| 研究課題/領域番号 |
24591369
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| 研究機関 | 長崎大学 |
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研究代表者 |
SAENKO Vladimir 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 |
中島 正洋 長崎大学, 原爆後障害医療研究所, 教授 (50284683)
光武 範吏 長崎大学, 原爆後障害医療研究所, 准教授 (50404215)
伊東 正博 独立行政法人国立病院機構(長崎医療センター臨床研究センター), 臨床検査科, 部長 (30184691)
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| キーワード | 内分泌学 / 甲状腺腫瘍 / 遺伝子多型 |
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| 研究概要 |
The first line of investigation was aimed at the assessment of the association of several single-nucleotide polymorphisms (SNPs), which have been previously reported to confer risk for thyroid cancer, with a benign tumor of the thyroid, follicular adenoma (FA), in Japanese patients.
The number of cases of pathologically verified FA in the study was increased from 841 to a total of 959. In addition, we considered genotyping of a group of patients with papillary thyroid carcinoma (PTC), which will be analyzed in parallel with the FAs. Expanding our previous study (Matsuse et al., 2011), 27 newly diagnosed cases were added to form a series of 535 PTCs. DNA was extracted from all FA and PTC tissues successfully.
Genotyping was performed using predesigned and functionally tested primer/probe mixes for FOXE1 rs965513 and NKX2-1 rs944289. The number of successfully genotyped samples is 931 FA and 517 PTC. Data on 2766 population controls were obtained by the collaborators from Kyoto university. The number of SNPs initially planned to be analyzed (2 SNPs mentioned above) was decided to be expanded (to 5) because of recent publications on novel associations reported on the genome-wide scale in thyroid cancer.
Mechanism of FOXE1 translocation to the cytoplasm, which is a hallmark of PTC (Bychkov et al., 2014), is being addressed using lentiviral transduction of several PTC cell lines and primary PTC cultures. One cell line was found to displayed moderate cytoplasmic expression, and will be used in further experiments.
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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
The overall progress is largely in line with initial planning and thus could be self-estimated at mark ②.
According to our planning, collection, pathological verification of follicular adenoma cases and DNA extraction were accomplished on time. The number of samples included in the study (959) corresponds well to the initially aimed sample size of 1000 cases and ensures the desired statistical power.
Measures have been taken to improve DNA quality in the samples that displayed poor PCR amplification on direct genotyping (presumably due to chemical modifications caused by extensive fixation in formalin, and long sample storage/age). Several techniques were tested eventually resulting in the adoption of treatment according to a protocol used for archived tissues from A-bomb hibakusha (Taga et al., 2013). Using it, genotype calls were successfully obtained for 95-97% samples in our series. This problem was solved timely and will not cause a delay in the work.
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| 今後の研究の推進方策 |
Future research strategy includes accomplishment of genotyping of FA and PTC patients for 3 SNPs: NRG1 rs2439302 (C_16238367_10), FOXE1 rs1867277 (C_11736668_10) and POU5FB rs6983267 (C_29086771_20). After these results are obtained, statistical analysis will be performed using logistic regression models or exact test for trend (Yamada et al., 2009). In case association are found, the relationship of rs2439302 to NRG1 and of rs944289 to NKX2-1 and PTCSC3 genes will be examined by RT-PCR in a series of about 100 normal thyroid and PTC tissues from surgical specimens.
Functional studies of the mechanism of FOXE1 translocation to the cytoplasm will include transient transduction of KTC-1 cell line with lentiviral vector carrying FLAG-tagged FOXE1 insert (vector is already available) and/or attempts to establish stable subclones which display not only nuclear but also cytoplasmic FOXE1 localization. Cells will be fractionated into the cytoplasmic and nuclear fractions for immunoprecipitation with anti-FLAG conjugated magnetic beads. In addition, we plan to use reversible cross-linking agents to identify proteins interacting with FOXE1. If these experiments are successful, the components of FOXE1 interactome will be identified using mass-spectrometry analysis.
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