| 研究課題/領域番号 |
15K09438
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| 研究機関 | 長崎大学 |
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研究代表者 |
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)
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| 研究分担者 |
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)
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| 研究期間 (年度) |
2015-04-01 – 2018-03-31
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| キーワード | 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 |
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| 研究実績の概要 |
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.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
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.
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| 今後の研究の推進方策 |
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”.
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