JP2004248508A - Hurp gene as molecular marker for bladder cancer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting bladder cancer using the expression of an HURP (hepatoma up-regulated protein) gene as a marker. <P>SOLUTION: The HURP gene serves as a molecular marker in detection, preliminary screening and monitoring of the bladder cancer in a human subject. The method comprises detecting the bladder cancer, especially TCC (transitional cell carcinoma) of the bladder in the human subject. The expression of the human HURP gene detected in a sample collected from the subject suspected to have the bladder cancer is indicative of the presence of the bladder cancer. The detection of the expression of the human HURP gene is carried out by detecting an mRNA transcript of the human HURP gene in a biological sample using an RT-PCR (reverse transcriptase-polymerase chain reaction) method, etc. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

【００４９】
第２プライマー対：
【００５０】
【化２】

【００５１】
フォーワードプライマー１はリバースプライマー２と一対になることができ、フォーワードプライマー２はリバースプライマー１と一対になることができると考えられる。
【００５２】
本発明によれば、ヒトＨＵＲＰ遺伝子の発現は、ヒトＨＵＲＰ遺伝子からのｍＲＮＡ転写物又はヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物から翻訳されたタンパク質の分析によって測定される。
【００５３】
ヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物からの翻訳されたタンパク質の検出は、バイオテクノロジーの分野でよく知られた慣用の方法論により行うことができる。
【００５４】
本発明の好ましい実施態様において、ヒトＨＵＲＰ遺伝子の発現の検出は、生物学的サンプル中のヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物の存在を検出することによって行われる。
【００５５】
好ましくは、ヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物の存在の検出は、以下の方法論：ハイブリダイゼーション、サイクリングプローブ反応（ｃｙｃｌｉｎｇ ｐｒｏｂｅ ｒｅａｃｔｉｏｎ）、ポリメラーゼ連鎖反応（ＰＣＲ）、ネスティッド（ｎｅｓｔｅｄ）ＰＣＲ、逆転写酵素ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）、マルチプレックスＰＣＲポリメラーゼ連鎖反応−一本鎖コンフォーメーション多型、リガーゼ連鎖反応（ＬＣＲ）、制限断片長多型核酸配列ベースの増幅（ＮＡＳＢＡ）、及び転写仲介増幅（ｔｒａｎｓｃｒｉｐｔｉｏｎ−ｍｅｄｉａｔｅｄ ａｍｐｌｉｆｉｃａｔｉｏｎ）（ＴＭＡ）
の少なくとも１つ用いて行われる。
【００５６】
本発明の好ましい実施態様において、ヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物の存在の検出は、ＲＴ−ＰＣＲを用いて行われる。
【００５７】
本発明のより好ましい実施態様において、ヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物の存在の検出は、以下：
（ａ）生物学的サンプルから全ＲＮＡを抽出すること；
（ｂ）工程（ａ）で抽出されたＲＮＡを、フォーワードプライマーとリバースプライマーの少なくとも１つのプライマー対を用いて、逆転写酵素ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）処理に供すること、各プライマーは、配列番号１に対応するヌクレオチド配列を有するヒトＨＵＲＰ遺伝子の少なくとも１３ヌクレオチドにハイブリダイズするヌクレオチド配列を有し；及び
（ｃ）ヒトＨＵＲＰ遺伝子のヌクレオチド配列の一部と同一又は相補的なヌクレオチド配列を有するＲＴ−ＰＣＲ産物が、工程（ｂ）のＲＴ−ＰＣＲ処理から産生されたかどうかを検出すること、前記ＲＴ−ＰＣＲ産物の存在が膀胱癌の存在を示す、
により行われる。
【００５８】
好ましくは、工程（ｂ）で使用される各プライマーが、ヒトＨＵＲＰ遺伝子の少なくとも１８ヌクレオチドにハイブリダイズする。
【００５９】
好ましくは、工程（ｂ）で使用される少なくとも１つのプライマー対は、配列番号２及び配列番号４で示される配列から選ばれるヌクレオチド配列を有するフォーワードプライマーと、配列番号３及び配列番号５で示される配列から選ばれるヌクレオチド配列を有するリバースプライマーを含む。
【００６０】
本発明の好ましい実施態様において、工程（ｂ）で使用される少なくとも１つのプライマー対は、配列番号４に対応するヌクレオチド配列を有するフォーワードプライマーと配列番号５に対応するヌクレオチド配列を有するリバースプライマーを含む。
【００６１】
本発明の好ましい実施態様において、工程（ｂ）におけるＲＴ−ＰＣＲ処理は、配列番号２に対応するヌクレオチド配列を有する第１フォーワードプライマーと配列番号３に対応するヌクレオチド配列を有する第１リバースプライマーとを含む第１プライマー対を用いる第１増幅反応、及び配列番号４に対応するヌクレオチド配列を有する第２フォーワードプライマーと配列番号５に対応するヌクレオチド配列を有する第２リバースプライマーとを含む第２プライマー対を用いる第２増幅反応を含む。
【００６２】
本発明によれば、ヒト被験者における膀胱癌の検出のためのプライマーセットが提供され、それは配列番号２及び配列番号４で示される配列から選ばれるヌクレオチド配列を有するフォーワードプライマーと配列番号３及び配列番号５で示される配列から選ばれるヌクレオチド配列を有するリバースプライマーとを含む。
【００６３】
本発明の好ましい実施態様において、フォーワードプライマーは配列番号２で示されるヌクレオチド配列を有する。
【００６４】
本発明の他の好ましい実施態様において、フォーワードプライマーは配列番号４で示されるヌクレオチド配列を有する。
【００６５】
本発明の好ましい実施態様において、リバースプライマーは配列番号３で示されるヌクレオチド配列を有する。
【００６６】
本発明の他の好ましい実施態様において、リバースプライマーは配列番号５で示されるヌクレオチド配列を有する。
【００６７】
本発明によれば、ヒト被験者における膀胱癌の検出のための診断キットが提供され、それはフォーワードプライマーとリバースプライマーの少なくとも１つのプライマー対を含み、各プライマーは、配列番号１に対応するヌクレオチド配列を有するヒトＨＵＲＰ遺伝子の少なくとも１３ヌクレオチドにハイブリダイズするヌクレオチド配列を有する。
【００６８】
本発明の他の好ましい実施態様において、診断キットは、配列番号２に対応するヌクレオチド配列を有する第１フォーワードプライマーと配列番号３に対応するヌクレオチド配列を有する第１リバースプライマーとを含む第１プライマー対、及び配列番号４に対応するヌクレオチド配列を有する第２フォーワードプライマーと配列番号５に対応するヌクレオチド配列を有する第２リバースプライマーを含む第２プライマー対を含む。
【００６９】
前述したことに加え、本発明の範囲内における各種の用途があらゆるタイプの評価アッセイを使用することを含むことは、当業者なら理解するだろう。
【００７０】
例えば、ＲＮＡ及びタンパク質は、その分野において公知の技術を用いてテストサンプルから単離及びアッセイされ得る。それらは、例えば、新鮮な又は凍結させた生検、ホルマリンで固定された組織、そして血液、血漿、血清、尿又は痰のような体液から単離され得る。
【００７１】
ＲＴ−ＰＣＲが使用できるが、他の技術も考えられる。これらは、ＰＣＲ、ネスティッドＰＣＲ インサイチュ（ｉｎ ｓｉｔｕ）ハイブリダイゼーション、ノーザンブロッティング、高密度発現アレイ、マイクロアレイ、並びにＥＬＩＳＡ、ウェスタンブロッティング及び酵素アッセイのような特定のタンパク質産物をアッセイするための技術を含む特定のｍＲＮＡ種をアッセイするための他の技術を含む。
【００７２】
本発明は以下の実施例を参照してより詳細に述べられ、それらは単に例証の目的であって、本発明の範囲を制限しようとするものではない。
【００７３】
実施例１：ＨＵＲＰ遺伝子発現のＰＴ−ＰＣＲ分析
材料及び方法：
患者の特徴
１９９８年３月から２００１年９月までＣｈｉ Ｍｅｉメディカルセンターにおいて、膀胱切開術（ｃｙｓｔｅｃｔｏｍｙ）及び経尿道的切除術（ｔｒａｎｓｕｒｅｔｈｒａｌ ｒｅｓｅｃｔｉｏｎ）を用いて尿路から８０のＴＣＣサンプルを得た。遠くの肉眼的に正常な組織もまた分析のために得られ、それらは腫瘍に近接する組織サンプルとしてみなされた。全組織サンプルは液体窒素中で凍結され、−８６℃で様々な期間貯蔵された。各凍結ブロックの代表的な切片は、パラフィン中に埋め込まれ、ヘマトキシリン−エオシンで染色され、サンプル中の腫瘍の状態を評価するために病理学者によって調べられた。
【００７４】
全ての標本は、世界保健機関の分類の修正を用いてグレードに分けられ、病理学的なステージング（ｓｔａｇｉｎｇ）は、ＴＮＭ病理学ステージングシステムに従った（Ｅｐｓｔｅｉｎ ＪＩ， Ａｍｉｎ ＭＢ， Ｒｅｕｔｅｒ ＶＲ， ｅｔ ａｌ： Ｔｈｅ Ｗｏｒｌｄ Ｈｅａｌｔｈ Ｏｒｇａｎｉｚａｔｉｏｎ／Ｉｎｔｅｒｎａｔｉｏｎａｌ Ｓｏｃｉｅｔｙ ｏｆ Ｕｒｏｌｏｇｉｃａｌ Ｐａｔｈｏｌｏｇｙ ｃｏｎｓｅｎｓｕｓ ｃｌａｓｓｉｆｉｃａｔｉｏｎ ｏｆ ｕｒｏｔｈｅｌｉａｌ （ｔｒａｎｓｉｔｉｏｎａｌ ｃｅｌｌ） ｎｅｏｐｌａｓｍｓ ｏｆ ｔｈｅ ｕｒｉｎａｒｙ ｂｌａｄｄｅｒ． Ｂｌａｄｄｅｒ Ｃｏｎｓｅｎｓｕｓ Ｃｏｎｆｅｒｅｎｃｅ Ｃｏｍｍｉｔｔｅｅ． Ａｍ Ｊ Ｓｕｒｇ Ｐａｔｈｏｌ ２２： １４３５−１４４８， １９９８；及びＣｈｉｓｈｏｌｍ ＧＤ， Ｈｉｎｄｍａｒｓｈ ＪＲ， Ｈｏｗａｔｓｏｎ ＡＧ， ｅｔ ａｌ： ＴＮＭ （１９７８） ｉｎ ｂｌａｄｄｅｒ ｃａｎｃｅｒ： ｕｓｅ ａｎｄ ａｂｕｓｅ． Ｂｒ Ｊ Ｕｒｏｌ ５２： ５００−５０５， １９８０）。
【００７５】
膀胱腫瘍の段階は、どれほど深く腫瘍が浸潤しているかを示す。表在性腫瘍はＴａと呼ばれ、Ｔ１−４は筋肉への浸潤の増加の程度を述べるのに使用される。膀胱腫瘍のグレードはＩ−ＩＶ（１−４）のスケールで表される。グレードは細胞の細胞学的外観を反映し、グレードＩの細胞はほとんど正常であり、グレードＩＩの細胞はわずかに正常から外れており、グレードＩＩＩの細胞は明らかに異常であり、グレードＩＶの細胞は高度に異常である。
【００７６】
ＲＮＡの単離：
全ＲＮＡは、Ｕｌｔｒａｓｐｅｃ（登録商標）ＲＮＡ単離システム（Ｂｉｏｔｅｃｘ Ｌａｂｏｒａｔｏｒｉｅｓ Ｉｎｃ．， Ｈｏｕｓｔｏｎ， Ｔｅｘａｓ）を用いて凍結した組織標本又は尿のペレットから単離された。ハンドヘルドのガラス−テフロン中で、約０．５−１．０グラムの組織を１ｍｌのＵｌｔｒａｓｐｅｃ（登録商標）試薬を用いてホモジナイズした。ホモジナイゼーションの後、ホモジネートを４℃で５分間保存し、０．２ｍｌのクロロホルムで抽出し、その後、１２，０００ｇ（４℃）で１５分間遠心分離した。水相のＲＮＡを同量のイソプロパノールで沈殿させ、７５％のエタノールで２回洗浄した。ＲＮＡの含有量を分光学的に評価し（Ａ２６０の１ ＯＤは４０μｇ／ｍｌのＲＮＡに等しい。）、抽出物の純度はＡ２６０／２８０比を用いて評価し、それは全ての場合において１．７５を超えた。尿の回収のために、５０ミリリットルのきれいに採取された尿は、全ての患者からその日の最初の排尿で得られた。サンプルは３，０００ｇ（４℃）で２０分間遠心分離され、細胞のペレットは１ｍｌのＵｌｔｒａｓｐｅｃ（登録商標）試薬と混合され、上述したのと同じ方法を用いて抽出された。
【００７７】
逆転写酵素−ポリメラーゼ連鎖反応
一本鎖の相補的ＤＮＡ（ｃＤＮＡ）は、１μｌのＳｕｐｅｒＳｃｒｉｐｔ ＩＩ逆転写酵素（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ Ｉｎｃ．， Ｇａｉｔｈｅｒｓｂｕｒｇ， ＭＤ）とともに１μｇの全ＲＮＡのｏｌｉｇｏ−ｄＴプライミングを用いて、４２℃で５０分間合成された。７０℃で１５分間加熱した後、第１増幅反応は、１０分の１の逆転写されたＲＮＡ、２００μｍｏｌ／ＬのｄＮＴＰｓを含むＴａｑポリメラーゼバッファー、１ＵのＤｙＮＡｚｙｍｅ（登録商標）ＩＩ ＤＮＡポリメラーゼ（Ｆｉｎｎｚｙｍｅｓ Ｉｎｃ．， Ｆｉｎｌａｎｄ）、及び１０μＭの各ヒトＨＵＲＰプライマー５’−ＧＧＡＴＣＣＡＡＴＡＧＡＣＡＣＴＴＴＧＧＴＴＴＧ−３’（フォーワード）及び５’−ＧＧＡＴＣＣＣＡＣＣＴＴＴＣＣＴＴＣＴＧＧＴＴＣ−３’（リバース）を用いて、９４℃で１分間変性、５５℃で１分間アニーリングおよび７２℃で１分間伸長を３０サイクルについて、その後、７２℃で５分間インキュベートすることにより行われた。
【００７８】
尿標本のネスティッドＰＣＲのために、第１増幅産物の１０分の１を、ネスティッドＨＵＲＰプライマー５’−ＣＡＡＣＧＡＡＡＡＣＡＧＡＴＧＣＴＣ−３’（フォーワード）及び５’−ＴＧＡＧＴＡＧＣＴＧＡＴＣＧＡＧＴＣ−３’（リバース）とともに、９４℃で１分間変性、６０℃で１分間アニーリング、および７２℃で１分間の伸長を３０サイクルについて、その後、７２℃で５分間インキュベートを行う増幅の第２ラウンドに供した。
【００７９】
テンプレートとしてＲＴを有さないルーチンのＲＴ−ＰＣＲのコントロールは陰性であり、テンプレートとしてｐＥＴ３２ａ（＋）ベクター（Ｎｏｖａｇｅｎ Ｉｎｃ．から購入した）に全長のＨＵＲＰヌクレオチド配列を挿入することにより図２に示すように構築されたｐＥＴ３２ａ（＋）−ＨＵＲＰを用いたものは陽性であった。増幅産物は、１．５％アガロースゲル中０．１μｇのＧｅｎｅＲｕｌｅｒ（登録商標）１００ｂｐ ＤＮＡラダー（ＭＢＩ Ｆｅｒｍｅｎｔａｓ， Ｌｉｔｈｕａｎｉａ）を用いる電気泳動を使用して分離され、エチジウムブロマイド染色を用いて可視化された。
【００８０】
データの定量化及び標準化
ＨＵＲＰ遺伝子のｍＲＮＡ発現レベルを定量化するために、全てのゲルがＩｍａｇｅ Ｍａｓｔｅｒ（登録商標）ＶＤＳビデオイメージングシステム（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ Ｉｎｃ．， Ｐｉｓｃａｔａｗａｙ）を用いて撮影され、ＬＩＳＣＡＰイメージキャプチャーソフトウェア（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ Ｉｎｃ．， バージョン１．０）を用いて書類に記録した。ＰＣＲ産物の光学的画素は、ＩｍａｇｅＭａｓｔｅｒ（登録商標）ＴｏｔａｌＬａｂソフトウェア（Ａｍｅｒｓｈａｍ Ｐｈａｒｍａｃｉａ Ｂｉｏｔｅｃｈ Ｉｎｃ．， バージョン１．１１）を用いて任意の単位に定量化された。
【００８１】
試験サンプルを比較するために、データの標準化は必要であった。この目的のために、β−アクチンのＰＣＲ増幅産物は、ＰＣＲにかけられたｃＤＮＡテンプレートと相対的に等しい量を表すために内部標準として使用された。平均値は平均±ＳＤ値として表され、Ｓｔｕｄｅｎｔ’ｓ ｔ検定を用いて比較された。
【００８２】
結果：
ＴＣＣ組織サンプル及びコントロール被験者におけるＨＵＲＰ遺伝子の代表的な発現パターンを図３Ａに示す。腫瘍に近接する部分よりも１１対の腫瘍部分（患者番号：２９４Ｎ／Ｔ， ３０６Ｎ／Ｔ， ３０７Ｎ／Ｔ， ３１５Ｎ／Ｔ， ３１７Ｎ／Ｔ， ３１９Ｎ／Ｔ， ３２０Ｎ／Ｔ， ３２１Ｎ／Ｔ， ３２７Ｎ／Ｔ， ３３５Ｎ／Ｔ， ３３７Ｎ／Ｔ）において、相対的により多くのヒトＨＵＲＰ遺伝子のｍＲＮＡ転写物が増幅された（図３Ａ参照。）。組織サンプルの１対（患者番号：３２２Ｎ／Ｔ）のみが、両方ともヒトＨＵＲＰ遺伝子の検出できない転写物を示した。内部標準として、β−アクチンのＰＣＲ増幅産物（図３Ｂ参照。）が、ＰＣＲに供されたｃＤＮＡテンプレートと相対的に等しい量を表すために使用された。
【００８３】
実施例２：ＲＴ−ＰＣＲ分析によるＨＵＲＰ遺伝子発現の腫瘍特異性の評価
ＨＵＲＰの過剰発現が腫瘍特異性を示すかどうかを決定するために、良性前立腺過形成（ＢＰＨ）の患者由来の前立腺尿路上皮のヒトＨＵＲＰ転写物が、逆転写酵素−ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）を用いて増幅された。
【００８４】
実験は、良性前立腺過形成（ＢＰＨ）の患者由来の前立腺尿路上皮のＨＵＲＰ転写物が、ＨＵＲＰ遺伝子発現が腫瘍特異性を示すかどうかを決定するために逆転写酵素−ポリメラーゼ連鎖反応（ＲＴ−ＰＣＲ）を用いて増幅された以外は、上の実施例１に示された手順に従って行われた。
【００８５】
標準の膀胱鏡検査法を用いたＢＰＨ患者由来の前立腺尿路上皮の１５の標本は、集められ、ＲＴ−ＰＣＲに供された。図４を参照すると、β−アクチン転写物はＢＰＨ尿路上皮の５つの代表的なサンプルから等しく増幅されたが、これらのサンプル中にはＨＵＲＰ転写物は検出されなかった。ＢＰＨサンプルに加えて、肝臓由来の４サンプル及び心臓由来の４サンプルを含む膀胱でないコントロールが試験され、これらの組織中にはＨＵＲＰ転写物は検出されなかった（データは示していない。）。
【００８６】
実施例３：ＨＵＲＰ遺伝子発現のＲＴ−ＰＣＲ分析による腫瘍段階の評価
ＨＵＲＰ遺伝子の過剰発現と腫瘍グレードとの間の可能性のある相関関係を決定するために、ＴＣＣを含む組織のサンプル及び対応の腫瘍近接組織サンプルの４５対のヒトＨＵＲＰ転写物が分析され、β−アクチン転写物を用いて標準化された。
【００８７】
表１を参照すると、３５の腫瘍近接部分（３５／４５， ７７．８％）が腫瘍の部分よりもＨＵＲＰ遺伝子の発現量が少ないこと（グループＩ， ｐ＜０．０００１）、及び１０の腫瘍近接部分（１０／４５， ２２．２％）が腫瘍部分とほとんど等しい量のＨＵＲＰ遺伝子を示したこと（グループＩＩ）は、注目すべきことである。
【００８８】
ゼロのグレードＩ、３７のグレードＩＩ、３１のグレードＩＩＩ、１２のグレードＩＶの病理学的同定を有する８０人のＴＣＣ患者が集められ、組織サンプルのヒトＨＵＲＰ発現比を表ＩＩにリストする。ＨＵＲＰ発現と腫瘍グレードとの間に有意な相関関係は存在しないようである。それらの中で、ＨＵＲＰ陰性の９の腫瘍、６はグレードＩＩ、１はグレードＩＩＩそして２はグレードＩＶであった。
【００８９】
【表１】

【００９０】
【表２】

【００９１】
実施例４：ＴＣＣの検出のためのバイオマーカーとしてのＨＵＲＰ遺伝子発現の評価尿ＴＣＣ検出のための新規な尿の分子マーカーとしての、尿ＨＵＲＰの潜在的な適用性を評価するために、新たな又は再発したＴＣＣの７人の更なる患者が、ＲＴ−ＰＣＲを用いて尿ＨＵＲＰについて分析された。全ＲＮＡは尿の細胞ペレットから抽出され、ｏｌｉｇｏ−ｄＴプライミングによって逆転写された。増幅反応は、ＨＵＲＰ特異的プライマー又はβ−アクチン特異的プライマーを用いて行われた。３７０塩基対のｃＤＮＡは、ＴＣＣの全患者の尿の細胞ペレットから増幅された（図５Ａ）。対照的に、７人の更なる個人（３人は尿路感染症、２人は膀胱の慢性炎症、１人は腎細胞癌、１人はＢＨＰを有した。）由来の尿の細胞ペレットは、ＨＵＲＰ転写物を有しなかった。コントロールの実験において、３０７塩基対のβ−アクチンｃＤＮＡ断片は、コントロールの被験者及びＴＣＣ患者の尿から見分けのつかないほど増幅された（図５Ｂ）。
【００９２】
本明細書中に引用された全ての特許及び参考文献は、参考として本明細書に全体として援用される。矛盾する場合は、定義を含めた本発明の説明が優先される。
【００９３】
本発明は、上記の特定の実施態様を参照して述べられたが、本発明の範囲及び精神から逸脱することなく数多くの変更と変形を行うことができるのは明らかである。従って、本発明は添付のクレームに示されたようにのみ限定しようとするものである。
【００９４】
【配列表】

【図面の簡単な説明】
【図１】図１はヒトＨＵＲＰ遺伝子のヌクレオチド配列の全長（配列番号１）を示し、ここで本発明に従ってデザインされた４つのプライマーに相補的なヌクレオチド配列は、下線が引かれ、太字になっている；
【図２】図２は上記の実施例で用いられているベクターｐＥＴ３２ａ（Ｎｏｖａｇｅｎ Ｉｎｃ．）の制限地図を示し、ＢａｍＨ Ｉ制限部位はヒトＨＵＲＰ遺伝子の全長核酸配列を挿入するために使用される。
【図３】図３Ａ及び３Ｂはそれぞれ、ＴＣＣ組織におけるＨＵＲＰ及びβ−アクチンの発現のＲＴ−ＰＣＲ分析を示し、Ｎ＝腫瘍近接組織、Ｔ＝腫瘍組織、および２９４，３０６，３０７，３１５，３１７，３１９，３２０，３２１，３２２，３２７，３３５及び３３７＝患者の番号である。
【図４】図４は５人の良性の前立腺過形成の患者から採取した前立腺の尿路上皮サンプルにおけるヒトＨＵＲＰ発現のＲＴ−ＰＣＲ分析を示し、ここで左はβ−アクチン（３０７ｂｐ）および右はＨＵＲＰ（３７０ｂｐ）であり、Ｎ１，Ｎ２，Ｎ３，Ｎ４及びＮ５＝５つの良性前立腺過形成（ＢＰＨ）サンプル；ＮＣ＝ネガティブコントロール；そして、Ｍは塩基対における分子量マーカーを示す。
【図５】図５Ａ及び５Ｂはそれぞれ、排泄された尿サンプル中のＨＵＲＰ及びβ−アクチンの発現のＲＴ−ＰＣＲ分析を示し、ここで２，６，７，８，１４，１５，２０，５，１０，１１，１２，１３，１６及び１８＝患者の番号；ＮＣ：ネガティブコントロール；Ｍは塩基対における分子量マーカーを示す。[0001]
Background of the Invention
1) Field of the invention
The present invention relates to the use of the human hepatoma up-regulated protein (HURP) gene as a molecular marker in the detection, preliminary screening or monitoring of bladder cancer in human subjects, wherein the bladder cancer is present. Detected expression of the human HURP gene in a sample taken from a suspected human subject indicates the presence of bladder cancer. Thus, the present invention provides an effective method for detecting bladder cancer in human subjects, particularly transitional cell carcinoma of the bladder (TCC). The present invention further provides a non-invasive method for the detection, preliminary screening or monitoring of urinary TCC in human subjects with high accuracy and convenience, wherein a urine sample collected from a suspected human subject is Analyzed to measure expression of the human HURP gene, the presence of which is indicative of the presence of urinary TCC.
[0002]
2) Description of related technology
Urothelial carcinoma is the second most common malignant disorder of the genitourinary tract and the second most common cause of death in all genitourinary tumors (Konety BR and Gottenberg R.). H (Getzenberg RH): Urine based markers of urological molecularity. J Urol 165: 600-611, 2001, Non-Patent Document 1). Transitional cell carcinoma of the bladder (TCC) is responsible for over 90% of urothelial neoplasms and exists as a low-grade occult papillary superficial lesion or a high-grade tumor that can be invasive and fatal I do. If bladder cancer is detected early during the localization phase, the 5-year survival rate is 94%. Once the disease has spread locally or distantly, the 5-year survival rate drops to 49% and 6%, respectively (Droller MJ: Individualizing the approach to invasive blader cancer. Conte. Moon, pp. 54-61, 1990, Non-Patent Document 2). It is therefore important to detect early events in the recurrence of superficial cancers before the cancer cells have time to change their behavior to invasive.
[0003]
Classical cytology and cystoscopy under planned follow-up protocols are the primary methods of surveillance for patients with urinary TCC. Low sensitivity to low-grade tumors using the cytology of excreted urine often requires invasive cystoscopy, and therefore the discomfort and potential infections associated with urethral instrumentation during cystoscopy Induce the risk of. The development of a sensitive, non-invasive diagnostic test that can specifically detect bladder cancer at an early stage will improve clinical outcomes by starting treatment earlier.
[0004]
U.S. Patent No. 6,376,188 entitled "Methods and probe set for detecting cancer";"Methods for detection of nucleic acid sequences in urine"; U.S. Pat. No. 6,287,820 entitled Methods 2) and "Methods for protection of nucleic acid sequences in urine" each disclose the use of a set of chromosomal probes to detect urine samples in bladder cancer. The diagnosis was disclosed.
[0005]
U.S. Patent No. 6,335,167, entitled "Comparative genomic hybridization (CGH)," discloses detecting the copy number of a nucleic acid sequence, particularly q21 of human chromosome 8 in a test sample, It is selected from the group consisting of q31-qter of chromosome 13, p15-pter of human chromosome 7, q24-qter of human chromosome 8, cen-p13 of human chromosome 11, and q13-qter of human chromosome 9. Disclosed is the diagnosis of bladder cancer by detecting amplification of a unique sequence in at least one position.
[0006]
US Patent No. 6,291,163, entitled "Method for detecting cell proliferative disorders", discloses the diagnosis of cancer (including bladder cancer) and precancerous cancer in a subject by detecting nucleic acid sequences. Characteristic consists in the following steps: (a) amplifying a test sample DNA of a locus in which the subject is heterozygous, wherein the locus includes the first and second alleles, wherein the locus is DNA, wherein the test sample DNA is derived from cells of an organ that has flowed into the test sample, wherein the test sample is selected from the group consisting of: urine, sputum, bile, stool, saliva, tears, serum and plasma of the subject; And (b) the level of microsatellite DNA present in the second allele Detecting allelic imbalance at the locus by measuring and comparing the level of microsatellite DNA present in the first allele to which the allele imbalance is cancer or precancerous. Show.
[0007]
WO 01/86288, entitled "Method and Apparatus for Early Diagnosis of Blader Tumor in Urine Samples", discloses a method for the early diagnosis of bladder tumors in urine samples, which is a family of cytokeratins of the protein marker. And at least one additional marker selected from the group comprising lymphocyte markers suitable for detecting inflammatory cells associated with neoplastic infiltration to determine RNA accessibility as a standard for estimating abundance. Amplification of RNA extracted from cells present in urine by using a marker for β-actin, a marker for messenger RNA of the catalytic component of telomerase (hTRT) to prove Extent, and it includes a final step of detecting the amplified material.
[0008]
WO 99/63110 entitled "Diagnostics and treatment of cancer" describes (i) obtaining a sample containing nucleic acid and / or protein from the patient's bladder, preferably urothelium; and A method for diagnosing bladder cancer in a human patient, comprising the step of determining whether the sample contains levels of Pax5 nucleic acid or protein associated with bladder cancer, has been disclosed.
[0009]
WO 02/27329 (Patent Document 8) entitled "Biomarkers of translational cell carcinoma of the blader" uses a marker that distinguishes between a sample of a TCC patient and a sample of a control (eg, a subject in whom TCC cannot be detected) to obtain a bladder. Disclosed are protein markers, methods and kits that can be used as an aid to the diagnosis of transitional cell carcinoma (TCC).
[0010]
U.S. Patent No. 6,335,170 discloses a method for measuring the expression pattern of urothelial or bladder cancer cells, comprising: a sample comprising urothelial or bladder cancer cells. Measuring the expression of one or more genes therein, thereby forming a first pattern of expression; subtracting the second pattern of expression from the first pattern of expression, wherein the second pattern is one or more. Formed using a sample containing cells of the submucosal tissue, smooth muscle or connective tissue, the subtraction step forming a third pattern of expression, which is the submucosal, smooth muscle present in the sample. Or it reflects the expression of urothelial or bladder cancer cells independent of the proportion of cells in the connective tissue.
[0011]
Other relevant studies on bladder cancer include the following:
Konety BR and Goetzenberg RH: Urine based markers of urological medical. J Urol 165: 600-611, 2001 (Non-Patent Document 1); Droller MJ: Individualizing the approach to invasive blader cancer. Contemp. Urol. , July / August, pp. 54-61, 1990 (Non-Patent Document 2); Nomura N, Miyajima N, Sazuka T, et al .: Prediction of the coding sequences. of unidentified human genes. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) reduced by analysis of randomized sampled cDNA clones from humane imagery DNA Res 1: 27-35, 1994 (Non-Patent Document 3); Bassall S, Nomura N, Venter D, et al .: Characterization of a novel human cell-regulated. homologue of Drosophila dlg1. Genomics, 77: 5-7, 2001 (Non-Patent Document 4); Ellis WJ, Blumenstein BA, Isshak LM, et al .: Clinical evaluation of TAB and compared to voided urine cytology and the Bird BTA test in patients with the recurrent blader tutors. The Multi Center Study Group. Urology 50: 882-887, 1997 (Non-Patent Document 5); Sarrosdy MF, Hudson MA, Hudson MA, Ellis WJ, et al .: Implemented debourde anced edron der d er d e r a b e s s s s s BTA stat Test. Urology 50: 349-353, 1997 (Non-patent Document 6); Seripa D (Paripla P), Gallucci M (Gallucci M), et al .: Sensitivity detection of translational transmission. microstellite analysis of cells exfoliated in urine. Int J Cancer 95: 364-369, 2001 (Non-Patent Document 7); Ponsky LE, Sharma S, Pandrangi L, et al .: Screening and monitoring fordancing the use of NMP22. J Urol 166: 75-78, 2001 (Non Patent Literature 8); Connectivity BR (Konty BR), Nguyen TS (Nguyen TS), Dyr R (Dhir R), et al .: Detection of blade trading carousel trading navigation protein, BLCA-4. Clin Cancer Res 6: 2618-2625, 2000 (Non-Patent Document 9); Rotem D, Cassel A, Lindenfeld N, et al .: Urinary cytokeratin 20 as a maker translational cell carcinoma. Eur Urol 37: 601-604, 2000 (Sanchez-Carbayo M), Urutia M, Gonzales de Butrago et al. : Evaluation of two new tumour markers: blader tumour fibrontin and cytokeratin 18 for the diagnosis of blader cancer. Clin Cancer Res. 6: 3585-3594, 2000 (Non-Patent Document 11); Sanchez-Carbayo M, Urrutia M, Silva JM (Silva JM) et al. the diagnosis of blader cancer. Urology 55: 526-532, 2000 (Non-Patent Document 12); Smith SD (Smith SD), Wheeler M.A. (Wheeler, MA), Plesia J (Plescia J), et al. JAMA 285: 324-328, 2001 (Non-Patent Document 13).
[0012]
However, to our knowledge, none of the above references report human HURP gene expression in bladder or urothelial carcinoma. Applicants have surprisingly discovered that TCC tissue samples showed reproducible and significant expression of HURP mRNA transcripts.
[0013]
Based on this finding, by analyzing the expression of the human HURP gene in tumor tissue samples and biological samples such as excreted urine, detection, preliminary screening or monitoring of bladder cancer in human subjects is possible. An accurate, convenient and non-invasive diagnostic method can be developed, and the expression of the human HURP gene detected indicates the presence of bladder cancer.
[0014]
[Patent Document 1]
US Patent No. 6,376,188
[0015]
[Patent Document 2]
US Patent No. 6,251,638
[0016]
[Patent Document 3]
US Patent No. 6,287,820
[0017]
[Patent Document 4]
US Patent No. 6,335,167
[0018]
[Patent Document 5]
US Patent No. 6,291,163
[0019]
[Patent Document 6]
WO 01/86288
[0020]
[Patent Document 7]
WO 99/63110
[0021]
[Patent Document 8]
WO 02/27329
[0022]
[Patent Document 9]
US Patent No. 6,335,170
[0023]
[Non-patent document 1]
Konety BR and Goetzenberg RH: Urine based markers of urological medical. J Urol 165: 600-611, 2001.
[0024]
[Non-patent document 2]
Droller MJ: Individifying the approach to invasive blader cancer. Contemp. Urol. , July / August, pp. 54-61, 1990.
[0025]
[Non-Patent Document 3]
Nomura N, Miyajima N, Sazuka T, et al: Prediction of the coding sequences of unified humanities. I. The coding sequences of 40 new genes (KIAA0001-KIAA0040) reduced by analysis of randomized sampled cDNA clones from humane imagery DNA Res 1: 27-35, 1994
[0026]
[Non-patent document 4]
Bassall S, Nomura N, Venter D, et al .: Characterization of a novel human cell-cycle-regulatedhomology of Drosophila. Genomics, 77: 5-7, 2001.
[0027]
[Non-Patent Document 5]
Ellis AW over Jay (Ellis WJ), Blue Men stain Bie (Blumenstein BA), Ishaku-Eruemu (Ishak LM), et al: Clinical evaluation ofthe BTA TRAK assay and comparison to voided urine cytology and the BardBTA test in patients with recurrent bladder tumors. The Multi Center Study Group. Urology 50: 882-887, 1997.
[0028]
[Non-Patent Document 6]
Salosdy MF, Hudson MA, Ellis WJ, et al .: Implemented detection of current blade tasting the best ard. Urology 50: 349-353, 1997.
[0029]
[Non-Patent Document 7]
Seripa Dee (Seripa D), Parera plc (Parrella P), Garutchi M. (Gallucci M), et al: Sensitive detection of transitional cell carcinoma of the bladder by microsatellite analysis of cells exfoliated in urine. Int
J Cancer 95: 364-369, 2001.
[0030]
[Non-Patent Document 8]
Ponsky LE, Sharma S, Pandrangi L et al .: Screening and monitoring for blader cancer: refining the use of NMP22. J Urol 166: 75-78, 2001.
[0031]
[Non-Patent Document 9]
Connectivity BR, Nguyen TS, Dhir R, et al .: Detection of blader cancer using a novel nuclear matrix, BL-4. Clin Cancer Res 6: 2618-2625, 2000
[0032]
[Non-Patent Document 10]
Rotem D, Cassel A, Lindenfeld N, et al .: Urinary cytokeratin 20 as a marker for translational carcinoma. Eur Urol 37: 601-604, 2000
[0033]
[Non-Patent Document 11]
Sanchez-Carbayo M, Urutia M, Gonzalez de Buitragon Jerm, etc. the diagnosis of blader cancer. Clin Cancer Res. 6: 3585-3594, 2000
[0034]
[Non-Patent Document 12]
Sanchez-Carbayo M, Urutia M, Silva JM (Silva JM), et al .: Urinary tissue polypeptidogen identificantogenidogenesis agent Urology 55: 526-532, 2000
[0035]
[Non-patent document 13]
Smith SD, Wheeler, MA, Plescia J, et al .: Urine detection of survivin and diagnosis of bladedder cancer. JAMA 285: 324-328, 2001 Summary of the Invention
Accordingly, in a first aspect of the present invention, the present invention provides:
Obtaining a biological sample from a subject suspected of having bladder cancer; and
Detecting expression of the human HURP gene in the sample, wherein the detected expression of the human HURP gene indicates the presence of bladder cancer;
And a method for detecting bladder cancer in a human subject.
[0036]
In a second aspect, the present invention provides:
Periodically obtaining a biological sample from a subject suspected of having bladder cancer; and
Detecting the expression of the human HURP gene in the sample, wherein the detected expression of the human HURP gene indicates the presence of bladder cancer;
There is provided a method of monitoring bladder cancer in a human subject, comprising:
In a third aspect, the present invention provides a primer set for detecting bladder cancer in a human subject, comprising a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, And a reverse primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 5.
[0037]
In a fourth aspect, the present invention provides a diagnostic kit for detecting bladder cancer in a human subject, comprising at least one primer pair of a forward primer and a reverse primer, each primer corresponding to SEQ ID NO: 1. Having a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having the nucleotide sequence of
[0038]
The above and other objects, features and advantages of the present invention will become apparent by reference to the following detailed description and preferred embodiments, taken in conjunction with the accompanying drawings.
[0039]
Detailed description of the invention
Genes and proteins involved in cell cycle control and apoptosis have been discovered to be important in cancer growth. There is a continuing need in the art in identifying components of cells that control the cell cycle and apoptosis.
[0040]
The human liver cancer up-regulated protein (HURP) gene (NCBI accession number AB076695) having the full-length nucleotide sequence as shown in SEQ ID NO: 1 was developed by Chen-Kung Chou et al. First identified as a related gene. In the study of Chou's hepatocellular carcinoma (HCC), a novel cell cycle regulatory gene (CCRG) is present in cell cycle-dependent expression profiles, particularly in cDNA libraries and microarray databases of human HCC tissues. A search for a unique gene identified it as a liver cancer up-regulated protein (HURP) (Chou et al., Manuscript submitted for publication).
[0041]
Surprisingly, the human HURP gene can be a potential molecular maker for bladder cancer, and that the human HURP gene is sensitive enough to detect low-grade bladder tumors and is not cancerous or clinical Applicants have for the first time found that they can be used in non-invasive urine diagnostics, which are specific for eliminating the majority of trivial patients.
[0042]
According to the present invention, there is provided a method of detecting, preliminary screening or monitoring of bladder cancer in a human subject, comprising:
Obtaining a biological sample from a subject suspected of having bladder cancer; and
Detecting the expression of the human HURP gene in the sample, wherein the detected expression of the human HURP gene indicates the presence of bladder cancer.
[0043]
The method of the present invention may be applied to the detection, preliminary screening and monitoring of transitional cell carcinoma (TCC) of the bladder.
[0044]
Preferably, the method is performed using a biological sample comprising cells selected from the group consisting of urothelial cancer cells, bladder cancer cells, or a combination thereof.
[0045]
Preferably, said biological sample is selected from the group consisting of urine, urothelial biopsy, blood, plasma and serum. In a preferred embodiment of the invention, the biological sample is urine.
[0046]
The human HURP gene has a nucleotide sequence as shown in FIG. 1 and SEQ ID NO: 1, on the basis of which oligonucleotides that hybridize to at least 13 nucleotides of the human HURP gene are commonly used in the art of biotechnology. Can be designed to serve as diagnostic probes or primers for detecting the expression of said genes using the methodology of
[0047]
By way of example, referring to FIG. 1, Applicants have designed four primers that are useful in the methods of the present invention for detecting bladder cancer and include the following:
First primer pair:
[0048]
Embedded image

[0049]
Second primer pair:
[0050]
Embedded image

[0051]
It is believed that forward primer 1 can be paired with reverse primer 2, and forward primer 2 can be paired with reverse primer 1.
[0052]
According to the present invention, expression of the human HURP gene is measured by analysis of the mRNA transcript from the human HURP gene or the protein translated from the mRNA transcript of the human HURP gene.
[0053]
Detection of the translated protein from the mRNA transcript of the human HURP gene can be performed by conventional methodologies well known in the biotechnology field.
[0054]
In a preferred embodiment of the present invention, the detection of the expression of the human HURP gene is performed by detecting the presence of a human HURP gene mRNA transcript in a biological sample.
[0055]
Preferably, the detection of the presence of the mRNA transcript of the human HURP gene comprises the following methodologies: hybridization, cycling probe reaction, polymerase chain reaction (PCR), nested PCR, reverse transcriptase polymerase chain. Reaction (RT-PCR), multiplex PCR polymerase chain reaction-single-stranded conformation polymorphism, ligase chain reaction (LCR), restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification (transcription- medium amplification (TMA)
Is performed using at least one of the following.
[0056]
In a preferred embodiment of the present invention, the detection of the presence of the mRNA transcript of the human HURP gene is performed using RT-PCR.
[0057]
In a more preferred embodiment of the present invention, the detection of the presence of the mRNA transcript of the human HURP gene comprises:
(A) extracting total RNA from a biological sample;
(B) subjecting the RNA extracted in step (a) to reverse transcriptase polymerase chain reaction (RT-PCR) treatment using at least one primer pair of a forward primer and a reverse primer; Having a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having a nucleotide sequence corresponding to SEQ ID NO: 1; and
(C) detecting whether an RT-PCR product having a nucleotide sequence identical or complementary to a part of the nucleotide sequence of the human HURP gene was produced from the RT-PCR treatment in step (b); The presence of the PCR product indicates the presence of bladder cancer,
It is performed by.
[0058]
Preferably, each primer used in step (b) hybridizes to at least 18 nucleotides of the human HURP gene.
[0059]
Preferably, at least one primer pair used in step (b) has a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and a forward primer shown in SEQ ID NO: 3 and SEQ ID NO: 5. And a reverse primer having a nucleotide sequence selected from the sequences to be prepared.
[0060]
In a preferred embodiment of the present invention, at least one primer pair used in step (b) comprises a forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5. Including.
[0061]
In a preferred embodiment of the present invention, the RT-PCR treatment in the step (b) comprises a first forward primer having a nucleotide sequence corresponding to SEQ ID NO: 2 and a first reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 3. And a second primer comprising a second forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a second reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5 Includes a second amplification reaction using pairs.
[0062]
According to the present invention, there is provided a primer set for detecting bladder cancer in a human subject, which comprises a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 and SEQ ID NO: 3 and SEQ ID NO: 3. And a reverse primer having a nucleotide sequence selected from the sequence represented by No. 5.
[0063]
In a preferred embodiment of the present invention, the forward primer has the nucleotide sequence shown in SEQ ID NO: 2.
[0064]
In another preferred embodiment of the present invention, the forward primer has the nucleotide sequence shown in SEQ ID NO: 4.
[0065]
In a preferred embodiment of the present invention, the reverse primer has the nucleotide sequence shown in SEQ ID NO: 3.
[0066]
In another preferred embodiment of the invention, the reverse primer has the nucleotide sequence shown in SEQ ID NO: 5.
[0067]
According to the present invention there is provided a diagnostic kit for the detection of bladder cancer in a human subject, comprising at least one primer pair of a forward primer and a reverse primer, each primer comprising a nucleotide sequence corresponding to SEQ ID NO: 1. Has a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having
[0068]
In another preferred embodiment of the present invention, the diagnostic kit comprises a first primer comprising a first forward primer having a nucleotide sequence corresponding to SEQ ID NO: 2 and a first reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 3 And a second primer pair comprising a second forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a second reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5.
[0069]
In addition to the foregoing, those skilled in the art will appreciate that various uses within the scope of the present invention include using any type of evaluation assay.
[0070]
For example, RNA and proteins can be isolated and assayed from test samples using techniques known in the art. They can be isolated, for example, from fresh or frozen biopsies, formalin-fixed tissues, and body fluids such as blood, plasma, serum, urine or sputum.
[0071]
RT-PCR can be used, but other techniques are also contemplated. These include specific techniques including PCR, nested PCR in situ hybridization, Northern blotting, high density expression arrays, microarrays, and techniques for assaying specific protein products such as ELISA, Western blotting and enzyme assays. Other techniques for assaying mRNA species are included.
[0072]
The present invention will be described in more detail with reference to the following examples, which are for illustrative purposes only and are not intended to limit the scope of the invention.
[0073]
Example 1: PT-PCR analysis of HURP gene expression
Materials and methods:
Patient characteristics
From March 1998 to September 2001, 80 TCC samples were obtained from the urinary tract using a cystotomy and a transurethral resection at the Chi Mei Medical Center. Distant macroscopically normal tissues were also obtained for analysis and they were considered as tissue samples close to the tumor. All tissue samples were frozen in liquid nitrogen and stored at -86 C for various periods of time. A representative section of each frozen block was embedded in paraffin, stained with hematoxylin-eosin, and examined by a pathologist to assess the status of the tumor in the sample.
[0074]
All specimens were graded using a World Health Organization classification correction, and pathological staging was according to the TNM pathology staging system (Epstein JI, Amin MB, Reuter VR, et al.). :.. The World Health Organization / International Society of Urological Pathology consensus classification of urothelial (transitional cell) neoplasms of the urinary bladder Bladder Consensus Conference Committee Am J Surg Pathol 22: 1435-1448, 19 98; and Chisholm GD, Hindmarsh JR, Howatson AG, et al: TNM (1978) in blader cancer: use and absolute. Br J Urol 52: 500-505, 1980).
[0075]
The stage of the bladder tumor indicates how deep the tumor has invaded. Superficial tumors are called Ta and T1-4 is used to describe the degree of increased muscle invasion. Bladder tumor grades are expressed on a scale of I-IV (1-4). The grade reflects the cytological appearance of the cells, Grade I cells are almost normal, Grade II cells are slightly out of normal, Grade III cells are clearly abnormal, Grade IV cells Is highly abnormal.
[0076]
RNA isolation:
Total RNA was isolated from frozen tissue specimens or urine pellets using an Ultraspec® RNA isolation system (Biotecx Laboratories Inc., Houston, Tex.). Approximately 0.5-1.0 grams of tissue was homogenized in 1 ml Ultraspec® reagent in a handheld glass-Teflon. After homogenization, the homogenate was stored at 4 ° C. for 5 minutes, extracted with 0.2 ml of chloroform, and then centrifuged at 12,000 g (4 ° C.) for 15 minutes. The RNA in the aqueous phase was precipitated with the same amount of isopropanol and washed twice with 75% ethanol. The RNA content was assessed spectrophotometrically (1 OD of A260 equals 40 μg / ml RNA) and the purity of the extract was assessed using the A260 / 280 ratio, which in all cases was 1.75. Exceeded. For urine collection, 50 milliliters of neatly collected urine was obtained at the first void of the day from all patients. The sample was centrifuged at 3,000 g (4 ° C.) for 20 minutes, and the cell pellet was mixed with 1 ml of Ultraspec® reagent and extracted using the same method as described above.
[0077]
Reverse transcriptase-polymerase chain reaction
Single-stranded complementary DNA (cDNA) was synthesized with 1 μl of SuperScript II reverse transcriptase (Life Technologies Inc., Gaithersburg, Md.) Using oligo-dT priming of 1 μg of total RNA at 42 ° C. for 50 minutes. Was. After heating at 70 ° C. for 15 minutes, the first amplification reaction was performed with 1/10 reverse transcribed RNA, Taq polymerase buffer containing 200 μmol / L dNTPs, 1 U DyNAzyme® II DNA polymerase (Finnzymes Inc.). And 10 μM of each human HURP primer 5′-GGATCC. AATAGACACTTTGGTTTG -3 '(forward) and 5'-GGATCCCACCTTTCCTTTCT GGTTC Using -3 '(reverse), denaturation at 94 ° C for 1 minute, annealing at 55 ° C for 1 minute and extension at 72 ° C for 1 minute were performed for 30 cycles, followed by incubation at 72 ° C for 5 minutes.
[0078]
For nested PCR of urine specimens, one-tenth of the first amplification product was combined with nested HURP primers 5′-CAACGAAAACAGATGCTC-3 ′ (forward) and 5′-TGAGTAGCTGATCGAGGTC-3 ′ (reverse) at 94 ° C. Thirty cycles of denaturation for 1 minute, annealing at 60 ° C. for 1 minute, and extension at 72 ° C. for 1 minute, followed by a second round of amplification with incubation at 72 ° C. for 5 minutes.
[0079]
Routine RT-PCR controls without RT as template were negative, as shown in FIG. 2 by inserting the full-length HURP nucleotide sequence into the pET32a (+) vector (purchased from Novagen Inc.) as template. Those using pET32a (+)-HURP constructed as described above were positive. Amplification products were separated using electrophoresis with 0.1 μg GeneRuler® 100 bp DNA ladder (MBI Fermentas, Lithuania) in a 1.5% agarose gel and visualized using ethidium bromide staining.
[0080]
Data quantification and standardization
To quantify the mRNA expression level of the HURP gene, all gels were photographed using an Image Master® VDS video imaging system (Amersham Pharmacia Biotech Inc., Piscataway) and the LISCAP image capture software (AmershamPhashaPamhashaBamshaPamway). Inc., version 1.0). Optical pixels of the PCR product were quantified to arbitrary units using ImageMaster® TotalLab software (Amersham Pharmacia Biotech Inc., version 1.11).
[0081]
Normalization of the data was necessary to compare the test samples. To this end, the PCR amplification product of β-actin was used as an internal standard to represent a relative equivalent amount of the cDNA template subjected to PCR. Mean values were expressed as mean ± SD values and compared using Student's t-test.
[0082]
result:
Representative expression patterns of the HURP gene in TCC tissue samples and control subjects are shown in FIG. 3A. Eleven pairs of tumor parts (patient number: 294 N / T, 306 N / T, 307 N / T, 315 N / T, 317 N / T, 319 N / T, 320 N / T, 321 N / T, 327 N / T, 335 N / T, 337 N / T), relatively more human HURP gene mRNA transcripts were amplified (see FIG. 3A). Only one pair of tissue samples (patient number: 322 N / T) both showed undetectable transcripts of the human HURP gene. As an internal standard, the PCR amplification product of β-actin (see FIG. 3B) was used to represent a relative equivalent amount to the cDNA template subjected to PCR.
[0083]
Example 2: Evaluation of tumor specificity of HURP gene expression by RT-PCR analysis
To determine whether overexpression of HURP shows tumor specificity, human HURP transcripts of prostatic urothelium from patients with benign prostatic hyperplasia (BPH) were analyzed using reverse transcriptase-polymerase chain reaction (RT-RT). PCR).
[0084]
Experiments were performed to determine whether HURP transcripts of prostatic urothelium from patients with benign prostatic hyperplasia (BPH) show reverse transcriptase-polymerase chain reaction (RT-RT) to determine whether HURP gene expression is tumor specific. The procedure was as described in Example 1 above, except that amplification was carried out using PCR).
[0085]
Fifteen specimens of prostatic urothelium from BPH patients using standard cystoscopy were collected and subjected to RT-PCR. Referring to FIG. 4, β-actin transcript was equally amplified from five representative samples of BPH urothelium, but no HURP transcript was detected in these samples. In addition to BPH samples, non-bladder controls containing 4 samples from liver and 4 samples from heart were tested and no HURP transcript was detected in these tissues (data not shown).
[0086]
Example 3 Evaluation of Tumor Stage by RT-PCR Analysis of HURP Gene Expression
To determine a possible correlation between HURP gene overexpression and tumor grade, 45 pairs of human HURP transcripts of TCC-containing tissue samples and corresponding tumor-proximal tissue samples were analyzed and β -Normalized with actin transcript.
[0087]
Referring to Table 1, 35 tumor-proximal portions (35/45, 77.8%) had lower expression of the HURP gene than tumor portions (Group I, p <0.0001), and 10 tumors It is noteworthy that the contiguous portion (10/45, 22.2%) showed almost the same amount of HURP gene as the tumor portion (Group II).
[0088]
Eighty TCC patients with a pathological identification of zero Grade I, 37 Grade II, 31 Grade III, 12 Grade IV were recruited and the ratio of human HURP expression in tissue samples is listed in Table II. There appears to be no significant correlation between HURP expression and tumor grade. Among them, 9 tumors negative for HURP, 6 were grade II, 1 was grade III and 2 were grade IV.
[0089]
[Table 1]

[0090]
[Table 2]

[0091]
Example 4: Evaluation of HURP gene expression as a biomarker for detection of TCC To evaluate the potential applicability of urinary HURP as a novel urinary molecular marker for urinary TCC detection, a new Alternatively, seven additional patients with relapsed TCC were analyzed for urinary HURP using RT-PCR. Total RNA was extracted from urine cell pellets and reverse transcribed by oligo-dT priming. Amplification reactions were performed using HURP-specific primers or β-actin-specific primers. A 370 bp cDNA was amplified from urine cell pellets of all patients with TCC (FIG. 5A). In contrast, cell pellets from urine from seven additional individuals (three had urinary tract infections, two had chronic inflammation of the bladder, one had renal cell carcinoma, and one had BHP) , Had no HURP transcript. In control experiments, a 307 base pair β-actin cDNA fragment was indistinguishably amplified from the urine of control subjects and TCC patients (FIG. 5B).
[0092]
All patents and references cited herein are hereby incorporated by reference in their entirety. In case of conflict, the present description, including definitions, will control.
[0093]
Although the present invention has been described with reference to the above specific embodiments, it is evident that many modifications and variations can be made without departing from the scope and spirit of the invention. Accordingly, the invention is only intended to be limited as set forth in the appended claims.
[0094]
[Sequence list]

[Brief description of the drawings]
FIG. 1 shows the full length of the nucleotide sequence of the human HURP gene (SEQ ID NO: 1), wherein the nucleotide sequences complementary to the four primers designed according to the invention are underlined and bolded ing;
FIG. 2 shows a restriction map of the vector pET32a (Novagen Inc.) used in the above example, wherein the BamHI restriction site is used to insert the full-length nucleic acid sequence of the human HURP gene.
FIGS. 3A and 3B show RT-PCR analysis of HURP and β-actin expression in TCC tissue, respectively, where N = tumor-proximal tissue, T = tumor tissue, and 294,306,307,315,317. , 319, 320, 321, 322, 327, 335 and 337 = patient number.
FIG. 4 shows RT-PCR analysis of human HURP expression in prostate urothelial samples taken from five patients with benign prostatic hyperplasia, where left is β-actin (307 bp) and right Is HURP (370 bp), N1, N2, N3, N4 and N5 = 5 benign prostate hyperplasia (BPH) samples; NC = negative control; and M indicates molecular weight markers in base pairs.
FIGS. 5A and 5B show RT-PCR analysis of HURP and β-actin expression in excreted urine samples, respectively, where 2,6,7,8,14,15,20,5. , 10, 11, 12, 13, 16, and 18 = patient number; NC: negative control; M indicates molecular weight marker in base pairs.

Claims (34)

(1) obtaining a biological sample from a subject suspected of having bladder cancer; and (2) detecting the expression of the human HURP gene in the sample, wherein the detected expression of the human HURP gene is A method for detecting bladder cancer in a human subject, comprising indicating the presence of bladder cancer. 2. The method of claim 1, wherein the bladder cancer is a transitional cell carcinoma of the bladder (TCC). 2. The method of claim 1, wherein the biological sample comprises cells selected from the group consisting of urothelial cancer cells, bladder cancer cells, or a combination thereof. The method of claim 1, wherein the biological sample is selected from the group consisting of urine, urothelial biopsy, blood, plasma, and serum. 5. The method according to claim 4, wherein the biological sample is urine. 2. The method according to claim 1, wherein the human HURP gene has the nucleotide sequence shown in SEQ ID NO: 1. 7. The method of claim 6, wherein detecting expression of the human HURP gene is performed by detecting the presence of an mRNA transcript of the human HURP gene in the biological sample. The detection of the presence of the mRNA transcript of the human HURP gene involves the following methodologies: hybridization, cycling probe reaction, polymerase chain reaction (PCR), nested PCR, reverse transcriptase polymerase chain reaction (RT- PCR), multiplex PCR polymerase chain reaction-single-stranded conformation polymorphism, ligase chain reaction (LCR), restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification ( TMA)
The method of claim 7, wherein the method is performed using at least one of the following. The method according to claim 7, wherein the detection of the presence of the mRNA transcript of the human HURP gene is performed using RT-PCR. The method according to claim 7, wherein the detection of the presence of the mRNA transcript of the human HURP gene is performed by:
(A) extracting total RNA from a biological sample;
(B) subjecting the RNA extracted in step (a) to reverse transcriptase polymerase chain reaction (RT-PCR) treatment using at least one primer pair of a forward primer and a reverse primer; Having a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having a nucleotide sequence corresponding to No. 1;
(C) detecting whether an RT-PCR product having a nucleotide sequence identical or complementary to a part of the nucleotide sequence of the human HURP gene was produced from the RT-PCR treatment in step (b); -The presence of the PCR product indicates the presence of bladder cancer. At least one primer pair used in the step (b) comprises a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and a sequence shown in SEQ ID NO: 3 and SEQ ID NO: 5. And a reverse primer having a nucleotide sequence of choice. The at least one primer pair used in step (b) comprises a forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5. the method of. The RT-PCR treatment in the step (b) uses a first primer pair including a first forward primer having a nucleotide sequence corresponding to SEQ ID NO: 2 and a first reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 3. A first amplification reaction and a second amplification reaction using a second primer pair including a second forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a second reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5 The method of claim 10, comprising: (1 ′) periodically obtaining a biological sample from a subject suspected of having bladder cancer; and
(2 ′) A method for monitoring bladder cancer in a human subject, comprising the step of detecting the expression of the human HURP gene in the sample, wherein the detected expression of the human HURP gene indicates the presence of bladder cancer. 15. The method of claim 14, wherein the bladder cancer is a transitional cell carcinoma of the bladder (TCC). 15. The method of claim 14, wherein the biological sample comprises cells selected from the group consisting of urothelial cancer cells, bladder cancer cells, or a combination thereof. 15. The method according to claim 14, wherein the biological sample is selected from the group consisting of urine, urothelial biopsy, blood, plasma and serum. 18. The method according to claim 17, wherein the biological sample is urine. The method according to claim 14, wherein the human HURP gene has the nucleotide sequence shown in SEQ ID NO: 1. 20. The method of claim 19, wherein detecting the expression of the human HURP gene is performed by detecting the presence of an mRNA transcript of the human HURP gene in the biological sample. Detection of the presence of the mRNA transcript of the human HURP gene involves the following methodology:
Hybridization, cycling probe reaction, polymerase chain reaction (PCR), nested PCR, reverse transcriptase polymerase chain reaction (RT-PCR), multiplex PCR polymerase chain reaction-single-stranded conformation Template, ligase chain reaction (LCR), restriction fragment length polymorphism nucleic acid sequence-based amplification (NASBA), and transcription-mediated amplification (TMA)
21. The method of claim 20, which is performed using at least one of the following. 21. The method according to claim 20, wherein the detection of the presence of the mRNA transcript of the human HURP gene is performed using RT-PCR. The method according to claim 20, wherein the detection of the presence of the mRNA transcript of the human HURP gene is performed as follows:
(A ′) extracting total RNA from a biological sample;
(B ′) subjecting the RNA extracted in step (a ′) to a reverse transcriptase polymerase chain reaction (RT-PCR) treatment using at least one primer pair of a forward primer and a reverse primer; Having a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having a nucleotide sequence corresponding to SEQ ID NO: 1;
(C ′) detecting whether an RT-PCR product having a nucleotide sequence identical or complementary to a part of the nucleotide sequence of the human HURP gene was produced by the RT-PCR treatment in step (b ′); The presence of the RT-PCR product indicates the presence of bladder cancer. At least one primer pair used in the step (b ′) comprises a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 and the sequence shown in SEQ ID NO: 3 and SEQ ID NO: 5 24. The method of claim 23, comprising a reverse primer having the selected nucleotide sequence. 24. The method according to claim 23, wherein the at least one primer pair used in step (b ') comprises a forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5. The described method. The RT-PCR treatment in the step (b ′) uses a first primer pair including a first forward primer having a nucleotide sequence corresponding to SEQ ID NO: 2 and a first reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 3. And a second amplification using a second primer pair comprising a second forward primer having a nucleotide sequence corresponding to SEQ ID NO: 4 and a second reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5 24. The method of claim 23, comprising a reaction. A DNA sequence having a nucleotide sequence selected from the sequences shown in SEQ ID NOs: 2 to 5. A human subject comprising a forward primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, and a reverse primer having a nucleotide sequence selected from the sequences shown in SEQ ID NO: 3 and SEQ ID NO: 5 Primer set for detecting bladder cancer. The primer set according to claim 28, wherein the forward primer has a nucleotide sequence represented by SEQ ID NO: 2. The primer set according to claim 28, wherein the forward primer has a nucleotide sequence represented by SEQ ID NO: 4. The primer set according to claim 28, wherein the reverse primer has a nucleotide sequence represented by SEQ ID NO: 3. The primer set according to claim 28, wherein the reverse primer has a nucleotide sequence represented by SEQ ID NO: 5. Bladder cancer in a human subject, comprising at least one primer pair of a forward primer and a reverse primer, each primer having a nucleotide sequence that hybridizes to at least 13 nucleotides of the human HURP gene having a nucleotide sequence corresponding to SEQ ID NO: 1 Diagnostic kit for the detection of A first primer pair including a first forward primer having a nucleotide sequence corresponding to SEQ ID NO: 2 and a first reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 3, and a first primer pair having a nucleotide sequence corresponding to SEQ ID NO: 4 35. The diagnostic kit according to claim 34, comprising a second primer pair comprising a two forward primer and a second reverse primer having a nucleotide sequence corresponding to SEQ ID NO: 5.

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