The analysis of microRNA localization using fluorescence resonance energy transfer based in situ hybridization

• Project/Area Number: 16K08471
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: General anatomy (including histology/embryology)
• Research Institution: University of Miyazaki
• Principal Investigator: 菱川 善隆 宮崎大学, 医学部, 教授 (60304276)
• Co-Investigator(Kenkyū-buntansha): 石塚 匠 宮崎大学, 医学部, 助教 (50700085) ; 川口 真紀子 宮崎大学, 医学部, 助教 (90405598) ; チョウジョウフ ナランツオツク 宮崎大学, 医学部, 講師 (90640962) ; ピュー シンウー 宮崎大学, 医学部, 助教 (90718123) [Withdrawn]
• Project Period (FY): 2016-04-01 – 2019-03-31
• Project Status: Completed (Fiscal Year 2018)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2018: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2017: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2016: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: 蛍光共鳴エネルギー移動 / in situ hybridization / 28S rRNA / microRNA / miR122 / ラット肝切除モデル / パラフィン切片 / 凍結切片 / in situ hybidization / miR-122 / ラット肝臓 / 解剖学 / 細胞組織化学 / in situハイブリダイゼーション

Outline of Annual Research Achievements

高感度蛍光発色（Fluorescence Resonance Energy Transfer: FRET；蛍光共鳴エネルギー移動）を利用して、組織切片上で特定の細胞での小分子非翻訳RNAであるmicroRNA (miRNA)発現動態を解析する新たな検出法として、FRET based in situハイブリダイゼーション(FRET-ISH)法による、組織・細胞レベルでの新たなmiRNA 局在検出法を開発することを目的とした。標的とするmiRNA自体が25塩基以下と非常に短いことより、まずmiRNA用プローブのハイブリダイゼーション条件の最適化を行った。具体的には、大量に組織内に発現している28S rRNAについて、通常ISH陽性コントロールに使う28ＳrRNA検出プローブ配列（39塩基数）から、miRNAの塩基数に対応した22塩基数の短いプローブ配列を選択し、蛍光色素を標識したFRET標識プローブを作製し、ラット肝臓パラフィン及び凍結切片を用いて①プローブ濃度、②ハイブリダイゼーション温度、③ハイブリダイゼーション溶液組成、④ハイブリダイゼーション反応時間と洗浄条件、を検討して小分子RNA検出のためのFRET-ISH法の最適な条件検討を行った。その結果、通常のISH法と比較して、プローブ濃度は１/10に、ハイブリダイゼーション反応時間は3時間（通常17時間）で検出可能であった。この条件を基に、肝臓に豊富に存在するmiR-122のFRETプローブを作製しマウス肝組織内発現を検討した。その結果、miR-122は、正常肝組織内の中心静脈周囲の肝細胞での発現が強く認められ、miRNA発現が組織内の部位により異なっている可能性があることが判明した。本研究で、FRET-ISH法が組織細胞内でのmiRNAの発現局在の検出に関する新たな解析法として有用であることを明らかにした。

Research Products

• [Int'l Joint Research] Department of Medical Research/Pathein University/Department of Traditional Medicine(ミャンマー)
• [Int'l Joint Research] Mongolian Nat. Univ. of Med. Sciences(モンゴル)
• [Journal Article] The HDAC Inhibitor, SAHA, Combined with Cisplatin Synergistically Induces Apoptosis in Alpha-fetoprotein-producing Hepatoid Adenocarcinoma Cells (2019)
  • Author(s): Kyaw MTH, Yamaguchi Y, Choijookhuu N, Yano K, Takagi H, Takahashi N, Synn Oo P, Sato K, Hishikawa Y.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 52 Issue: 1 Pages: 1-8
  • DOI: 10.1267/ahc.18044
  • NAID: 130007605543
  • Peer Reviewed / Open Access
• [Journal Article] HMGB2 is a novel adipogenic factor that regulates ectopic fat infiltration in skeletal muscles. (2018)
  • Author(s): Lee D, Taniguchi N, Sato K, Choijookhuu N, Hishikawa Y, Kataoka H, Morinaga H, Lotz M, Chosa E.
  • Journal Title: Sci. Rep. Volume: 8 Pages: 9601-9601
  • DOI: 10.1038/s41598-018-28023-7
  • Peer Reviewed / Open Access
• [Journal Article] Pivotal role of IL-22BP in the epithelial autoregulation of IL-22 signaling in the control of skin inflammation. (2018)
  • Author(s): Tomohiro Fukaya, Takehito Fukui, Tomofumi Uto, Hideaki Takagi, Junta Nasu, Noriaki, Miyanaga, Keiichi Arimura, Takeshi Nakamura, Haruhiko Koseki, Narantsog Choijookhuu, Yoshitaka Hishikawa, Katsuaki Sato
  • Journal Title: Front. Immunol. Volume: 9 Pages: 1-13
  • DOI: 10.3389/fimmu.2018.01418
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The HDAC Inhibitor, SAHA, Prevents Colonic Inflammation by Suppressing Pro-inflammatory Cytokines and Chemokines in DSS-induced Colitis (2018)
  • Author(s): Ali MN, Choijookhuu N, Takagi H, Srisowanna N, Nguyen Nhat Huynh M, Yamaguchi Y, Synn Oo P, Tin Htwe Kyaw M, Sato K, Yamaguchi R, Hishikawa Y.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 51 Issue: 1 Pages: 33-40
  • DOI: 10.1267/ahc.17033
  • NAID: 130006405039
  • Peer Reviewed / Open Access
• [Journal Article] Estrogen Regulates Mitochondrial Morphology through Phosphorylation of Dynamin-related Protein 1 in MCF7 Human Breast Cancer Cells (2018)
  • Author(s): Oo PS, Yamaguchi Y, Sawaguchi A, Kyaw MTH, Choijookhuu N, Ali MN, Srisowanna N, Hino S, Hishikawa Y.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 51 Issue: 1 Pages: 21-31
  • DOI: 10.1267/ahc.17034
  • NAID: 130006405038
  • Peer Reviewed / Open Access
• [Journal Article] Critical role of plasmacytoid dendritic cells in induction of oral tolerance. (2018)
  • Author(s): Uto T, Takagi H, Fukaya T, Nasu J, Fukui T, Miyanaga N, Arimura K, Nakamura T, Choijookhuu N, Hishikawa Y, Sato K.
  • Journal Title: J Allergy Clin. Immunol. Volume: 印刷中
  • DOI: 10.1016/j.jaci.2017.11.048
  • Peer Reviewed
• [Journal Article] Photodynamic therapy of human biliary cancer cell line using combination of phosporus porphyrins and light emitting diode. (2017)
  • Author(s): Jin Matsumoto, Kou Suzuki, Masahide Yasuda, Yuya Yamaguchi, Yoshitaka Hishikawa, Naoya Imamura, Atsushi Nanashima
  • Journal Title: Bioorg Med Chem. Volume: 25 Pages: 6536-6541
  • DOI: 10.1016/j.bmc.2017.10.031
  • Peer Reviewed
• [Journal Article] Novel role of Rac-Mid1 signaling in medial cerebellar development (2017)
  • Author(s): Nakamura T, Ueyama T, Ninoyu Y, Sakaguchi H, Choijookhuu N, Hishikawa Y, Kiyonari H, Kohta M, Sakahara M, de Curtis I, Kohmura E, Hisa Y, Aiba A, Saito N.
  • Journal Title: Development Volume: 144 Pages: 1863-1875
  • DOI: 10.1242/dev.147900
  • NAID: 120006363303
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Flexible adaptation of male germ cells from female iPS cells of endangered Tokudaia osimensis (2017)
  • Author(s): Arata Honda, Narantsog Choijookhuu, Haruna Izu, Yoshihiro Kawano, Mizuho Inokuchi, Kimiko Honsho, Ah-Reum Lee, Hiroki Nabekura, Hiroshi Ohta, Tomoyuki Tsukiyama, Yasuhide Ohinata, Asato Kuroiwa, Yoshitaka Hishikawa, Mitinori Saitou, Takamichi Jogahara, Chihiro Koshimoto
  • Journal Title: Sci Adv. Volume: -
  • DOI: 10.1126/sciadv.1602179
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Arsenic trioxide induces ROS activity and DNA damage, leading to G0/G1 extension in skin fibroblasts through the ATM-ATR-associated Chk pathway. (2017)
  • Author(s): Chayapong J, Madhyastha H, Madhyastha R, Nurrahmah QI, Nakajima Y, Choijookhuu N, Hishikawa Y, Maruyama M.
  • Journal Title: Environ Sci Pollut Res Int. Volume: 24 Pages: 5316-5325
  • DOI: 10.1007/s11356-016-8215-7
  • Peer Reviewed
• [Journal Article] Discrimination of Stem Cell Status after Subjecting Cynomolgus Monkey Pluripotent Stem Cells to Naive Conversion (2017)
  • Author(s): Honda A, Kawano Y, Izu H, Choijookhuu N, Honsho K, Nakamura T, Yabuta Y, Yamamoto T, Takashima Y, Hirose M, Sankai T, Hishikawa Y, Ogura A, Saitou M
  • Journal Title: Sci Rep. Volume: 7 Pages: 45285-45285
  • DOI: 10.1038/srep45285
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Frequency of CD4+CD161+ T Cell and Interleukin-10 Expression in Inflammatory Bowel Diseases (2017)
  • Author(s): Tsuchiya K, Ikeda T, Batmunkh B, Choijookhuu N, Ishizaki H, Hotokezaka M, Hishikawa Y, Nanashima A.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 50 Issue: 1 Pages: 21-28
  • DOI: 10.1267/ahc.16035
  • NAID: 130005399467
  • Peer Reviewed / Open Access
• [Journal Article] Estrogen Accelerates Cell Proliferation through Estrogen Receptor α during Rat Liver Regeneration after Partial Hepatectomy (2017)
  • Author(s): Batmunkh B, Choijookhuu N, Srisowanna N, Byambatsogt U, Synn Oo P, Noor Ali M, Yamaguchi Y, Hishikawa Y.
  • Journal Title: ACTA HISTOCHEMICA ET CYTOCHEMICA Volume: 50 Issue: 1 Pages: 39-48
  • DOI: 10.1267/ahc.17003
  • NAID: 130005399468
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] The role of estrogen receptors in intestinal homeostasis and disease. (2016)
  • Author(s): Choijookhuu N, Hino SI, Hishikawa Y.
  • Journal Title: Receptors & Clinical Investigation Volume: in press
  • DOI: 10.14800/rci.1109
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Arsenic acid inhibits proliferation of skin fibroblasts, and increases cellular senescence through ROS mediated MST1-FOXO signaling pathway (2016)
  • Author(s): Yamaguchi Y, Madhyastha H, Madhyastha R, Choijookhuu N, Hishikawa Y, Pengjam Y, Nakajima Y, Maruyama M
  • Journal Title: J. Toxicol. Sci. Volume: 41 Issue: 1 Pages: 105-113
  • DOI: 10.2131/jts.41.105
  • NAID: 130005119779
  • Peer Reviewed / Open Access
• [Journal Article] Plasmacytoid dendritic cells orchestrate TLR7-mediated innate and adaptive immunity for the initiation of autoimmune inflammation (2016)
  • Author(s): Hideaki Takagi, Keiichi Arimura, Tomofumi Uto, Tomohiro Fukaya, Takeshi Nakamura, Narantsog Choijookhuu, Yoshitaka Hishikawa, Katsuaki Sato
  • Journal Title: Sci Rep Volume: 6 Pages: 1-18
  • DOI: 10.1038/srep24477
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] 遺伝子の局在を見るーin situ hybridization (2016)
  • Author(s): 菱川善隆
  • Journal Title: Clinical Neuroscience Volume: 34 Pages: 625-628
• [Presentation] A new approach for FRET based in situ hybridization (2019)
  • Author(s): Narantsog Choijookhuu、菱川善隆
  • Organizer: 第13回日中合同組織細胞化学セミナー
  • Int'l Joint Research / Invited
• [Presentation] FRETを用いたin situハイブリダイゼーションによる遺伝子発現解析 (2018)
  • Author(s): 菱川善隆
  • Organizer: 日本顕微鏡学会第74回学術講演会
  • Invited
• [Presentation] A new strategy of FRET based molecular beacon fluorescent probe for in situ hybridization. (2017)
  • Author(s): Choijookhuu N ,石塚匠,徐岩,小路武彦,菱川善隆
  • Organizer: 第122回日本解剖学会総会・全国学術集会
  • Place of Presentation: 長崎市
  • Year and Date: 2017-03-28
• [Presentation] FRET(蛍光共鳴エネルギー移動)標識プローブによる高感度ISH法の有用性 (2017)
  • Author(s): 菱川善隆
  • Organizer: 第58回日本組織細胞化学会総会・学術集会
  • Invited
• [Presentation] The effect of estrogen and its receptor(ERα)in the liver regeneration after partial hepatectomy. (2017)
  • Author(s): 菱川善隆
  • Organizer: The12th China-Japan joint Seminar on Histochemistry and Cytochemistry
  • Int'l Joint Research / Invited
• [Presentation] Role of estrogen on mitochondrial dynamic through Drp1 and P-Drp1 in human breast cancer cell line. (2016)
  • Author(s): Phyu Synn Oo, Akira Sawaguchi,山口優也, Narantsog Choijookhu, Baatarsuren Batmunk,菱川善隆
  • Organizer: 第3回ミャンマーー日本シンポジウム in パテイン大学
  • Place of Presentation: パテイン、ミャンマー
  • Year and Date: 2016-12-03
  • Int'l Joint Research
• [Presentation] FRET based in situ hybridization: Advanced molecular histochemical approach (2016)
  • Author(s): Hishikawa Y
  • Organizer: 第3回ミャンマーー日本シンポジウム in パテイン大学
  • Place of Presentation: パテイン、ミャンマー
  • Year and Date: 2016-12-03
  • Int'l Joint Research
• [Presentation] A new strategy: FRET based molecular beacon fluorescent probe for in situ hybridization. (2016)
  • Author(s): Choijookhuu N ,石塚匠,徐岩,小路武彦,菱川善隆
  • Organizer: 第58回 日本顕微鏡学会九州支部学術講演会
  • Place of Presentation: 北九州市
  • Year and Date: 2016-12-03
• [Presentation] Possible regulation of estrogen and estrogen receptor alpha during liver regeneration (2016)
  • Author(s): Baatarsuren Batmunkh, Narantsog Choijookhuu, Phyu Synn Oo, Naparee Srisowanna,山口優也, 菱川善隆
  • Organizer: 第48回日本臨床分子形態学会・学術集会
  • Place of Presentation: 熊本市
  • Year and Date: 2016-09-23
• [Presentation] A new approach: FRET based molecular beacon fluorescent probe for in situ hybridization. (2016)
  • Author(s): Choijookhuu N ,石塚匠,徐岩,小路武彦,菱川善隆
  • Organizer: 第57回日本組織細胞化学会総会・学術集会
  • Place of Presentation: 三鷹市
  • Year and Date: 2016-09-03
• [Presentation] RET Based Molecular Beacon Fluorescent Probe For In Situ Hybridization. (2016)
  • Author(s): Choijookhuu N ,石塚匠,徐岩,小路武彦,菱川善隆
  • Organizer: 日本顕微鏡学会第72回学術講演会
  • Place of Presentation: 仙台市
  • Year and Date: 2016-06-14
• [Remarks] 宮崎大学医学部解剖学講座組織細胞化学分野
  • URL: http://www.med.miyazaki-u.ac.jp/home/anatomy1/