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血漿エクソソーム由来microRNA を用いたグリオブラストーマ診断バイオマーカーの探索
山口, 響子;森口, 徹生;山下, 大介;大西, 丘倫;金子, 貞男;的場, 亮;鄭, 漢忠;近藤, 亨
Permalink : http://hdl.handle.net/2115/73622
KEYWORDS : グリオブラストーマ（GBM）;エクソソーム;microRNA（miR）;バイオマーカー;glioblastoma multiform (GBM);exosome;biomaker
Abstract
最も悪性度の高い脳腫瘍の１つであるグリオブラストーマ（GBM）は，標準治療（外科手術および化学放射線療法）を施しても平均生存期間中間値（約15か月）が極めて短い難治性疾患である．この難治性の原因の１つは，腫瘍細胞の強い組織浸潤能と増殖能により神経症状発症時には摘出不可能な範囲に腫瘍細胞が拡散しているためである．つまり，簡便かつ検出感度の高いGBM診断用バイオマーカーがあれば，早期腫瘍摘出が可能となり，予後の改善が期待できる．分泌小胞体エクソソームは，その産生細胞が発現しているmRNA，microRNA (miR)，タンパクなどを含み，様々な体液中に安定に存在することから，新たなバイオマーカー探索の標的としてその解析と利用が注目されている．今回，私たちはGBM患者と健常人の血漿中のエクソソームに含まれるmiRを比較分析することで，診断マーカーとなりうるmiRの同定を目的として解析を行った．GBM患者６人の凍結血漿８検体（同一患者の再発術前１検体と術後１検体を含む）と健常人２人の凍結血漿を用いて解析を行った．凍結血漿から調整したエクソソーム内miRについて，RNA-seqを用いた網羅的な解析を行い，GBM患者血漿エクソソームに多く含まれる34種のmiRと健常人血漿エクソソームに多く含まれる47種のmiRを同定した．GBM患者血漿エクソソームに多く含まれていたmiRの中で，エクソソームバイオマーカーとして報告のないmiR-186とmiR-20aについて，定量PCRを用いてGBM患者と健常人血漿エクソソーム内miR量を検討した．その結果，miR-20aはGBM患者と健常人間で有意な差は認められなかったが，GBM患者５人中４人の血漿エクソソームにmiR-186が豊富に含まれていることを確認した．加えて，術後患者ではmiR-186量が健常人レベルまで減少していることを発見した．さらに，標準治療を行った再発例においても，術前血漿エクソソーム内miR-186の上昇を認めた．これらの結果は，血漿エクソソーム内miR-186がGBMの病状に即した新規バイオマーカーである可能性を示唆している．
Glioblastoma multiform (GBM), the most malignant brain tumor with a median survival rate of about 15 months, frequently recurs despite multimodal treatment with surgery and chemo/radiotherapies. One of the reasons why GBM is incurable is that it is impossible to remove all of GBM cells invading into the large area of brain, when patients are diagnosed. It is, therefore, crucial to identify a simple and sensitive biomarker that diagnoses GBM at its earliest stage. Exosomes are extracellular vesicles secreted by all cells including tumor cells. Since exosomes contain mRNA, microRNA (miR) and proteins specific for the cell of origin, and are relatively stable in body fluids including blood, they are attracting more attention as targets for identifying new biomarkers. Here we addressed to identify new diagnostic exosomal miR in the plasma of GBM patients, by comparing with those of healthy volunteers. Plasma samples were collected from six GBM patients and two healthy controls. We have performed RNA sequence analysis, using plasma exosomes from one preoperative GBM patient and two healthy volunteers and identified that thirty four miRs were predominantly included in the plasma exosomes of GBM patients, while forty seven ones did in those of healthy volunteers. Among the GBM plasma exosome miRs, we focused on miR-186 and miR-20a, as they have not yet shown as exosome biomarkers. Using plasma exosomes from five GBM patients and two volunteers, we performed qRT-PCR and verified the increase amount of miR-186 in the GBM plasma exosomes, whereas miR-20a showed no significant difference between GBM plasma exosomes and control ones. It should be notable that miR-186 in the GBM plasma exosomes decreased after operation, and significantly increased in recurrence. Taken together, our results identified miR-186 as a potentially useful GBMdiagnostic biomarker.
Glioblastoma multiform (GBM), the most malignant brain tumor with a median survival rate of about 15 months, frequently recurs despite multimodal treatment with surgery and chemo/radiotherapies. One of the reasons why GBM is incurable is that it is impossible to remove all of GBM cells invading into the large area of brain, when patients are diagnosed. It is, therefore, crucial to identify a simple and sensitive biomarker that diagnoses GBM at its earliest stage. Exosomes are extracellular vesicles secreted by all cells including tumor cells. Since exosomes contain mRNA, microRNA (miR) and proteins specific for the cell of origin, and are relatively stable in body fluids including blood, they are attracting more attention as targets for identifying new biomarkers. Here we addressed to identify new diagnostic exosomal miR in the plasma of GBM patients, by comparing with those of healthy volunteers. Plasma samples were collected from six GBM patients and two healthy controls. We have performed RNA sequence analysis, using plasma exosomes from one preoperative GBM patient and two healthy volunteers and identified that thirty four miRs were predominantly included in the plasma exosomes of GBM patients, while forty seven ones did in those of healthy volunteers. Among the GBM plasma exosome miRs, we focused on miR-186 and miR-20a, as they have not yet shown as exosome biomarkers. Using plasma exosomes from five GBM patients and two volunteers, we performed qRT-PCR and verified the increase amount of miR-186 in the GBM plasma exosomes, whereas miR-20a showed no significant difference between GBM plasma exosomes and control ones. It should be notable that miR-186 in the GBM plasma exosomes decreased after operation, and significantly increased in recurrence. Taken together, our results identified miR-186 as a potentially useful GBMdiagnostic biomarker.
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