JP2023505849A - 腫瘍マーカー及び治療ターゲットとしての長鎖ノンコーディングrna letn - Google Patents
腫瘍マーカー及び治療ターゲットとしての長鎖ノンコーディングrna letn Download PDFInfo
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Abstract
Description
本発明は癌分野に関する。より具体的には、本発明は、長鎖ノンコーディングRNA LETNを腫瘍マーカー及び治療ターゲットとする使用に関する。
近年、シークエンシング技術の急速な発展に伴い、ヒトゲノム上の大量のノンコーディング領域で、翻訳活性がない長鎖ノンコーディングRNA(lnc RNA)を転写し、かつ数がタンパク質を大幅に超えることを発見し、ヒトの様々な疾患又は生理機能に関連する数百種類のlncRNAを発見した。特に癌において、ますます多くの証拠はlncRNAが腫瘍抑制又は腫瘍発生において相乗効果を果たすことを証明した[1]。しかし、大多数のlncRNAの分子及び生物学的機能についてはあまり知られていない。
本発明者らは、理論研究と実験ツールを組み合わせることで、マルチオミクスデータマイニングツールを用いて、がんとの関連で潜在的な機能を持つ可能性のあるlncRNAの網羅的解析を行った。肝細胞がん(LIHC)において、本発明者らは、本実験室で設計されたアルゴリズムにより、癌ゲノムアトラス(TCGA)のデータを利用してlncRNAに対して機能調査を行い、これまで研究されていなかったlncRNA RP11-196G18.22(LETNと命名する)を発見した。本発明者らは、LETNが191対の転写因子および標的遺伝子を調節し、肝臓癌の発生・進展に広く強力な制御力を持つ可能性があると予測し、実験によって検証した。
1)lncRNA RP11-196G18.22(LETN)を過剰発現している細胞におけるlncRNA RP11-196G18.22(LETN)の発現レベルを測定するステップと、
2)候補化合物をステップ1)の細胞と接触させるステップと、
3)ステップ2)の後、細胞におけるlncRNA RP11-196G18.22(LETN)の発現レベルを測定するステップと、
4)ステップ1)とステップ3)で測定されたlncRNA RP11-196G18.22(LETN)の発現レベルを比較するステップとを含み、lncRNA RP11-196G18.22(LETN)の発現レベルの低下は、前記候補化合物が抗癌性を有することを示し、好ましくは、前記細胞が癌細胞である。
1)腫瘍又は腫瘍細胞サンプルにおけるLETNの発現は、対照(正常又は健康組織/細胞)に対して増加するか否かを測定するステップと、
2)前記腫瘍が治療に適するか否かを特定するステップとを含み、増加された発現がLETN発現抑制剤による治療に適することを示す。
前記薬剤が腫瘍又は腫瘍細胞サンプルにおけるLETNの発現を低減させることができるか否かをテストすることを含み、低減させることができると、前記薬剤が癌の治療及び/予防に適することを示す。一実施形態において、前記癌は固形腫瘍であり、好ましくは、肝臓癌、肺癌、前立腺癌、乳癌、前立腺癌、膵臓癌、腎臓癌、胃癌、軟部組織癌、胆道癌、膀胱癌、直腸癌、子宮内膜癌、頭頸部癌、結腸癌、食道癌及び甲状腺癌からなる群から選ばれる。好ましい一実施形態において、前記LETNのヌクレオチド配列は、SEQ ID NO: 1に示す(Ensembl登録番号ENST00000564237.1)。
本明細書で使用される用語は、特に断りのない限り、当業者に理解される通常の技術的意味を有する。当技術分野における定義および用語については、Sambrookら、Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Plainsview, New York (1989); およびAusubelら、Current Protocols in Molecular Biology (Supplement 47), John Wiley & Sons, New York (1999)を参照することが、当業者には特に推奨される。
肝細胞癌(LIHC)において、癌ゲノムアトラス(TCGA)のデータ(https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga)を利用してlncRNAに対して機能調査を行い、本実験室の設計したアルゴリズムにより、調節転写因子及びその標的遺伝子機能を有するlncRNAを予測した。調節対数の多少、癌とパラ癌(para-carcinoma)が発現相違があるか否か、及びゲノムコピー数変異という3つの要因から、総合的にクリーニングして最終的に22本のlncRNAs(UBE2SP2,BMS1P8,RP11-443P15.2,LINC01296/DUXAP10,LL22NC03-N14H11.1,RP11-284F21.10,DUXAP8,CRNDE,CTD-2227E11.1,LINC00853,LINC00665,RP11-196G18.22,GOLGA2P7,RP11-14N7.2,PVT1,LINC00511,RP11-396C23.2,MIR4435-2HG,AL450992.2,HCG25,PCAT6,LINC00152)を選択した。いくつかの非特異的配列のlncRNA(その配列は完全に遺伝子のエクソン内に位置し、機能的に検証するための特異的なsiRNAを設計することができない)を除去して、16本のlncRNAsが残り、次に実験によりこれらのlncRNAについて表型検証を行った。具体的に、これらのlncRNAsに特異的なsiRNAを設計し(下記表1参照)、リポソームLipofectamine 2000 (Thermo Fisher,11668019)により、肝臓癌細胞系HUH7(中科院上海セルバンク)のトランスフェクションを行い、対応するlncRNAをノックダウンした後に細胞増殖への影響を検出した結果(図5参照)、7本のlncRNA(DUXAP8、PCAT6,LINC00511、LINC00152、RP11-198G18.22、PVT1、CRNDE、図5の右側7本のlncRNA)のみHUH7細胞の増殖を抑制することができるが、このうち現在研究されていないlncRNAはわずか1つであった。このlncRNAは、lncRNA RP11-196G18.22(LETNと命名する)であり、また、以前にアルゴリズム予測で同定したそれによって制御される転写因子や標的遺伝子の数が最も多いlncRNAであり、肝臓癌の発生及び進行において幅広く強力な制御能を持つ可能性が示唆された。これらの知見に基づき、TCGAデータベースの肝臓癌とパラ癌におけるLETNの発現を解析したところ、肝臓癌組織ではパラ癌組織よりもLETNの発現が非常に高いことがわかった。これを他の癌組織に拡張したところ、ほとんどの癌でLETN発現がパラ癌組織よりも高いことがわかった(図1,A)。
HUH7(中科院上海セルバンク)とSMMC-7721(中科院上海セルバンク)の2種類の肝臓癌細胞系を選択し、LETNに対してsiRNAノックダウン(siRNAプライマーは下記に示す)を行うことにより、(細胞を35mmペトリ皿に接種し細胞接着後にトランスフェクションを行い、250ml培地を取って、それぞれ2ulリポソームLipofectamine 2000 (Thermo Fisher,11668019)及び20nMのsiRNAを希釈し、その後、それをよく混ぜ、20分間インキュベートし、その後、それを細胞含有ペトリ皿内にゆっくり滴って、48時間後に、RT-qPCR(プライマー対配列は下記の表2を参照)によりノックダウンレベルを検出し、ノックダウン効率について図4、Aを参照し、LETNのノックダウンが細胞の増殖を明らかに低下させることができる(図1,C)ことが発見された(siNC及びsiLMNAは2種類の異なる陰性対照であり、オフターゲットを防止するために、LETNに対して、2つのノックダウンsiRNA、即ちsiLETN-1及びsiLETN-2を設計した)。
アンチセンス 5'-3' AAGAAGACAUGGAGACAGC (SEQ ID NO: 35)
siLETN-2 センス 5'-3' GCUCUCUGCUCAAGUAUUA (SEQ ID NO: 36)
アンチセンス 5'-3' UAAUACUUGAGCAGAGAGC (SEQ ID NO: 37)
siNC(陰性対照) センス 5'-3' ACGUGACACGUUCGGAGAA (SEQ ID NO: 38)
アンチセンス 5'-3' UUCUCCGAACGUGUCACGU (SEQ ID NO: 39)
siLMNA センス 5'-3' AUCUCAUCCUGAAGUUGCUUC (SEQ ID NO: 40)
アンチセンス 5'-3' GAAGCAACUUCAGGAUGAGAU (SEQ ID NO: 41)
sgLETN-2 センス 5'-3' GAGACGATATGCTACGGGTG (SEQ ID NO: 71)
sgEV-1 センス 5'-3' GAACGTTGGCACTACTTCAC (SEQ ID NO: 72)
sgEV-2 センス 5'-3' GCGCCTTAAGAGTACTCATC (SEQ ID NO: 73)
さらに、LETNの腫瘍細胞の腫瘍形成能力への影響を研究した。まず、ウイルスでパッケージされたshRNAにより、LETNノックダウンの安定細胞株を構築した(shRNA配列は表3を参照。まず、ウイルスパッケージングし、293T細胞(中科院上海セルバンク)を100mm平皿に接種し、2日目トランスフェクションを行い、1ml培地を取って12ulリポソームLipofectamine 2000 (Thermo Fisher,11668019)を希釈し、1ml培地を取ってパッケージングベクター7.1ugΔ8.9(清華大学ライブラリプラットフォーム)及び3.55ug VSVG(清華大学ライブラリプラットフォーム)、並びに発現ベクター3ug plv-LETNを希釈し、その後、それをよく混ぜて、20分間インキュベートし、その後、それを細胞含有ペトリ皿内にゆっくり滴って、培地を加えて10mlまで補い、48時間後に上清を回収し、3000rpmで10分間遠心分離し、この上清はウイルス液であり、後で使用するために-80°Cで保存。LETNノックダウン細胞株の構築について、まず細胞を35mmペトリ皿に接種し、2日目500ulウィルス液を加え、培地を加えて2mlまで補い、48時間後にピューロマイシンを添加してウイルスが発現されていない細胞を除去し、ピューロマイシンを3日に1回添加し、このようにして必要な安定細胞株を取得した)、LETNノックダウンHUH7細胞系(RT-qPCRによりそのノックダウン効率を検出し、ノックダウン効率は図4,Bを参照)を、無胸腺のヌードマウス(BALB / cヌードマウス、オス、清華大学動物センター)の皮下に注入し、5週間の培育の後、腫瘍ブロックのサイズをバーニアキャリパーで毎週測定した。腫瘍を取り出してその重量及び体積を測定した結果、LETNノックダウン細胞で形成された腫瘍ブロックは対照組よりも明らかに小さく、その腫瘍の体積及び重量は対照組よりも大幅に小さいことが発見された(図1,H)。対応に、HUH7を選択してLETN過剰発現の安定細胞株を構築し、上記無胸腺ヌードマウスの皮下に注入し、5週間育成した後、同じ操作処理が行われた結果、LETN過剰発現の細胞で形成された腫瘍ブロックは対照組よりも明らかに高く、その腫瘍の体積及び重量も対照組の腫瘍よりも大幅に大きいことが発見された(図1,I)。要するに、細胞増殖、クローン形成、及び皮下腫瘍形成等の腫瘍指標により詳細な実験検証を行い、lncRNA LETNが、肝臓癌の発生及び進行を抑制する顕著な機能を有し、潜在的な治療ターゲットであると考えられる。
まず、RNAインサイチュハイブリダイゼーション[6]及び細胞核細胞質分離技術(Nuclear/Cytosol Fractionation Kit (Biovision,K266-25)) により、鑑定して、LETNが主に細胞核内に位置し、且つクラスターを形成していることが発見された。LETNがタンパク質と結合することにより機能する可能性が高いことが示唆される。RNA pull downにより、プルダウンされたタンパク質を質量分析して、LETNと互いに作用するタンパク質を見つけて、最終的に、NPM1がそれと結合される機能タンパク質であることを見つけた。その後、RNA pull down技術[7]により、NPM1抗体Anti-NPM1 (Abcam,ab10530)を利用してプルダウンを行い、NPM1が確かにlncRNA LETNをプルダウンすることができることも発見された。細胞蛍光共局在実験も、さらにLETNとNPM1が互いに結合し核小体内に位置することを証明した。
本発明は、上記実施形態を参照して具体的に説明されているが、本発明はこれらの特定の実施形態に限定されないことは、当業者には理解されるはずである。本明細書で教示された方法および技術的解決策に基づいて、当業者は、本発明の精神から逸脱することなく適切な修正または改良を行うことができ、その結果得られる同等の実施形態は本発明の範囲内である。
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Claims (10)
- lncRNA RP11-196G18.22(LETN)の発現レベルを検出する試薬の、癌の診断薬又は診断キットの製造における使用。
- 前記試薬が、lncRNA RP11-196G18.22に特異的なプローブ、遺伝子チップ又はPCRプライマーである、請求項1に記載の使用。
- 前記癌が固形腫瘍であり、好ましくは、肝臓癌、肺癌、前立腺癌、乳癌、前立腺癌、膵臓癌、腎臓癌、胃癌、軟部組織癌、胆道癌、膀胱癌、直腸癌、子宮内膜癌、頭頸部癌、結腸癌、食道癌及び甲状腺癌からなる群から選ばれるものである、請求項1に記載の使用。
- 前記lncRNA RP11-196G18.22のヌクレオチド配列が、SEQ ID NO: 1(Ensembl登録番号ENST00000564237.1)に示すものである、請求項1~3のいずれか一項に記載の使用。
- lncRNA RP11-196G18.22(LETN)の発現を低減又は抑制する薬剤の、癌治療薬の製造における使用。
- lncRNA RP11-196G18.22(LETN)の発現を低減又は抑制する薬剤が、Gapmer、アンチセンスRNA、siRNA、esiRNA、shRNA、miRNA、RNAアプタマー、TALEN、CRISPR及びジンクフィンガーヌクレアーゼからなる群から選ばれるものである、請求項5に記載の使用。
- 前記癌が固形腫瘍であり、好ましくは、肝臓癌、肺癌、前立腺癌、乳癌、前立腺癌、膵臓癌、腎臓癌、胃癌、軟部組織癌、胆道癌、膀胱癌、直腸癌、子宮内膜癌、頭頸部癌、結腸癌、食道癌及び甲状腺癌からなる群から選ばれるものである、請求項5に記載の使用。
- 前記lncRNA RP11-196G18.22のヌクレオチド配列が、SEQ ID NO:1(Ensembl登録番号ENST00000564237.1)に示すものである、請求項5に記載の使用。
- 前記癌治療薬が、他の抗癌剤、例えば化学療法薬、例えばNPM1発現又は変異を低減又は抑制する薬剤、或いはLETNとNPM1との結合を抑制する薬剤をさらに含む、請求項5~8のいずれか一項に記載の使用。
- 抗癌剤をスクリーニングする方法であって、
1)lncRNA RP11-196G18.22(LETN)を過剰発現している細胞におけるlncRNA RP11-196G18.22(LETN)の発現レベルを測定するステップと、
2)候補化合物をステップ1)の細胞と接触させるステップと、
3)ステップ2)の後の細胞におけるlncRNA RP11-196G18.22(LETN)の発現レベルを測定するステップと、
4)ステップ1)とステップ3)で測定されたlncRNA RP11-196G18.22(LETN)の発現レベルを比較するステップと、を含み、
lncRNA RP11-196G18.22(LETN)の発現レベルの低下は、前記候補化合物が抗癌性を有することを示し、
好ましくは、前記細胞は、癌細胞である、方法。
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