JP5884219B2 - Method for detecting urothelial cancer based on miRNA expression profiling - Google Patents
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Description
本発明は、尿検体中のmiRNAの発現量の変動を測定することから成る、尿路上皮癌の検出方法等に関する。 The present invention relates to a method for detecting urothelial cancer, etc., comprising measuring a change in the expression level of miRNA in a urine sample.
成熟型マイクロRNA（miRNA）とは１９〜２１塩基のnon-coding RNA分子であり、70〜100塩基ほどのヘアピン構造をとる前駆体マイクロRNA（pre-miRNA）から成る。2007年3月時点で、500種類のヒトmiRNAがゲノム中に存在することが報告されている（miRBase::Sequences, Sanger Institute）。 A mature microRNA (miRNA) is a 19- to 21-base non-coding RNA molecule, and consists of a precursor microRNA (pre-miRNA) having a hairpin structure of about 70 to 100 bases. As of March 2007, 500 human miRNAs have been reported to exist in the genome (miRBase :: Sequences, Sanger Institute).
miRNAの機能は、遺伝子の非翻訳領域の配列依存的に作用し、遺伝子発現を翻訳後あるいは転写レベルで制御していることから、発生、分化およびアポトーシスに関与していることが示唆されるが、その機能は依然不明である。近年、miRNAの間違った遺伝子制御機構がヒト疾患に関与していることが報告されている。特に最近、ヒト癌細胞において発現が変動しているmiRNAの報告が相次いでなされている (非特許分献１)。 The miRNA function acts in a sequence-dependent manner in the untranslated region of the gene and regulates gene expression after translation or at the transcriptional level, suggesting that it is involved in development, differentiation and apoptosis. The function is still unknown. In recent years, it has been reported that the wrong gene regulation mechanism of miRNA is involved in human diseases. Recently, miRNAs whose expression is varied in human cancer cells have been reported one after another (Non-patent distribution 1).
本発明者は、既にこのようなmiRNAの発現量の変動を測定することから成る膀胱癌の検出方法等を確立した（特許文献１）。 The present inventor has already established a method for detecting bladder cancer and the like, which comprises measuring such a change in the expression level of miRNA (Patent Document 1).
これまで尿路上皮癌は侵襲的な内視鏡検査および生検にて診断されてきた。また、上部尿路（腎盂・尿管）の癌は内視鏡的アプローチが困難なため特に診断が難渋する場合が多い。非侵襲的検査として尿細胞検査が有用とされているが、特異度（90-95％）が高いが感度は低い（20-30％）ため、検査が陰性であっても癌の存在を否定できないという弱点がある。尿中ＮＭＰ-22やＢＴＡなどの新しいマーカーは感度・特異度共に50％ぐらいであり、補助的診断に過ぎず殆ど普及はしていない。このような理由から、尿路上皮癌の新規の診断・検出用マーカー・キットを開発することは急務である。 So far, urothelial cancer has been diagnosed by invasive endoscopy and biopsy. In addition, cancer of the upper urinary tract (renal pelvis and ureter) is often difficult to diagnose because it is difficult to perform an endoscopic approach. Urine cell testing is considered useful as a non-invasive test, but the specificity is high (90-95%) but the sensitivity is low (20-30%), so even if the test is negative, the presence of cancer is denied There is a weakness that can not be. New markers such as urinary NMP-22 and BTA have both sensitivity and specificity of about 50%, and are only auxiliary diagnoses and are not widely used. For these reasons, it is urgent to develop a novel marker kit for diagnosis and detection of urothelial cancer.
今回、本発明者は、２種類のmiRNAが尿路上皮癌患者の尿中で健常人と比較して有意に発現変動していることを初めて見出した。これらmiRNAは尿路上皮癌の癌化プロセスに重要な役割を担っていると考えられる。本発明者はこのような知見に基づき本発明を完成した。 This time, the present inventor has found for the first time that the expression of two types of miRNAs is significantly changed in the urine of patients with urothelial cancer compared to healthy individuals. These miRNAs are thought to play an important role in the canceration process of urothelial cancer. The present inventor has completed the present invention based on such findings.
即ち、本発明は、主に以下の各態様を含むものである。
［１］尿検体中の細胞中のmiR-96及びmiR-183から成る群より選択された少なくとも一つのmiRNAの発現量の変動を測定する方法、及び、該測定方法から成る尿路上皮癌の検出方法。
［２］細胞中のmiR-96及びmiR-183から成る群より選択された少なくとも一つのmiRNAの発現量の変動に基づく、尿路上皮癌の治療又は予防に有効な物質のスクリーニング方法。
［３］本発明方法に用いるキット。
［４］上記検出方法と尿細胞検査とを組み合わせることを特徴とする、尿路上皮癌の検出方法。
That is, the present invention mainly includes the following aspects.
[1] A method for measuring a change in the expression level of at least one miRNA selected from the group consisting of miR-96 and miR-183 in cells in a urine sample, and urothelial cancer comprising the method Detection method.
[2] A screening method for a substance effective for treating or preventing urothelial cancer based on a change in the expression level of at least one miRNA selected from the group consisting of miR-96 and miR-183 in cells.
[3] A kit used in the method of the present invention.
[4] A method for detecting urothelial cancer, comprising combining the above detection method and a urine cell test.
本発明において、尿検体中の特定のmiRNA の発現量の変動を測定することによって、尿路上皮癌の非侵襲的検出が可能となり、更に、これらmiRNA の発現量の変動に基づき、尿路上皮癌の治療又は予防に有効な物質をスクリーニングすることが可能となった。
本発明で使用するmiRNAは小さい（22塩基対）ために、酵素や酸による変性が少なく検体採取から24時間後でも安定した検出が可能である。又、これらmiRNAは尿検体中の細胞成分のみでなく可溶性分画にも存在するので、本発明方法は従来の尿細胞検査と較べて高い特異度に加えて高い感度を有しており、尿細胞検査で陰性の検体でも検出し得る。
更に、リアルタイムＰＣＲを応用した場合には、検出にかかる時間は約3時間以下と迅速かつ信頼性に優れた検出系が可能となる。
又、上記検出方法と尿細胞検査とを組み合わせることによって、高い検出率で非侵襲的な尿路上皮癌の判定が可能である。
In the present invention, non-invasive detection of urothelial cancer becomes possible by measuring the variation in the expression level of a specific miRNA in a urine sample, and further, based on the variation in the expression level of these miRNAs, It has become possible to screen for substances effective for the treatment or prevention of cancer.
Since the miRNA used in the present invention is small (22 base pairs), there is little denaturation by enzymes and acids, and stable detection is possible even 24 hours after sample collection. In addition, since these miRNAs are present not only in cell components in urine specimens but also in soluble fractions, the method of the present invention has high sensitivity in addition to high specificity compared to conventional urine cell tests, and urine Even a negative sample in a cell test can be detected.
Furthermore, when real-time PCR is applied, the time required for detection is about 3 hours or less, and a detection system that is quick and highly reliable is possible.
In addition, by combining the detection method and the urine cell test, it is possible to determine noninvasive urothelial cancer with a high detection rate.
本発明の方法において測定されるmiR-96及びmiR-183に関する情報（例えば、塩基配列等）は、既に記載したデータベース（miRBase::Sequences, Sanger Institute）から容易に入手可能である。 Information on miR-96 and miR-183 measured in the method of the present invention (for example, base sequence and the like) can be easily obtained from a database (miRBase :: Sequences, Sanger Institute) already described.
本願明細書の実施例で示されるように、尿検体中の上記miRNAの発現量が正常細胞と比較して尿路上皮癌細胞において有意に亢進していることが判明した。従って、より具体的には、上記miRNAの少なくとも一つのmiRNAの尿検体中の発現量の亢進（増加）を測定する方法を用いることによって、尿路上皮癌を検出することが可能である。ここで、miRNAの発現量が有意に亢進しているとは、例えば、本願明細書の実施例２におけるROC曲線分析におけるカットオフ値に基づき判定した場合に、尿路上皮癌の細胞におけるmiRNAの発現量が正常組織由来の細胞に比べて、約10倍以上、好ましくは約20倍以上、更に好ましくは約30倍以上であり、これを（判定）基準として尿路上皮癌であるか否かを検出することができる。 As shown in the Examples of the present specification, it was found that the expression level of the miRNA in the urine specimen was significantly increased in urothelial cancer cells as compared with normal cells. Therefore, more specifically, urothelial cancer can be detected by using a method for measuring the increase (increase) in the expression level of at least one miRNA in the urine sample. Here, when the expression level of miRNA is significantly enhanced, for example, when it is determined based on the cut-off value in the ROC curve analysis in Example 2 of the present specification, miRNA expression in cells of urothelial carcinoma The expression level is about 10 times or more, preferably about 20 times or more, more preferably about 30 times or more, compared to normal tissue-derived cells. Can be detected.
従って、上記miRNAの少なくとも一つの発現量の亢進に基づき、尿路上皮癌の治療又は予防に有効な物質をスクリーニングすることができる。より具体的には、以下の工程：
(a) 被検物質の存在下に細胞を培養する工程、
(b)該細胞における各miRNAの発現量を測定する工程、及び
(c)発現量の亢進を抑制するか又は発現量を減少させる物質を選択する工程、を含む方法でスクリーニングすることができる。
Therefore, a substance effective for the treatment or prevention of urothelial cancer can be screened based on the increased expression level of at least one miRNA. More specifically, the following steps:
(a) culturing cells in the presence of a test substance,
(b) measuring the expression level of each miRNA in the cell, and
(c) screening by a method comprising a step of selecting a substance that suppresses the increase in the expression level or decreases the expression level.
このようなスクリーニング方法において、例えば、尿路上皮癌由来の細胞中を使用した場合には、尿路上皮癌の治療に有効な物質をスクリーニングすることが可能である。一方、癌化が促進されるような状況下（例えば、公知の発癌物質の共存下、放射線の照射等）におかれた正常組織由来の細胞を使用した場合には、被検物質は尿路上皮癌の予防に有効な物質をスクリーニングすることが可能である。 In such a screening method, for example, when a cell derived from urothelial cancer is used, it is possible to screen a substance effective for the treatment of urothelial cancer. On the other hand, when cells derived from normal tissue placed under conditions that promote canceration (for example, coexistence of known carcinogens, irradiation of radiation, etc.), the test substance is on the urinary tract. It is possible to screen for substances effective for the prevention of skin cancer.
本発明の検出方法及びスクリーニング方法において、miRNAの発現量は当業者に公知の任煮の方法で測定することが出来る。例えば、上記の各miRNAに特異的にハイブリダイズするプライマー又はプローブを用いてmiRNAの発現量を測定することが出来る。このようなプライマー又はプローブは、当業者であれば、上記のデータベースの情報等を参考にして、これらのmiRNAの塩基配列に基づき適宜設計することが可能である。尚、尿検体からのmiRNAの抽出及び調製も当業者に公知の任意の方法で行うことが出来る。 In the detection method and screening method of the present invention, the expression level of miRNA can be measured by a method known to those skilled in the art. For example, the expression level of miRNA can be measured using a primer or probe that specifically hybridizes to each miRNA. Such a primer or probe can be appropriately designed by those skilled in the art based on the base sequences of these miRNAs with reference to the information in the above database. Extraction and preparation of miRNA from urine specimens can also be performed by any method known to those skilled in the art.
このようなプライマー又はプローブを用いる測定方法としては、Northern blot法が古典的方法である。最近では、miRNAを搭載したマイクロアレイ（Liu CG et al, (2004)An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues, PNAS, U S A, 101, 9740-9744; Lim LP et al, (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs, Nature, 433, 769-773）、改良型インベーダー法（Allawi HT et al, (2004) Quantitation of microRNAs using a modified Invader assay, Rna., 10, 153-1161）、ビーズを基にしたフローサイトメータ法（Lu J et al, (2005) MicroRNA expression profiles classify human cancers, Nature, 435, 834-838）などが報告されている。また、最も定量性のある方法としてreal-time PCRがある（Chen C et al, (2005) Real-time quantification of microRNAs by stem-loop RT-PCR, Nucleic Acids Res, 33, e179）。 As a measuring method using such a primer or probe, the Northern blot method is a classic method. Recently, microarrays with miRNA (Liu CG et al, (2004) An oligonucleotide microchip for genome-wide microRNA profiling in human and mouse tissues, PNAS, USA, 101, 9740-9744; Lim LP et al, (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs, Nature, 433, 769-773), modified invader method (Allawi HT et al, (2004) Quantitation of microRNAs using a modified Invader assay, Rna., 10, 153 -1161), a flow cytometer method based on beads (Lu J et al, (2005) MicroRNA expression profiles classify human cancers, Nature, 435, 834-838) and the like have been reported. The most quantitative method is real-time PCR (Chen C et al, (2005) Real-time quantification of microRNAs by stem-loop RT-PCR, Nucleic Acids Res, 33, e179).
上記のプライマー又はプローブの塩基配列は、鋳型との特異的な結合が可能となるような適当な塩基数、例えば、数十ｂｐ、１０〜３０ｂｐ程度を有することが好ましく、更には、プライマー内でヘアピン構造をとったり、センス鎖とアンチセンス鎖とが互いにアニーリングしないような塩基配列とすることも重要である。例えば、OligoTM(National Bioscience Inc.製)のような市販のプライマー設計用のソフトウェアを使用することも可能である。 The base sequence of the above-mentioned primer or probe preferably has an appropriate number of bases capable of specific binding to the template, for example, several tens of bp, about 10 to 30 bp. It is also important to have a hairpin structure or a base sequence that does not anneal the sense strand and the antisense strand to each other. For example, commercially available primer design software such as Oligo ™ (National Bioscience Inc.) can be used.
本発明の検出方法又はスクリーニング方法に使用されるキットは、測定対象又は測定原理等に応じて、適当な構成をとることが出来る。該キットは、その構成要素として、例えば、上記のｍＲＮＡ（ｃＤＮＡ）の増幅用プライマー及びＤＮＡチップ等で使用するハイブリダイゼーション用のプローブを含むことが出来る。更に、上記キットには、その構成・使用目的などに応じて、当業者に公知の他の要素又は成分、例えば、各種試薬、酵素、緩衝液、反応プレート（容器）等が含まれる。尚、ＰＣＲ反応後の検出を容易にするために、これらプライマーの少なくともいずれかの末端に、当業者に公知の任意の蛍光物質等の標識物質が結合していることが好ましい。例えば、適当な蛍光物質として、６−カルボキシフルオレッセイン（ＦＡＭ）、４，７，２’，４’，５’，７’−ヘキサクロロー６−カルボキシフルオレッセイン（ＨＥＸ）、ＮＥＤ（アプライドシステムズジャパン社）及び６−カルボキシ−Ｘ−ローダミン（Ｒｏｘ）等を挙げることが出来る。 The kit used in the detection method or screening method of the present invention can have an appropriate configuration according to the measurement object or measurement principle. The kit can contain, for example, the above-described primers for amplification of mRNA (cDNA) and a probe for hybridization used in a DNA chip or the like. Further, the kit includes other elements or components known to those skilled in the art, such as various reagents, enzymes, buffers, reaction plates (containers), and the like, depending on the configuration and purpose of use. In order to facilitate detection after the PCR reaction, it is preferable that a labeling substance such as an arbitrary fluorescent substance known to those skilled in the art is bound to at least one end of these primers. For example, suitable fluorescent substances include 6-carboxyfluorescein (FAM), 4,7,2 ′, 4 ′, 5 ′, 7′-hexachloro-6-carboxyfluorescein (HEX), NED (Applied Systems Japan) And 6-carboxy-X-rhodamine (Rox).
更に、以下の実施例に示すように、上記検出方法と尿細胞検査とを組み合わせる（併用する）ことによって、高い検出率で非侵襲的な尿路上皮癌の判定が可能となる。尿細胞検査は、例えば、「細胞診標本作成マニュアル（泌尿器）」（細胞検査士会（編）2004年発行等に記載されている、当業者に公知の方法・手段で行うことが出来る。例えば、約50ｃｃ以上の採尿をして、遠心分離にて尿沈渣をとりパパニコロウ染色をして顕微鏡で観察し、核の異型度から癌の判定をする。通常５段階法でclass I, IIを陰性、class IIIを擬陽性、class IV, Vを陽性と判定する。従って、本発明は、尿検体中のmiR-96の発現量の変動を測定することから成る尿路上皮癌の検出方法と尿細胞検査とを組み合わせることを特徴とする、尿路上皮癌の検出方法にも係るものである。 Furthermore, as shown in the following examples, by combining (using in combination) the above detection method and urine cell examination, it becomes possible to determine noninvasive urothelial cancer with a high detection rate. The urine cell test can be performed by a method / means known to those skilled in the art described in, for example, “Cytodiagnosis Specimen Preparation Manual (Urology)” (issued in 2004 by Cytologists Association (ed.)). Collect urine of about 50 cc or more, collect urinary sediment by centrifugation, stain with Papanicolaou and observe with a microscope, and judge cancer from the degree of nuclear atypia. Class III is determined as false positive, and class IV and V are determined as positive.Therefore, the present invention relates to a method for detecting urothelial cancer and measuring urinary cells, comprising measuring changes in the expression level of miR-96 in a urine sample. The present invention also relates to a method for detecting urothelial cancer, characterized by combining with a test.
以下、本発明を実施例によって詳細に説明するが、本発明の技術的範囲は以下の実施例の記載によって何ら限定して解釈されるものではない。当業者であれば、本明細書の記載に基づき、本発明の技術的範囲を逸脱せずに、多くの変形及び修飾を実施することが可能である。又、特に記載のない場合には、以下の実施例は、例えば、Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, F. M. et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y, 1995等に記載されている、当業者に公知の標準的な遺伝子工学及び分子生物学的技術に従い実施することが出来る。尚、本明細書中に引用された文献の記載内容は本明細書の開示、及び、内容の一部を構成するものである。 EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, the technical scope of this invention is limited and is not interpreted at all by description of a following example. A person skilled in the art can make many variations and modifications based on the description of the present specification without departing from the technical scope of the present invention. Also, unless otherwise specified, the following examples are described in, for example, Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols. It can be carried out according to standard genetic engineering and molecular biology techniques known to those skilled in the art, as described in Molecular Biology, John Wiley & Sons, New York, NY, 1995, etc. In addition, the description content of the literature referred in this specification comprises the indication of this specification, and a part of content.
（方法）
miRNA特異的逆転写反応および定量PCR
膀胱癌患者臨床検体19検体（癌部14検体、非癌部5検体）及び膀胱癌由来細胞株３種（BOY、KK47、T24）よりtotal RNAをtrizol（Gibco BRL, Life Technologies, Gaitherburg, MD, USA）用いて抽出した。cDNAは得られたtotal RNAからmiRNA特異的なプライマー（TaqMan microRNA Assay Protocol, PE, Applied Biosystems, Foster City, CA, UAS）を用いて合成した。逆転写反応は以下のとおり行った。10nM RNA samples, 50nM stem-loop RT primer, 1 x RT buffer, 0.25mM each of dNTPs, 3.33 U/μl MultiScribe reverse transcriptase 及び 0.25 U/μl RNase Inhubitor。また、これら7.5μlの反応液を以下のプロトコールでインキュベートした。16℃・30分、42℃・30分、85℃・5分、反応後4℃で保存した。定量PCRは、ABI 7300 Seqence Detection Systemを用いて以下の反応系で行った。95℃・10分インキュベートした後、95℃・15秒、60度・10分の反応を40回行った。
(Method)
miRNA-specific reverse transcription and quantitative PCR
Trizol (Gibco BRL, Life Technologies, Gaitherburg, MD) total RNA from 19 clinical specimens of bladder cancer patients (14 specimens in cancer, 5 specimens in non-cancerous part) and 3 types of cell lines derived from bladder cancer (BOY, KK47, T24) USA) and extracted. cDNA was synthesized from the obtained total RNA using miRNA-specific primers (TaqMan microRNA Assay Protocol, PE, Applied Biosystems, Foster City, CA, UAS). The reverse transcription reaction was performed as follows. 10 nM RNA samples, 50 nM stem-loop RT primer, 1 x RT buffer, 0.25 mM each of dNTPs, 3.33 U / μl MultiScribe reverse transcriptase and 0.25 U / μl RNase Inhubitor. Further, these 7.5 μl reaction solutions were incubated according to the following protocol. It was stored at 4 ° C after the reaction at 16 ° C for 30 minutes, 42 ° C for 30 minutes, 85 ° C for 5 minutes. Quantitative PCR was performed in the following reaction system using ABI 7300 Sequence Detection System. After incubation at 95 ° C for 10 minutes, the reaction was carried out 40 times at 95 ° C for 15 seconds and at 60 ° C for 10 minutes.
miRNA１156種類の定量化と膀胱癌で発現変動するmiRNAの選び出し
膀胱癌患者臨床検体19例（癌部14検体および非癌部5検体）及び膀胱癌由来細胞株３種について、既に記載したデータベース（miRBase::Sequences, Sanger Institute）から選んだ156種類のmiRNA及びコントロール遺伝子の解析を行った。解析結果について、マイクロアレイで用いられているglobal median normalization法を用いてデータの標準化を行った。この解析データから、癌部および非癌部で優位に（P<0.01）発現差のある遺伝子を選択した。その結果、27種類のmiRNAが癌部で有意に発現変動していることが判明した（８種類のmiRNAは癌部で発現亢進、１９種類のmiRNAは癌部で発現低下）。以下の表１及び表２にそのリストを示す。
Quantification of 1156 miRNAs and selection of miRNAs that change in expression in bladder cancer <br/> 19 clinical specimens of bladder cancer patients (14 specimens for cancer and 5 specimens for non-cancerous part) and 3 types of cell lines derived from bladder cancer have already been described 156 miRNAs and control genes selected from the database (miRBase :: Sequences, Sanger Institute) were analyzed. The analysis results were normalized using the global median normalization method used in microarrays. From this analysis data, genes having a difference in expression predominantly (P <0.01) in cancerous and non-cancerous parts were selected. As a result, it was found that the expression of 27 types of miRNAs was significantly changed in the cancerous part (eight kinds of miRNAs increased in expression in the cancerous part and 19 kinds of miRNAs decreased in expression in the cancerous part). The list is shown in Table 1 and Table 2 below.
更に、膀胱癌で有意（P<0.01）に発現変動している27種類のmiRNAを用いてクラスター解析した結果、正常移行上皮５検体および癌細胞株３種類は同一のクラスターに帰属した。また、癌部14検体は、11検体と3検体の２つのクラスターに分かれたが、正常部のクラスターとは異なっていることが判明した。 Furthermore, as a result of cluster analysis using 27 miRNAs whose expression was significantly changed in bladder cancer (P <0.01), 5 normal transitional epithelia and 3 cancer cell lines belonged to the same cluster. The 14 cancer samples were divided into two clusters, 11 samples and 3 samples, but they were found to be different from the normal cluster.
癌部で発現は亢進しているmiRNAは、癌遺伝子としての機能を有していると考えられる。すなわち、miRNAが過剰に発現することにより、ターゲットとなる遺伝子をより制御してloss of functionを引き起こしている可能性が高い。上記の解析では8種類のmiRNAが癌部で発現上昇が認められた。この中で、miR-96およびmiR-183は大腸癌においても発現が亢進することが報告されている（Bandres E et al, (2006) Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer and non-tumoral tissues, Molecular Cancer, 5, 29.）。この2つのmiRNAは染色体7q32.2にクラスターを形成していることが知られている。 MiRNA whose expression is enhanced in the cancerous part is considered to have a function as an oncogene. That is, it is highly possible that miRNA is excessively expressed, thereby causing a loss of function by further controlling the target gene. In the above analysis, the expression of 8 types of miRNAs was increased in the cancerous part. Among them, miR-96 and miR-183 have been reported to be upregulated in colorectal cancer (Bandres E et al, (2006) Identification by Real-time PCR of 13 mature microRNAs differentially expressed in colorectal cancer. and non-tumoral tissues, Molecular Cancer, 5, 29.). These two miRNAs are known to form a cluster on chromosome 7q32.2.
尿検体におけるmiRNAの検出（１）
上記リストの中で膀胱癌で発現が上昇している上位３つのmiRNA（miR-96, miR-190, miR-183）について 尿中での検出が可能かどうかを検討した。尿路上皮癌（ＵＣ）患者３０名、尿路感染症（ＵＴＩ）患者１５名および健常人（ＨＣ）１６名から得られた尿検体からmiRNA分画をmirVana（商標）PARISTM（商標）キット（Ambion, Tokyo, Japan）を用いて抽出して、リアルタイムＰＣＲ法により、各miRNAの検出を行った。
Detection of miRNA in urine samples (1)
We examined whether the top three miRNAs (miR-96, miR-190, miR-183) whose expression is elevated in bladder cancer in the above list can be detected in urine. MiRNA fractions were collected from urine specimens obtained from 30 urothelial cancer (UC) patients, 15 urinary tract infection (UTI) patients, and 16 healthy persons (HC) mirVana ™ PARISTM ™ kit ( Ambion, Tokyo, Japan) and each miRNA was detected by real-time PCR.
その結果、miR-96とmiR-183の発現量はＵＣ検体においてはＨＣ検体と比べて有意に高かった（P=0.0027とP=0.0002）（図１、上）。またmiR-96とmiR-183の発現量は筋層浸潤癌（≧pT2）においては筋層非浸潤癌（≦pT1）と比べて有意に高かった（P=0.0045とP=0.0177）（図１、下）。ここで、「pT1」及び「pT2」は、夫々、腫瘍の浸潤が膀胱筋層に到達していない（筋層非浸潤）と膀胱筋層に到達している（筋層浸潤）を意味する。 As a result, the expression levels of miR-96 and miR-183 were significantly higher in UC samples than in HC samples (P = 0.0027 and P = 0.0002) (FIG. 1, top). The expression levels of miR-96 and miR-183 were significantly higher in muscle invasive cancer (≧ pT2) than in non-muscle invasive cancer (≦ pT1) (P = 0.0045 and P = 0.0177) (FIG. 1). ,under). Here, “pT1” and “pT2” mean that tumor invasion does not reach the bladder muscle layer (muscle layer non-invasion) and bladder muscle layer (muscle layer infiltration), respectively.
ROC曲線分析においてmiR-96（感度63.3％；特異度93.6％）、miR-183（感度63.3％；特異度83.9％）の検出は良好に尿路上皮腫瘍を非癌病変と区別可能であり、有用な検出（診断）マーカーとなる可能性が示唆された（図２）。さらに尿細胞検査で陰性と判定された10症例中で５症例でmiRNA検出にて癌陽性と判定された。尿細胞検査の感度は30％程度とされており、miRNAによる尿路上皮癌の検出は尿細胞検査を凌駕するものと考えられた。 In the ROC curve analysis, detection of miR-96 (sensitivity 63.3%; specificity 93.6%) and miR-183 (sensitivity 63.3%; specificity 83.9%) can distinguish urothelial tumors from non-cancerous lesions, The possibility of being a useful detection (diagnostic) marker was suggested (FIG. 2). Furthermore, in 10 cases judged negative by urine cell test, 5 cases were judged as cancer positive by miRNA detection. The sensitivity of the urine cell test is considered to be about 30%, and detection of urothelial cancer by miRNA was considered to surpass the urine cell test.
尿検体におけるmiRNAの検出（２）
更に別の集団を用いて、miRNA（miR-96， miR-183）について尿中での検出が可能かどうかを検肘した。尿路上皮癌（ＵＣ）患者８５名、尿路感染症（ＵＴＩ）患者１５名および健常人（ＨＣ）４９名から得られた尿検体からmiRNA分画をmirVana（商標）PARISTM（商標）キット（AInbion，Tokyo，Japan）を用いて抽出して、リアルタイムＰＣＲ法により、各miRNAの検出を行った。
Detection of miRNA in urine samples (2)
Furthermore, another group was used to examine whether miRNA (miR-96, miR-183) could be detected in urine. MiRNA fractions were collected from urine specimens obtained from 85 urothelial cancer (UC) patients, 15 urinary tract infection (UTI) patients, and 49 healthy persons (HC) mirVana ™ PARISTM ™ kit ( AInbion, Tokyo, Japan), and each miRNA was detected by real-time PCR.
その結果、miR-96とmiR-183の発現量はＵＣ検体においてはＨＣ検体と比べて有意に高かった（P<0.0001）（図３）。又、尿路上皮癌の悪性度（G1, G2, G3）が上昇するにつれてmiR-96とmiR-183の発現量は上昇していた（図４，上）。またmiR-96とmiR-183の発現量は浸潤癌（≧pT1）においては非浸潤癌（≦pTa）と比べて有意に高かった（P=0.0110とP=0.0072）（図４，左下）。ここで、「pTa」及び「pT1」は、夫々、腫瘍の浸潤が尿路上皮粘膜下層に到達していない（非浸潤）と尿路上皮粘膜下層に到達している（浸潤）を意味する。さらに根治的手術がなされた１５例においてはこれらのmiRNAの発現は有意に減少していた（P=0.0310とP=0.0037）（図４，右下）。 As a result, the expression levels of miR-96 and miR-183 were significantly higher in UC samples than in HC samples (P <0.0001) (FIG. 3). In addition, the expression levels of miR-96 and miR-183 increased as the malignancy of urothelial cancer (G1, G2, G3) increased (Fig. 4, upper). The expression levels of miR-96 and miR-183 were significantly higher in invasive cancer (≧ pT1) than in noninvasive cancer (≦ pTa) (P = 0.0110 and P = 0.0072) (FIG. 4, lower left). Here, “pTa” and “pT1” mean that tumor invasion does not reach the urothelial submucosa (non-infiltration) and reaches the urothelial submucosa (infiltration), respectively. Furthermore, in 15 cases undergoing radical surgery, the expression of these miRNAs was significantly reduced (P = 0.0310 and P = 0.0037) (FIG. 4, lower right).
ROC曲線分析においてmiR-96（感度64.7％；特異度92.2％）、miR-183（感度77.7％；特異度75.0％）の検出は良好に尿路上皮腫瘍を非癌病変と区別可能であり、有用な検出マーカーとなる可能性が示唆された（図５）。 In the ROC curve analysis, detection of miR-96 (sensitivity 64.7%; specificity 92.2%), miR-183 (sensitivity 77.7%; specificity 75.0%) can distinguish urothelial tumors from non-cancerous lesions, The possibility of becoming a useful detection marker was suggested (FIG. 5).
更に、尿細胞検査が施行されていた患者は６５例の中で陰性と判定された36症例中の20症例でmiRNA検査にて癌陽性と判定された。尿細胞検査による癌の検出率（感度）は45％と低く、miR-96の検出率はやや高く65％であったが、この両者を併用することにより、以下に示すように、癌の検出率は75％にまで増加した（図６）。
（１）尿細胞検査（陽性）、miR-96（陽性）(全65例中22例：33.8%)
（２）尿細胞検査（陰性）、miR-96（陽性）(尿細胞検査陰性36例中20例：55.6%)
（３）尿細胞検査（陽性）、miR-96（陰性）(尿細胞検査陽性29例中7例：24.1%)
即ち、尿細胞検査（陽性）のみだと29例しか見つけられないが、miR-96と尿細胞検査の併用により、全65例のうち49例までが少なくともいずれかの検査で陽性として検出可能になり（精度が上がった）、高い検出率で非侵襲的な尿路上皮癌の判定が可能となった。
Furthermore, of the 65 cases judged to be negative among 65 patients who had been subjected to urine cell examination, miRNA test determined that the cancer was positive. The detection rate (sensitivity) of cancer by urine cytology was as low as 45%, and the detection rate of miR-96 was slightly high at 65%. By using both of these, the detection of cancer is as shown below. The rate increased to 75% (Figure 6).
(1) Urine cell test (positive), miR-96 (positive) (22 of 65 cases: 33.8%)
(2) Urine cell test (negative), miR-96 (positive) (20 out of 36 urine cell test negative: 55.6%)
(3) Urine cell test (positive), miR-96 (negative) (7 out of 29 urine cell test positive: 24.1%)
In other words, only 29 urine cell tests (positive) can be found, but the combined use of miR-96 and urine cell test can detect up to 49 of 65 cases as positive in at least one of the tests. (Accuracy has improved), and non-invasive determination of urothelial cancer has become possible with a high detection rate.
本発明で使用するmiRNAは尿路上皮癌の検出のみならず治療の標的として大変有用である。更に、本発明を利用した血清および尿中のマイクロＲＮＡ検出が分析機器・キットの開発によって、1億5千万円といわれる本邦の尿路上皮癌診断薬の市場にブレイクスルーをもたらすことが可能となる。 The miRNA used in the present invention is very useful not only for detection of urothelial cancer but also as a therapeutic target. Furthermore, the detection of microRNA in serum and urine using the present invention can bring a breakthrough to the Japanese urothelial cancer diagnostic drug market, which is said to be 150 million yen, through the development of analytical instruments and kits. It becomes.
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