• Title/Summary/Keyword: SiRNA

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Effects of Transcription Factor AP2γ on Gene Expression of Desmosome Components in Mouse Embryos

  • Chung, Hak-Jae;Jeong, Jiyeon;Jeong, Yelin;Choi, Inchul
    • Reproductive and Developmental Biology
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    • v.40 no.2
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    • pp.23-26
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    • 2016
  • Transcription factor called activating enhancer binding protein 2C (AP2-gamma) is found in a variety of species and expressed from oocyte stage onwards, particularly restricted to the trophectoderm. Recent studies demonstrated that ablation of Tfap2c led to failure of tight junction biogenesis, particularly the knock-down embryos of Tfap2c did not form cavity from morula to blastocyst in mouse and pig. We speculated that the Tfa2pc may also be involved in desmosome biogenesis because blastocoel formation is coincident with the establishment of desmosome. To determine this, we depleted Tfap2c injecting siRNA into one-cell zygote and analysed the expression levels of genes that are required for desmosome complex such as PkP2, Pkp3, Dsc2, and Dsg2. We found only Pkp3 was up-regulated in the knockdowned morula embryos. Interestingly, upstream region of Pkp3 had putative Tfap2c binding sites. In conclusion, our results suggest that Tfap2c is not a crucial factor but somehow it might be involved in desmosome biogenesis directly or indirectly via Pkp3.

HIF-1α-Dependent Gene Expression Program During the Nucleic Acid-Triggered Antiviral Innate Immune Responses

  • Hong, Sun Woo;Yoo, Jae Wook;Kang, Hye Suk;Kim, Soyoun;Lee, Dong-ki
    • Molecules and Cells
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    • v.27 no.2
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    • pp.243-250
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    • 2009
  • Recent studies suggest a novel role of $HIF-1{\alpha}$ under nonhypoxic conditions, including antibacterial and antiviral innate immune responses. However, the identity of the pathogen-associated molecular pattern which triggers $HIF-1{\alpha}$ activation during the antiviral response remains to be identified. Here, we demonstrate that cellular administration of double-stranded nucleic acids, the molecular mimics of viral genomes, results in the induction of $HIF-1{\alpha}$ protein level as well as the increase in $HIF-1{\alpha}$ target gene expression. Whole-genome DNA microarray analysis revealed that double-stranded nucleic acid treatment triggers induction of a number of hypoxia-inducible genes, and induction of these genes are compromised upon siRNA-mediated $HIF-1{\alpha}$ knock-down. Interestingly, $HIF-1{\alpha}$ knock-down also resulted in down-regulation of a number of genes involved in antiviral innate immune responses. Our study demonstrates that $HIF-1{\alpha}$ activation upon nucleic acid-triggered antiviral innate immune responses plays an important role in regulation of genes involved in not only hypoxic response, but also immune response.

Up-Regulation of $p27^{Kip1}$ Protects hES Cells from Differentiation-Associated and Caspase 3-Dependent Apoptosis

  • Park, So-Hyun;Kim, Min Kyoung;Lee, Chul-Hoon
    • Journal of Microbiology and Biotechnology
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    • v.22 no.12
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    • pp.1790-1794
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    • 2012
  • Recently, it has been suggested that $p27^{Kip1}$, the cell cycle regulatory protein, plays a pivotal role in the progression of normal differentiation in murine embryonic stem (mES) cells. In the current study, we investigated the role of $p27^{Kip1}$ in the regulation of differentiation and apoptotic induction using Western blotting, quantitative real-time RT-PCR, and small interfering RNA (siRNA) assays and confocal laser scanning microscopic analysis of H9 human ES (hES) cells and H9-derived embryoid bodies (EBs) grown for 10 ($EB_{10}$) and 20 days ($EB_{20}$). Our results demonstrate that the proteins $p27^{Kip1}$ and cyclin D3 are strongly associated with cellular differentiation, and, for the first time, show that up-regulation of $p27^{Kip1}$ protects hES cells from inducing differentiation-associated and caspase 3-dependent apoptosis.

Curcumin-Induced Autophagy Augments Its Antitumor Effect against A172 Human Glioblastoma Cells

  • Lee, Jong-Eun;Yoon, Sung Sik;Moon, Eun-Yi
    • Biomolecules & Therapeutics
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    • v.27 no.5
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    • pp.484-491
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    • 2019
  • Glioblastoma is the most aggressive common brain tumor in adults. Curcumin, from Curcuma longa, is an effective antitumor agent. Although the same proteins control both autophagy and cell death, the molecular connections between them are complicated and autophagy may promote or inhibit cell death. We investigated whether curcumin affects autophagy, which regulates curcumin-mediated tumor cell death in A172 human glioblastoma cells. When A172 cells were incubated with $10{\mu}M$ curcumin, autophagy increased in a time-dependent manner. Curcumin-induced cell death was reduced by co-incubation with the autophagy inhibitors 3-methyladenine (3-MA), hydroxychloroquine (HCQ), and LY294002. Curcumin-induced cell death was also inhibited by co-incubation with rapamycin, an autophagy inducer. When cells were incubated under serum-deprived medium, LC3-II amount was increased but the basal level of cell viability was reduced, leading to the inhibition of curcumin-induced cell death. Cell death was decreased by inhibiting curcumin-induced autophagy using small interference RNA (siRNA) of Atg5 or Beclin1. Therefore, curcumin-mediated tumor cell death is promoted by curcumin-induced autophagy, but not by an increase in the basal level of autophagy in rapamycin-treated or serum-deprived conditions. This suggests that the antitumor effects of curcumin are influenced differently by curcumin-induced autophagy and the prerequisite basal level of autophagy in cancer cells.

Isolation of ten unrecorded yeasts from soil in Korea

  • Han, Joo Hyun;Oh, Hye Jin;Lee, Sang Eun;Kim, Myung Kyum
    • Journal of Species Research
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    • v.10 no.4
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    • pp.336-343
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    • 2021
  • In 2020, as a subset study to discover indigenous yeast species in Korea, a total of 22 yeast species were isolated from soil samples collected in Gwangju-si, Gyeonggi-do. Among them, 10 strains were unreported species. From the high 26S rRNA gene sequence similarity and formation of a robust phylogenetic clade with the closest species, it was determined that each strain belonged independent and predefined yeast species. The 22 strains were assigned to the genera Dothiora (1 strain), Sarocladium (1 strain), Tetrapisispora (1 strain) and Torulaspora (2 strains) of the phylum Ascomycota; the genera Erythrobasidium (1 strain), Leucosporidium (4 strains), Ustilentyloma (2 strains), Fellozyma (1 strain), Sampaiozyma (2 strains), Filobasidium (1 strain), Solicoccozyma (2 strains) and Vishniacozyma (4 strains) of the phylum Basidiomycota. This is the first official report of the following species in Korea: Dothiora cannabinae (1 strain), Sarocladium strictum (1 strain), Fellozyma inositophila (1 strain), Filobasidium magnum (1 strain), Solicoccozyma phenolicus (1 strain), Solicoccozyma terreus(1 strain), Vishniacozyma tephrensis(1 strain) and Vishniacozyma victoriae (3 strains). Cell morphology, phenotypic features and biochemical features are described in the Species Description section.

The Relationship between Prohibitin 1 Expression, Hepatotoxicity Induced by Acetaminophen, and Hepatoprotection by S-Adenosylmethionine in AML12 Cells

  • Eunhye Cho;Soohan Jung;Jina Kim;Kwang Suk Ko
    • Journal of Microbiology and Biotechnology
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    • v.32 no.11
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    • pp.1447-1453
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    • 2022
  • Prohibitin 1 (Phb1) is a pleiotropic protein, located mainly in the mitochondrial inner membrane and involved in the regulation of cell proliferation and the stabilization of mitochondrial protein. Acetaminophen (APAP) is one of the most commonly used over-the-counter analgesics worldwide. However, at high dose, the accumulation of N-acetyl-p-benzoquinone imine (NAPQI) can lead to APAP-induced hepatotoxicity. In this study, we sought to understand the regulation of mRNA expression in relation to APAP and GSH metabolism by Phb1 in normal mouse AML12 hepatocytes. We used two different Phb1 silencing levels: high-efficiency (HE, >90%) and low-efficiency (LE, 50-60%). In addition, the siRNA-transfected cells were further pretreated with 0.5 mM of Sadenosylmethionine (SAMe) for 24 h before treatment with APAP at different doses (1-2 mM) for 24 h. The expression of APAP metabolism-related and antioxidant genes such as Cyp2e1 and Ugt1a1 were increased during SAMe pretreatment. Moreover, SAMe increased intracellular GSH concentration and it was maintained after APAP treatment. To sum up, Phb1 silencing and APAP treatment impaired the metabolism of APAP in hepatocytes, and SAMe exerted a protective effect against hepatotoxicity by upregulating antioxidant genes.

Comprehensive Expression Analysis of Triterpenoid Biosynthesis Genes Using Pac-Bio Sequencing and rnaSPAdes assembly in Codonopsis lanceolata

  • Ji-Nam Kang;Si Myung Lee;Mi-Hwa Choi;Chang-Kug Kim
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.253-253
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    • 2022
  • Codonopsis lanceolata (C. lanceolata) has been widely used in East Asia as a traditional medicine to treat various diseases such as bronchitis, convulsions, cough, obesity, and hepatitis. C. lanceolata belonging to Campanulaceae contains bioactive compounds such as polyphenols, saponins, and steroids. However, despite the pharmacological significance of C. lanceolata, the genetic information of this plant is limited and there are few studies of its transcriptome. In this study, we constructed a unigene set of C. lanceolata using Pac-Bio sequencing. Furthermore, the reads generated from Pac-bio and Illumina sequencing were mixed and assembled using rnaSPAdes. All genes involved in the triterpenoid pathway, a major bioactive compounds of C. lanceolata, were searched from the two unigene sets and the expression profiles of these genes were analyzed. The results showed that lupeol, beta-amyrin, and dammarenediol synthesis genes were activated in the leaves and roots of C. lanceolata. In particular, the expression of genes related to lupeol synthesis was relatively high, suggesting that the main triterpenoid of C. lanceolata is lupeol. Transcriptome studies related to lupeol synthesis in C. lanceolata have been rarely reported. Lupeol has been reported to have pharmacological effects such as anti-inflammatory, anti-cancer, and anti-bacterial. This study suggests the importance of C. lanceolata as a lupeol producing plant.

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Application of Pac-Bio Sequencing, Trinity, and rnaSPAdes Assembly for Transcriptome Analysis in Medicinal Crop Astragalus membranaceus

  • Ji-Nam Kang;Si Myung Lee
    • Proceedings of the Korean Society of Crop Science Conference
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    • 2022.10a
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    • pp.254-254
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    • 2022
  • Astragalus membranaceus (A. membranaceus) has traditionally been used as a medicinal plant in East Asia for the treatment ofvarious diseases. A. membranaceus belongs to the legume family and is known to be rich in substances such as flavonoids and saponins. Recent pharmacological studies of A. membranaceus have shown that the plant has immunomodulatory, anti-oxidant, anti-cancer, and anti-inflammatory effects. However, knowledge of major biosynthetic pathways in A. membranaceu is still lacking. Recently developed sequencing techniques enable high-quality transcriptome analysis in plants, which is recognized as an important part in elucidating the regulatory mechanisms of many plant secondary metabolic pathways. However, it is difficult to predict the number of transcripts because plant transcripts contain a large number of isoforms due to alternative splicing events, which can vary depending on the assembly platform used. In this study, we constructed three unigene sets using Pac-Bio isoform sequencing, Trinity and rnaSPAdes assembly for detailed transcriptome analysis mA. membranaceus. Furthermore, all genes involved in the flavonoid biosynthetic pathway were searched from three unigene sets, and structural comparisons and expression profiles between these genes were analyzed. The isoflavone synthesis was active in most tissues. Flavonol synthesis was mainly active in leaves and flowers, and anthocyanin synthesis was specific in flowers. Gene structural analysis revealed structural differences in the flavonoid-related genes derived from the three unigene sets. This study suggests the need for the application of multiple unigene sets for the analysis of key biosynthetic pathways in plants.

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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.