Establishment of a synthetic promoter-based system for sensing oncogenic alteration in human live thyroid cells at a single-cell level

2015 Fiscal Year Research-status Report

• Project/Area Number: 15K09438
• Research Institution: Nagasaki University
• Principal Investigator: サエンコ ウラジミール 長崎大学, 原爆後障害医療研究所, 准教授 (30343346)
• Co-Investigator(Kenkyū-buntansha): ログノビッチ タチアナ 長崎大学, 原爆後障害医療研究所, 助教 (30423643) ; 光武 範吏 長崎大学, 原爆後障害医療研究所, 准教授 (50404215)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: 甲状腺癌 / 癌遺伝子 / 遺伝子プロモーター / 合成生物学 / 単一細胞レポーター

Outline of Annual Research Achievements

Thyroid-related RET/PTC1 and BRAFV600E oncogenes, and TRFP (Turbo Red fluorescent Protein) as a control were inserted into pLenti6/V5 vector and used to prepare lentiviral particles in the 293FT cells. Human epithelial thyroid-derived Nthyr-ori 3-1 cells were transduced with prepared lentiviruses with and efficacy of 70-80% as judged by V5-tag immunofluorescent staining for RET/PTC1 and BRAFV600E, and by the direct microscopic inspection for TRFP-carrying vector. Activation of MAP-kinase and AKT pathways and expression of transgenes was confirmed by Western blotting with anti-phospho-ERK, anti-phospho-AKt and anti-V5 antibodies, respectively. Cell morphology was microscopically monitored and documented. As expected, activated oncogenes induced mesenchymal cell-like changes while the control TRFP protein did not.
RNA from transduced cells was extracted with Isogene after 48-hour incubation, treated with DNAse and purified using Qiagen columns. RNA quality was RIN>8 as assessed on an Agilent 2100 Bioanalyzer.
Genome-wide expression profile was determined on Agilent SurePrint G3 Human GE 8x60K v2 Microarrays and analyzed using GeneSpring GX software. In total, 246 differentially expressed genes common for RET/PTC1 and BRAFV600E oncogenes, as compared with parent Nthyr-ori 3-1 cells or with those transduced with TRFP were identified with fold-change >2.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

According to our planning, we managed to prepare a set of plasmids carrying the inserts of thyroid cancer-specific RET/PTC1 and BRAFV600E oncogenes, and of a control TRFP gene. Lentiviruses were successfully obtained using 293FT cells.
Transduction of Nthyr-ori 3-1 cells with prepared lentiviruses with and efficacy of 70-80% was achieved. Corresponding V5-tagged oncogene or control protein expression was confirmed by immunofluorescence and Western blotting. Morphological changes in transduced cells were observed.
RNA was successfully collected from transduced or control cells, purified, subjected to quality analysis and analyzed using Agilent microarrays. Data were collected and analyzed resulting in the determination of 246 differentially expressed genes concordantly undergoing changes after transduction with RET/PTC1 and BRAFV600E oncogenes as compared to intact or TRFP-transduced Nthyr-ori 3-1 cells.
Overall, this phase of the study is performed timely and rather smoothly, in accordance to the initial planning.

Strategy for Future Research Activity

Sequences of the region from +2000 to -500bp from the transcription start site of 246 differentially expressed genes will be downloaded from the Eukaryotic Promoter Database (http://epd.vital-it.ch/human/human_database.php) and scanned for the presence of Transcription Factors Binding Sites (TFBS) using the JASPAR database (http://jaspar.genereg.net/cgi-bin/jaspar_db.pl) at the relative profile score threshold of 90%.
In addition, we will attempt TFBS scanning of the proximal promoter regions (+500 to -500bp from transcription start site) for the whole microarray dataset using a recently developed analytical platform ISMARA - Integrated System for Motif Activity Response Analysis (https://ismara.unibas.ch/fcgi/mara).
The results of manual promoter scanning/statistical analysis and those of ISMARA will be compared to determine the “positive” transcription factors with the highest probability.
A set of synthetic double-stranded oligonucleotides corresponding to “positive transcription factors” with 5’-phosphorylated ligatable EcoRI adapters will be prepared. A library will be obtained by overnight ligation of the annealed oligonucleotides into pEZX-ePG04 (GLuc/SeAP) plasmid. A set of 10-20 individual plasmids will be purified, sequenced and screened for promoter activity using Secrete-Pair Dual-Luminescence assay in 48-well format in HTori-3.1 cells cotransfected with RET/PTC1, BRAFV600E or control vectors to identify the “optimal promoter”.

Research Products

• [Journal Article] Targeted Foxe1 overexpression in mouse thyroid causes the development of multinodular goiter but does not promote carcinogenesis. (2016)
  • Author(s): Nikitski A, Saenko V, Shimamura M, Nakashima M, Matsuse M, Suzuki K, Rogounovitch T, Bogdanova T, Shibusawa N, Yamada M, Nagayama Y, Yamashita S, Mitsutake N
  • Journal Title: Endocrinology Volume: 印刷中 Pages: 印刷中
  • DOI: 10.1210/en.2015-2066
  • Peer Reviewed / Open Access
• [Journal Article] Major Factors Affecting Incidence of Childhood Thyroid Cancer in Belarus after the Chernobyl Accident: Do Nitrates in Drinking Water Play a Role? (2015)
  • Author(s): Drozd VM, Saenko VA, Brenner AV, Drozdovitch V, Pashkevich VI, Kudelsky AV, Demidchik YE, Branovan I, Shiglik N, Rogounovitch TI, Yamashita S, Biko J, Reiners C
  • Journal Title: PLoS One Volume: 10 Pages: e0137226
  • DOI: 10.1371/journal.pone.0137226.eCollection 2015
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] TCUKIN and CORGI Consortiums, Carmona LG; JTCMS Consortium: The 8q24 rs6983267G variant is associated with increased thyroid cancer risk. (2015)
  • Author(s): Sahasrabudhe R, Estrada A, Lott P, Martin L, Polanco Echeverry G, Velez A, Neta G, Takahasi M, Saenko V, Mitsutake N, Jaeguer E, Duque CS, Rios A, Bohorquez M, Prieto R, Criollo A, Echeverry M, Tomlinson I
  • Journal Title: Endocr Relat Cancer Volume: 22 Pages: 841-849
  • DOI: 10.1530/ERC-15-0081
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] BRAFV600E mutation is highly prevalent in thyroid carcinomas in the young population in Fukushima: a different oncogenic profile from Chernobyl. (2015)
  • Author(s): Mitsutake N, Fukushima T, Matsuse M, Rogounovitch T, Saenko V, Uchino S, Ito M, Suzuki K, Suzuki S, Yamashita S
  • Journal Title: Sci Rep Volume: 5 Pages: 16976
  • DOI: 10.1038/srep16976
  • Peer Reviewed / Open Access
• [Presentation] The combined influence of nitrates in drinking water and radiation on incidence of childhood thyroid cancer in Belarus after the Chernobyl accident (2015)
  • Author(s): Valentina M. Drozd, Vladimir A. Saenko, Alina V. Brenner, Vladimir Drozdovitch, Vasilii I. Pashkevich, Anatoliy V. Kudelsky, Yuri E. Demidchik, Igor Branovan, Nikolay Shiglik, Tatiana I. Rogounovitch, Shunichi Yamashita, Johannes Biko, Christoph Reiners
  • Organizer: Global Summit and Medicare Expo on Head & Neck Surgery
  • Place of Presentation: サンアントニオ（アメリカ）
  • Year and Date: 2015-11-30 – 2015-12-01
  • Int'l Joint Research
• [Presentation] Screening for Thyroid Nodular Disease in Brest Region of Belarus: 29 Years after The Chernobyl Catastrophy (2015)
  • Author(s): M. Lushchyk, V. Drozd, L. Danilova, A. Grigorovich, V. Sivuda, A. Romanovskyj, Y. Demidchik, T. Rogunovich, V. Saenko, I.D. Branovan, N. Takamura, S. Yamashita
  • Organizer: 15th International Thyroid Congress and 85th Annual Meeting of the ATA
  • Place of Presentation: オーランド（アメリカ）
  • Year and Date: 2015-10-18 – 2015-10-23
  • Int'l Joint Research
• [Presentation] Influence of Nitrate in Drinking Water in Open Wells on Childhood Thyroid Cancer Rates in Belarus and Ukraine after The Chernobyl Accident (2015)
  • Author(s): V. Drozd, V. Saenko, A. Brenner, V. Drozdovitch, M. Tronko, V. Shpak, T. Bogdanova, A. Serduk, I. Chernychenko, S. Tsymbaliuk, V. Pashkevich, A. Kudelsky, D. Branovan, N. Shiglik, T. Rogounovitch, S. Yamashita, J. Biko, C. Reiners
  • Organizer: 15th International Thyroid Congress and 85th Annual Meeting of the ATA
  • Place of Presentation: オーランド（アメリカ）
  • Year and Date: 2015-10-18 – 2015-10-23
  • Int'l Joint Research
• [Presentation] Gene rearrangements in radiation-induced thyroid cancer (2015)
  • Author(s): Vladimir Saenko
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: 国立京都国際会館（京都府・京都市）
  • Year and Date: 2015-05-25 – 2015-05-29
  • Int'l Joint Research
• [Presentation] Foxe1 Overexpression and Radiation Effects on Mouse Thyroid (2015)
  • Author(s): Alyaksandr Nikitski, Norisato Mitsutake, Keiji Suzuki, Vladimir Saenko, Tatiana Rogounovitch, Shunichi Yamashita
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: 国立京都国際会館（京都府・京都市）
  • Year and Date: 2015-05-25 – 2015-05-29
  • Int'l Joint Research
• [Presentation] Novel RET/PTC rearrangement identified by RNA-NGS in pediatric papillary thyroid carcinoma from Belarus (2015)
  • Author(s): T. Rogounovitch, V. Saenko, S. Mankouskaya, M. Fridman, Yu. Demidchik, S. Yamashita
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: 国立京都国際会館（京都府・京都市）
  • Year and Date: 2015-05-25 – 2015-05-29
  • Int'l Joint Research
• [Presentation] Potential Joint Effect of Nitrates in Drinking Water and Radiation Dose to the Thyroid on Incidence of Pediatric Thyroid Cancer in Belarus (2015)
  • Author(s): Valentina M Drozd, Vladimir A Saenko, Alina V Brenner, Vladimir Drozdovitch, Vasilii I Pashkevich, Alexander V Kudelsky, Yuri E Demidchik, Igor Branovan, Nikolay Shiglik, Tatiana I Rogounovitch, Shunichi Yamashita, Tamara Platonova, Johannes Biko, Christoph Reiners
  • Organizer: The 15th International Congress of Radiation Research
  • Place of Presentation: 国立京都国際会館（京都府・京都市）
  • Year and Date: 2015-05-25 – 2015-05-29
  • Int'l Joint Research
• [Remarks] 長崎大学原研疫学ホームページ
  • URL: http://www-sdc.med.nagasaki-u.ac.jp/dhrc/