2016 Fiscal Year Research-status Report
Mitochondrial DNA deletions as a bioindicator of radiation exposure in papillary thyroid carcinoma
| Project/Area Number |
16K09805
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| Research Institution | Nagasaki University |
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Principal Investigator |
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)
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| Co-Investigator(Kenkyū-buntansha) |
中沢 由華 長崎大学, 原爆後障害医療研究所, 助教 (00533902)
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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| Project Period (FY) |
2016-04-01 – 2019-03-31
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| Keywords | mtDNA / MiSeq NGS / mtDNA deletions |
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| Outline of Annual Research Achievements |
MtDNA is recognized to evolve 10-100 times faster than nuclear DNA due to the aggressive environment reach in oxygen species specific to mitochondrion, increased infidelity of mtDNA polymerase γ, slippage of mitochondrion systems of DNA repair, particulars of mtDNA structure, spatial proximity to mitochondrial membrane, and peculiarities of mtDNA replication and transcription. In our previous work we showed that in contrast to sporadic PTCs, a significant correlation between the prevalence of large-scale mtDNA deletions and relative mtDNA content was found in tumor tissues of radiation-associated PTCs. Additionally, using primary cultures of human thyrocytes exposed to ionizing radiation (IR) (from 0.5 to 5 Gy), we found a significant dose-dependent increase of mtDNA deletions, while mtDNA content did not change. Hence, IR can generate large-scale mtDNA deletions in thyrocytes after exposure, however we could not determine the particularities of deletions caused by IR.
Due to the availability of NGS, it was possible to detect sequence of each particular deletion of mtDNA arising as a result of irradiation.
Using MiSeq (Illumina) we already compared the mtDNA large-scale deletions in N (normal) and T (tumor) counterparts of radiation-induced PTCs from Belarus. In T counterparts we found significantly more deletions then in N counterparts.
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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
During the research period we prepared many primary cultures of thyrocytes, exposed them to 0.01, 0.025, 0.05 and 0.1 Gy (low-dose IR) and 0.25, 0.5, 1, 2 and 5 Gy, allowed to recover for 48h before DNA extractions. Using 2 sets of overlapping primers and Prime STAR GXL DNA polymerase (Takara Bio) we will perform PCR to reach amplicon size of 9kb. We purified PCR products using NucleoSpin Gel and PCR Clean-up (Takara Bio) before preparing library for sequencing using MiSeq (Illumina).
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| Strategy for Future Research Activity |
We plan to perform a comprehensive analysis of thyroid mtDNA deletions using MiSeq NGS (Illumina). Using the advantage and accuracy of NGS, we expect to discover radiation-specific mtDNA deletions in primary thyrocytes exposed to different doses (including low doses) of IR, and by comparing the patterns (size, localization in the mtDNA genome and sequence around breakpoints) of deletions in radiation-induced (post-Chernobyl) and sporadic PTCs in different age groups.
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| Causes of Carryover |
The fund-consuming parts of the study during the last year included preparing primary cultures of thyrocytes, exposed them to IR, extraction the DNA and PCR amplification. All reagents for these methods were not expensive, that’s budget was not fully used.
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| Expenditure Plan for Carryover Budget |
In this year we plan to use almost all budget for performing NGS using MiSeq (Illumina).
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| Remarks |
長崎大学原研医療
http://www-sdc.med.nagasaki-u.ac.jp/drms/
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Research Products
(8 results)
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[Journal Article] Genotype Analyses in the Japanese and Belarusian Populations Reveal Independent Effects of rs965513 and rs1867277 but Do Not Support the Role of FOXE1 Polyalanine Tract Length in Conferring Risk for Papillary Thyroid Carcinoma.2017
Author(s)
Nikitski AV, Rogounovitch TI, Bychkov A, Takahashi M, Yoshiura KI, Mitsutake N, Kawaguchi T, Matsuse M, Drozd VM, Demidchik Y, Nishihara E, Hirokawa M, Miyauchi A, Rubanovich AV, Matsuda F, Yamashita S, Saenko VA.
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Journal Title
Thyroid
Volume: 27
Pages: 224-235.
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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[Journal Article] Association between p53-binding protein 1 expression and genomic instability in oncocytic follicular adenoma in thyroid.2016
Author(s)
Mussazhanova Z, Akazawa Y, Matsuda K, Shichijo K, Miura S, Otsubo R, Oikawa M, Yoshiura K, Mitsutake N, Rogounovitch T, Saenko V, Kozykenova Z, Zhetpisbaev B, Shabdarbaeva D, Sayakenov N, Amantayev B, Kondo H, Ito M, Nakashima M.
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Journal Title
Endocr J
Volume: 63
Pages: 457-467
DOI
Peer Reviewed / Open Access
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[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.
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Journal Title
Endocrinology
Volume: 157
Pages: 2182-2195
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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