Ryu, Jong-Hoon;Min, Byung-Sun;Ko, Kwang-Ho;Shin, Chan-Young
    • Biomolecules & Therapeutics
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    • v.17 no.1
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    • pp.70-78
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    • 2009
  • Coptis japonica (C. japonica) is a perennial medicinal plant that has anti-inflammatory activity. C. japonica contains numerous biologically active alkaloids including berberine, palmatine, epi-berberine, and coptisine. The most well-known anti-inflammatory principal in C. japonica is berberine. For example, berberine has been implicated in the inhibition of iNOS induction by cytokines in microglial cells. However, the efficacies of other alkaloids components on microglial activation were not investigated yet. In this study, we investigated the effects of three alkaloids (palmatine, epi-berberine and coptisine) from C. japonica on lipopolysaccharide (LPS)-induced microglial activation. BV2 microglial cells were immunostimulated with LPS and then the production of several inflammatory mediators such as nitric oxide (NO), reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) were examined as well as the phosphorylation status of Erk1/2 mitogen activated protein kinase (MAPK). Palmatine and to a lesser extent epi-berberine and coptisine, significantly reduced the release of NO, which was mediated by the inhibition of LPS-stimulated mRNA and protein induction of inducible nitric oxide synthase (iNOS) from BV2 microglia. In addition to NO, palmatine inhibited MMP-9 enzymatic activity and mRNA induction by LPS. Palmatine also inhibited the increase in the LPS-induced MMP-9 promoter activity determined by MMP-9 promoter luciferase reporter assay. LPS stimulation increased Erk1/2 phosphorylation in BV2 cells and these alkaloids inhibited the LPS-induced phosphorylation of Erk1/2. The anti-inflammatory effect of palmatine in LPS-stimulated microglia may suggest the potential use of the alkaloids in the modulation of neuroinflammatory responses, which might be important in the pathophysiological events of several neurological diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease (PD) and stroke.

Effects of Zizyphi Spinosae Extract on Cisplatin and t-Butylhydroperoxide Induced Acute Renal Failure in Rabbits (토끼에서 cisplatin에 의해 유도된 급성 신부전시 산조인 추출물의 효과)

  • Kim, Jae Young;Kim, Chung Hui
    • Journal of Life Science
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    • v.24 no.7
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    • pp.777-783
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    • 2014
  • Cathepsin D (CtsD), an aspartyl peptidase, is involved in apoptosis, resulting in the release of cytochrome C from mitochondria in cells. Here, we investigated microRNA regulation of CtsD expression in 3T3-L1 cells. First, we observed the expression of CtsD in cells in response to doxorubicin (Dox). As expected, the level of CtsD mRNA increased in 3T3-L1 cells exposed to Dox in a dose-dependent manner. The cellular viability of ectopically expressed CtsD cells was decreased. Next, we used the miRanda program to search for particular microRNA targeting CtsD. MiR-145 was selected as a putative controller of CtsD because it had a high mirSVR score. In a reporter assay, the luciferase activity of cells containing the CtsD 3'-UTR region decreased in cells transfected with a miR-145 mimic compared to that of a control. The level of CtsD expression was down-regulated in preadipocytes ectopically expressing miR-145 and up-regulated by an miR-145 inhibitor. Cells also suppressed miR-145 expression when exposed to Dox. The miR-145 inhibitor reduced the cellular viability of 3T3-L1 cells. Taken together, these data suggest that miR-145 regulates CtsD-mediated cell death in adipocytes. These findings may have valuable implications concerning the molecular mechanism of CtsD-mediated cell death in obesity, suggesting that CtsD could be a useful therapeutic tool for the prevention and treatment of obesity by regulating fat cell numbers.

Induction of Nrf2/ARE-mediated cytoprotective genes by red ginseng oil through ASK1-MKK4/7-JNK and p38 MAPK signaling pathways in HepG2 cells

  • Bak, Min Ji;Truong, Van-Long;Ko, Se-Yeon;Nguyen, Xuan Ngan Giang;Jun, Mira;Hong, Soon-Gi;Lee, Jong-Won;Jeong, Woo-Sik
    • Journal of Ginseng Research
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    • v.40 no.4
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    • pp.423-430
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    • 2016
  • Background: The induction of cellular defensive genes such as phase II detoxifying and antioxidant enzymes is a highly effective strategy for protection against carcinogenesis as well as slowing cancer development. Transcription factor Nrf2 (nuclear factor E2-related factor 2) is responsible for activation of phase II enzymes induced by natural chemopreventive compounds. Methods: Red ginseng oil (RGO) was extracted using a supercritical $CO_2$ extraction system and chemical profile of RGO was investigated by GC/MS. Effects of RGO on regulation of the Nrf2/antioxidant response element (ARE) pathway were determined by ARE-luciferase assay, western blotting, and confocal microscopy. Results: The predominant components of RGO were 9,12-octadecadienoic acid (31.48%), bicyclo[10.1.0] tridec-1-ene (22.54%), and 22,23-dihydrostigmasterol (16.90%). RGO treatment significantly increased nuclear translocation of Nrf2 as well as ARE reporter gene activity, leading to upregulation of heme oxygenase-1 and NAD(P)H:quinone oxidoreductase 1. Phosphorylation of the upstream kinases such as apoptosis signal-regulating kinase (ASK)1, mitogen-activated protein kinase (MAPK) kinase (MKK)4/7, c-Jun N-terminal kinase (JNK), and p38 MAPK were enhanced by treatment with RGO. In addition, RGO-mediated Nrf2 expression and nuclear translocation was attenuated by JNK inhibitor SP600125 and p38 MAPK inhibitor SB202190. Conclusion: RGO could be used as a potential chemopreventive agent, possibly by induction of Nrf2/ARE-mediated phase II enzymes via ASK1-MKK4/7-JNK and p38 MAPK signaling pathways.

Wnt7b is Upregulated in Macrophages during Thymic Regeneration and Negatively Regulated by RANKL (흰쥐 가슴샘 재생과정 동안 대식세포에서 Wnt 7b의 발현증가 및 RANKL에 의한 발현조절)

  • Kim, Jong-Gab;Kim, Sung-Min;Kim, Bong-Seon;Kim, Jae-Bong;Yoon, Sik;Bae, Soo-Kyung
    • Journal of Life Science
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    • v.17 no.7 s.87
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    • pp.923-930
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    • 2007
  • Thymus can regenerate to its normal mass within 14 days after acute involution induced by cyclophosphamide (CY) in adult rat. Despite the established role of Wnt pathways in the process of thymus development, they have not yet been associated with the regeneration of adult thymus. The purpose of this study was to investigate whether Wnt7b, which is expressed in developing thymic epithelial cells rather than in thymocytes, is modulated during thymic regeneration in adult rat. Here, we show that Wnt7b expression was up-regulated in the regenerating thymus. Cells immunolabeled for the Wnt7b were identified as macrophages. Furthermore, Wnt7b gene expression was decreased by the treatment of receptor activator of NF-kappaB ligand (RANKL). Taken together, our results demonstrate that Wnt7b gene expression was increased in macrophages during thymic regeneration and negatively regulated by RANKL.

Transcriptional Regulation of MicroRNA-17 by PPARγ in Adipogenesis (지방분화시 PPARγ에 의한 microRNA-17의 발현 조절)

  • Bae, In-Seon;Kim, Hyun-Ji;Chung, Ki Yong;Choi, Inho;Kim, Sang Hoon
    • Journal of Life Science
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    • v.24 no.3
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    • pp.323-328
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    • 2014
  • MicroRNAs comprise a family of small noncoding RNAs that modulate physiological processes, including adipogenesis. MicroRNA-17 (miR-17) promotes adipocyte differentiation and enhances lipid accumulation. The transcriptional regulation of miR-17 during adipogenesis remains unknown. In this study, we investigated whether miR-17 is a target of peroxisome proliferator-activated receptor ${\gamma}$ ($PPAR{\gamma}$), which is a key regulator of adipogenesis. The levels of miR-17 and the expression of $PPAR{\gamma}$ increased after the induction of adipocyte differentiation. Three putative peroxisome proliferator response elements (PPREs) were identified in the miR-17 promoter region. Using chromatin immunoprecipitation and luciferase reporter assays, we observed the interaction of $PPAR{\gamma}$ with the miR-17 promoter. Mutagenesis experiments showed that the -677/-655 region of the miR-17 promoter could function as a PPRE site. These results suggest that $PPAR{\gamma}$ is essential for transcriptional activation of the miR-17 gene, thereby contributing to understanding the molecular mechanism of adipogenesis in adipocytes.

MicroRNA-766-3p Inhibits Tumour Progression by Targeting Wnt3a in Hepatocellular Carcinoma

  • You, Yu;Que, Keting;Zhou, Yun;Zhang, Zhen;Zhao, Xiaoping;Gong, Jianpin;Liu, Zuojin
    • Molecules and Cells
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    • v.41 no.9
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    • pp.830-841
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    • 2018
  • Recent studies have indicated that microRNAs (miRNAs) play an important role in hepatocellular carcinoma (HCC) progression. In this study, we showed that miR-766-3p was decreased in approximately 72% of HCC tissues and cell lines, and its low expression level was significantly correlated with tumour size, TNM stage, metastasis, and poor prognosis in HCC. Ectopic miR-766-3p expression inhibited HCC cell proliferation, colony formation, migration and invasion. In addition, we showed that miR-766-3p repressed Wnt3a expression. A luciferase reporter assay revealed that Wnt3a was a direct target of miR-766-3p, and an inverse correlation between miR-766-3p and Wnt3a expression was observed. Moreover, Wnt3a up-regulation reversed the effects of miR766-3p on HCC progression. In addition, our study showed that miR-766-3p up-regulation decreased the nuclear ${\beta}-catenin$ level and expression of Wnt targets (TCF1 and Survivin) and reduced the level of MAP protein regulator of cytokinesis 1 (PRC1). However, these effects of miR-766-3p were reversed by Wnt3a up-regulation. In addition, PRC1 upregulation increased the nuclear ${\beta}-catenin$ level and protein expression of TCF1 and Survivin. iCRT3, which disrupts the ${\beta}-catenin-TCF4$ interaction, repressed the TCF1, Survivin and PRC1 protein levels. Taken together, our results suggest that miR-766-3p down-regulation promotes HCC cell progression, probably by targeting the Wnt3a/PRC1 pathway, and miR-766-3p may serve as a potential therapeutic target in HCC.

Role of Sp in the Regulation of Notch1 Gene Expression by Curcumin (커큐민에 의한 노치발현 조절에서 Sp의 역할)

  • Park, Seon-Yeong;Kang, Yong-Gyu;Bae, Yun-Hee;Kim, Su-Ryun;Park, Hyun-Joo;Kang, Young-Soon;Kim, Mi-Kyoung;Wee, Hee-Jun;Jang, Hye-Ock;Bae, Moon-Kyoung;Woo, Jae Suk;Bae, Soo-Kyung
    • KSBB Journal
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    • v.28 no.1
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    • pp.1-6
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    • 2013
  • Curcumin has diverse anticancer activities that lead to tumor growth inhibition of cancer cells and induction of apoptosis. Curcumin is involved in the regulation of multiple genes via transcription factors including NF-${\kappa}B$, STATs, AP1, and SP. Notch signaling plays critical roles in maintaining the balance between cell proliferation, differentiation and apoptosis, and thereby may contribute to the development of various cancers involving breast cancer. This study was to investigate the effects of curcumin on Notch1 gene expression and to explore the underlying mechanism. Here, we found that curcumin decreased the levels of Notch1 mRNA and protein in MDA-MB-231 human breast cancer cells, along with the downregulation of Sp family genes (Sp1, Sp2, Sp3, and Sp4). The repressive effect of curcumin on Notch1 gene transcription was confirmed by performing Notch1 promoter-driven reporter assay and three Sp-binding sites were identified on Notch1 promoter that may act as curcumin-respose elements. Moreover, treatment with mitramycin A, a specific Sp inhibitor, decreased the levels of Notch1 mRNA and protein in human breast cancer cells. Taken together, our results indicate that Notch1 gene expression is downregulated by curcumin, at least in part, through the suppression of Sp family, which may lead to apoptosis in human breast cancer cells.

Effects of Amomi, Semen Extract on Synthesis of Insulin-like Growth Factor-1 and Anti-wrinkle in Skin (사인추출물의 인슐린 유사 성장인자-3의 합성과 피부 노화 개선에 대한 효과)

  • Choi Gyu Ho;Kim Su Nam;Lee So Hee;Sung Dae Seok;Son Eui Dong;Lee Chang Hoon;Lee Byeong Gon;Jang Ih Seop
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.30 no.3 s.47
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    • pp.389-392
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    • 2004
  • We screened several materials to stimulate IGF-1 promoter activity using luciferase reporter assay and found that Amomi Semen extract (ASE) among them is the most powerful stimulator We also studied about the anti-wrinkle effect of ethanolic extract of Amoni Semen in vitro and in vivo. Semi-quantitative RT-PCR showed that the extract elevated the presence level of IGF-1 mRNA. And $[^3H]$ proline incorporation and semi-quantitative RT-PCR showed that the extract increased the expression of type-I collagen compared with vehicle in vitro and in vivo, respectively. Significant inhibition of MMP-1 expression was determined by ELISA and Western blot. Finally, topical treatment of the extract on hairless mouse's dorsal skin expanded the volume of collagen and dermal thickness. These results suggest that Amomi Semen may be a good candidate for improving extracellular matrix through the increase of collagen expression and inhibition of MMP-1 expression. Moreover, this study enables us to guess that IGF-1 stimulated by the extract may be involved in the mechanism of anti-wrinkle effect of it.