2016 Fiscal Year Research-status Report
Establishment of a synthetic promoter-based system for sensing oncogenic alteration in human live thyroid cells at a single-cell level
| Project/Area Number |
15K09438
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| Research Institution | Nagasaki University |
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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)
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| 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, 特任研究員 (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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| Project Period (FY) |
2015-04-01 – 2018-03-31
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| 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 |
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| Outline of Annual Research Achievements |
Human thyroid epithelium-derived Nthyr-ori 3-1 cells were infected with lentiviruses carrying thyroid cancer-related oncogenes RET/PTC1 and BRAFV600E, and a control TRFP (Turbo Red fluorescent Protein). RNA was extracted from transduced cells 48h later and gene expression profiles were determined using Agilent SurePrint G3 Human GE 8x60K v2 Microarrays. Analysis with GeneSpring GX software identified 246 differentially expressed genes (fold-change >2 as compared to the control) simultaneously for RET/PTC1 and BRAFV600E-transduced cells
Sequences of the promoters from -2000 to +500bp from the transcription start site of these genes were downloaded from the Eukaryotic Promoter Database and scanned for the presence of Transcription Factors Binding Sites (TFBS) using the JASPAR database. In addition, the proximal promoter regions (-500 to +500bp from transcription start site) were analyzed using ISMARA platform for the whole microarray dataset.
Fifteen most significant TFBS were determined. Libraries containing single-copy or concatemerized individual TFBS were prepared and cloned into pEZX-ePG04 plasmid as artificial promoters. These plasmids were contransfected with RET/PTC1 and BRAFV600E expression vectors into Nthyr-ori 3-1 cells, and promoter activity was measured using dual-luminescent assay. As a results, 3 strongest TFBS were determined and confirmed to be able to respond on oncogenic stimulation individually or as a mixed-TFBS promoter.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
According to our planning, we transduced Nthyr-ori 3-1 cells with thyroid cancer-specific RET/PTC1 and BRAFV600E oncogenes with high efficacy. RNA was successfully collected from transduced or control cells and the expressiosome was analyzed using microarrays, 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.
Promoter analysis was performed using two independent approaches leading to the determination of 15 TFBS associated with gene upregulation in response to oncogenes. These TRBS were tested for the ability to serve as the elements of an artificial promoter driving the expression of a luciferase reporter for the screening. As a result, we identified 3 TBFS that can be used for the stable reporter cell system. A library of single-copy individual or concatemerized or mixed TFBS were prepared in the luciferase reporter plasmid and recloned into the pZsGreen1-DR reporter vector.
Overall, all phases of the study were performed timely and rather smoothly, in accordance to the initial planning.
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| Strategy for Future Research Activity |
Since our goal is to establish a fluorescent protein-based cell reported system, it is necessary to determine how the promoter activity in the luciferase assay is translated into the GFP signal. The following experiments are planned during this year.
1) From the library of artificial promoter/ZsGreen1-DR plasmids, 10 constructs that display the optimal signal-to-noise ratio in the luminescent assay will be chosen.
2) These plasmids will be stably transfected into the target Nthyr-ori 3-1 cells to establish clones via neomycin selection.
3) Clones will be tested for their ability to express the green fluorescent protein reporter in a background state and after challenging with RET/PTC1 and BRAFV600E-encoding vectors to determine the most sensitive and specific clones by live cell imaging in a Thermo Scientific Cellomics ArrayScan VTI 700 machine. We will establish 1-3 reporter cell lines reacting on the oncogene stimulation.
4) As it is known that radiation exposure can induce RET/PTC1 rearrangements in a dose-dependent manner in Nthyr-ori 3-1 cells, the reaction of reporter cell lines will be examined after irradiation with 0-10 Gy of γ-rays, apying particular attention at the low-dose interval (up to 200 mGy).
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| Causes of Carryover |
The fund-consuming parts of the study during the last year included cloning of the artificial promoters into the luciferase reporter plasmid and screening of the promoter activity using a dual reporter kit. First, we made a research and prepared self-made reagent mixes that could detect SEAP and GLuc substantially reducing the cost.
Second, after the initial screening of artificial promoters’ activities, we found that out of 15 individual TFBS-based promoters, 12 displayed very weak activity comparable with the background signal. Thus, there was no longer need to further investigate the effects of these TFBS repeat number, and we focused only on the 3 best-responding TFBS. This also resulted in the significant drop in the number of promoters to be tested and lead to the cost reduction.
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| Expenditure Plan for Carryover Budget |
Three major types of expense are planned as follows.
1) Microbiological and cell culture works. These include plasmid propagation, preparation of lentiviruses, cell transfection and infection, selection and experimentation with stable clones. Reagents and consumables will be purchased accordingly - 60%. 2) Other expense, including payment for using shared common use equipment and paper publication charge - 20%. 3) Travel expense to participate in the Japan Thyroid association meeting and other conferences and seminars inside the country - 20%. 4) No other expense such as payment to the third party or remuneration is planned (0%).
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Research Products
(4 results)
