JP2021075473A - Agents for preventing and/or treating mesothelioma - Google Patents
Agents for preventing and/or treating mesothelioma Download PDFInfo
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- JP2021075473A JP2021075473A JP2019201816A JP2019201816A JP2021075473A JP 2021075473 A JP2021075473 A JP 2021075473A JP 2019201816 A JP2019201816 A JP 2019201816A JP 2019201816 A JP2019201816 A JP 2019201816A JP 2021075473 A JP2021075473 A JP 2021075473A
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Abstract
Description
本発明は、中皮腫の予防及び／又は治療剤、並びに診断剤に関する。 The present invention relates to prophylactic and / or therapeutic agents for mesothelioma, and diagnostic agents.
中皮腫（悪性中皮腫）は、胸腔や腹腔の表面を覆う中皮由来の腫瘍である。中皮腫は、その発生部位によって胸膜中皮腫、腹膜中皮腫、心膜中皮腫等に分類される。悪性胸膜中皮腫（以下、「MPM」ともいう。）の原因は、アスベスト（石綿）の吸入であることが多い。アスベストは、耐熱性、耐摩耗性等に優れ、かつ加工が容易で安価であるため、日本では1970年代から1990年代に建材、摩擦材、断熱材等をはじめ、様々な用途に用いられていた。通常、MPMはアスベスト曝露から約20〜40年後に発症するため、MPM患者は今後増加すると考えられている。 Mesothelioma (mesothelioma) is a tumor derived from the mesothelium that covers the surface of the thoracic cavity and abdominal cavity. Mesothelioma is classified into pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and the like according to the site of occurrence. The cause of malignant pleural mesothelioma (hereinafter, also referred to as "MPM") is often inhalation of asbestos (asbestos). Asbestos has excellent heat resistance, wear resistance, etc., is easy to process, and is inexpensive. Therefore, asbestos was used in various applications in Japan from the 1970s to the 1990s, including building materials, friction materials, and heat insulating materials. .. Since MPM usually develops about 20 to 40 years after asbestos exposure, it is expected that the number of MPM patients will increase in the future.
MPMの悪性度は極めて高いにも関わらず、有効な薬物療法がない。また、外科的治療は容易ではなく、たとえ切除できても術後のquality of life (QOL) の低下や低くない再発率が問題となっている。手術ができない場合や、手術前にがんを小さくする、症状を緩和するなどの目的で、化学療法が用いられている。現在、MPMの治療に主に使用される化学療法剤はシスプラチンとペメトレキセドである。 Despite the extremely high malignancy of MPM, there is no effective drug therapy. In addition, surgical treatment is not easy, and even if it can be resected, the postoperative quality of life (QOL) is lowered and the recurrence rate is not low. Chemotherapy is used when surgery is not possible, to reduce cancer before surgery, or to relieve symptoms. Currently, the main chemotherapeutic agents used to treat MPM are cisplatin and pemetrexed.
ところで、ゲノム上にコードされた内在性の20〜25塩基程度の非コード（non-coding）RNAであるマイクロRNA（miRNA）は、ヒトでは2000種類以上が知られており、それぞれが複数の標的遺伝子の発現を調節し、細胞の増殖や分化など、様々な生命現象に関与している。がん細胞の増殖に関与するmiRNAについても数多くの報告があり、miRNAの発現を調節することでがん細胞の増殖を抑制するがん治療法が提案されている。しかしながら、実験レベルでは、いくつかのmiRNAがMPM細胞に対して抗腫瘍効果を示したことが報告されてはいるものの（例えば、非特許文献１参照）、miRNAを用いた中皮腫の治療薬は未だ実用化されていない。
また、hsa-miR-1290、has-miR-345-5p及びhas-miR-4732-5pと、中皮腫との関連性についても何ら知られていない。
By the way, more than 2000 types of microRNAs (miRNAs), which are endogenous non-coding RNAs encoded on the genome of about 20 to 25 bases, are known in humans, and each of them has a plurality of targets. It regulates gene expression and is involved in various biological phenomena such as cell proliferation and differentiation. There are many reports on miRNAs involved in the growth of cancer cells, and cancer treatment methods that suppress the growth of cancer cells by regulating the expression of miRNAs have been proposed. However, at the experimental level, although some miRNAs have been reported to have antitumor effects on MPM cells (see, for example, Non-Patent Document 1), miRNA-based therapeutic agents for mesothelioma. Has not been put into practical use yet.
Also, nothing is known about the association between hsa-miR-1290, has-miR-345-5p and has-miR-4732-5p and mesothelioma.
本発明の目的は、中皮腫細胞の増殖を抑制し得るmiRNAを同定し、該miRNAを用いて新規な中皮腫の予防及び／又は治療手段を提供することである。本発明の別の目的は、中皮腫細胞で発現が抑制されているmiRNAを同定し、該miRNAの発現量を指標として、中皮腫を診断する手段を提供することである。 An object of the present invention is to identify miRNAs capable of suppressing the growth of mesothelioma cells and to provide novel prophylactic and / or therapeutic means for mesothelioma using the miRNAs. Another object of the present invention is to identify a miRNA whose expression is suppressed in mesothelioma cells, and to provide a means for diagnosing mesothelioma using the expression level of the miRNA as an index.
本発明者は、上記の目的を達成すべく鋭意検討を重ねた結果、正常胸膜中皮細胞と比べてMPM細胞で発現が低下している3種のmiRNA（hsa-miR-1290、hsa-miR-345-5p及びhsa-miR-4732-5p）を同定し、これらmiRNAの発現を指標として、中皮腫の診断や抗中皮腫薬のスクリーニングが可能であることを明らかにした。さらに、これらのmiRNAをMPM細胞に導入し過剰発現させたところ、hsa-miR-1290を導入したMPM細胞では、細胞増殖が有意に抑制されることを見出した。
本発明者は、これらの知見に基づいてさらに研究を重ねた結果、本発明を完成するに至った。
As a result of diligent studies to achieve the above object, the present inventor has three types of miRNAs (hsa-miR-1290 and hsa-miR) whose expression is lower in MPM cells than in normal pleural mesothelial cells. -345-5p and hsa-miR-4732-5p) were identified, and it was clarified that the expression of these miRNAs can be used as an index to diagnose mesothelioma and screen for anti-mesothelioma drugs. Furthermore, when these miRNAs were introduced into MPM cells and overexpressed, it was found that cell proliferation was significantly suppressed in MPM cells into which hsa-miR-1290 was introduced.
The present inventor has completed the present invention as a result of further research based on these findings.
即ち、本発明は以下に関する。
［１］中皮腫の予防及び／又は治療剤であって、
(a) 配列番号１で表されるmiR-1290のヌクレオチド配列を含む核酸、又は
(b) 上記(a)のヌクレオチド配列と70%以上、好ましくは80%以上、より好ましくは90%以上の同一性を有するヌクレオチド配列からなり、且つmiR-1290と同等の機能を有するヌクレオチドを含む核酸
を含有する、剤。
［２］核酸が１本鎖または２本鎖である、［１］に記載の剤。
［３］核酸が配列番号１で表されるヌクレオチド配列又はその部分配列からなるRNA、或いはその修飾体である、［１］に記載の剤。
［４］核酸が配列番号１で表されるヌクレオチド配列からなるRNA又はその修飾体である、［１］に記載の治療剤。
［５］核酸が、miR-1290及びその前駆体からなる群から選ばれる少なくとも１種の核酸である、［１］に記載の治療剤。
［６］前駆体が、miR-1290のpri-miRNAまたはpre-miRNAである、［５］に記載の治療剤。
［７］中皮腫が胸膜中皮腫である、［１］〜［６］のいずれかに記載の治療剤。
［８］中皮腫の予防及び／又は治療方法であって、
(a) 配列番号１で表されるmiR-1290のヌクレオチド配列を含む核酸、又は
(b) 上記(a)のヌクレオチド配列と70%以上、好ましくは80%以上、より好ましくは90%以上の同一性を有するヌクレオチド配列からなり、且つmiR-1290と同等の機能を有するヌクレオチドを含む核酸
の有効量を、対象に投与することを含む、方法。
［９］miR-1290を特異的に検出し得る核酸プローブ及び／又は核酸プライマー、miR-345-5pを特異的に検出し得る核酸プローブ及び／又は核酸プライマー、並びにmiR-4732-5pを特異的に検出し得る核酸プローブ及び／又は核酸プライマーからなる群より選択される1以上の核酸プローブ及び／又は核酸プライマーを含む、中皮腫の診断剤。
［１０］以下の工程を含む、抗中皮腫活性を有する物質のスクリーニング方法：
（１）被検物質と中皮腫細胞とを接触させること；
（２）被検物質を接触させた細胞におけるmiR-1290の発現レベルを測定し、該発現レベルを、被検物質を接触させない対照細胞におけるmiR-1290の発現レベルと比較すること；並びに
（３）上記（２）の比較結果に基づいて、miR-1290の発現レベルを増大させた被検物質を、抗中皮腫活性を有する物質として選択すること。
That is, the present invention relates to the following.
[1] A prophylactic and / or therapeutic agent for mesothelioma.
(a) Nucleic acid containing the nucleotide sequence of miR-1290 represented by SEQ ID NO: 1 or
(b) Consists of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more identity with the nucleotide sequence of (a) above, and contains a nucleotide having a function equivalent to that of miR-1290. An agent containing a nucleic acid.
[2] The agent according to [1], wherein the nucleic acid is single-stranded or double-stranded.
[3] The agent according to [1], wherein the nucleic acid is RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a partial sequence thereof, or a modified product thereof.
[4] The therapeutic agent according to [1], wherein the nucleic acid is RNA consisting of the nucleotide sequence represented by SEQ ID NO: 1 or a modified product thereof.
[5] The therapeutic agent according to [1], wherein the nucleic acid is at least one nucleic acid selected from the group consisting of miR-1290 and its precursor.
[6] The therapeutic agent according to [5], wherein the precursor is miR-1290 pri-miRNA or pre-miRNA.
[7] The therapeutic agent according to any one of [1] to [6], wherein the mesothelioma is pleural mesothelioma.
[8] A method for preventing and / or treating mesothelioma.
(a) Nucleic acid containing the nucleotide sequence of miR-1290 represented by SEQ ID NO: 1 or
(b) Consists of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more identity with the nucleotide sequence of (a) above, and contains a nucleotide having a function equivalent to that of miR-1290. A method comprising administering to a subject an effective amount of nucleic acid.
[9] A nucleic acid probe and / or a nucleic acid primer capable of specifically detecting miR-1290, a nucleic acid probe and / or a nucleic acid primer capable of specifically detecting miR-345-5p, and a specific miR-4732-5p. A diagnostic agent for mesenteric tumors, which comprises one or more nucleic acid probes and / or nucleic acid primers selected from the group consisting of nucleic acid probes and / or nucleic acid primers that can be detected in.
[10] A method for screening a substance having anti-mesothelioma activity, which comprises the following steps:
(1) Contacting the test substance with mesothelioma cells;
(2) Measure the expression level of miR-1290 in cells contacted with the test substance and compare the expression level with the expression level of miR-1290 in the control cells not contacted with the test substance; and (3). ) Based on the comparison result of (2) above, a test substance having an increased expression level of miR-1290 should be selected as a substance having anti-mesothelioma activity.
本発明によれば、中皮腫細胞の増殖を抑制することができる。しかも、有効成分であるmiR-1290は、正常な胸膜中皮細胞で発現しているので、投与したmiR-1290が正常細胞に取り込まれても、タンパク質の発現量に大きな変化は起こらず、細胞障害が生じにくい、即ち、副作用が少ないことが期待できる。 According to the present invention, the proliferation of mesothelioma cells can be suppressed. Moreover, since the active ingredient miR-1290 is expressed in normal pleural mesothelial cells, even if the administered miR-1290 is taken up by normal cells, the protein expression level does not change significantly, and the cells It can be expected that the disorder is less likely to occur, that is, there are few side effects.
以下、本発明を説明する。本明細書において使用される用語は、特に言及しない限り、本発明が属する技術分野で通常に用いられる意味を有する。 Hereinafter, the present invention will be described. Unless otherwise specified, the terms used herein have meanings commonly used in the art to which the present invention belongs.
１．本発明の剤
本発明は、中皮腫の予防及び／又は治療剤であって、
(a) 配列番号１で表されるmiR-1290のヌクレオチド配列を含む核酸、又は
(b) 上記(a)のヌクレオチド配列と70%以上の同一性を有するヌクレオチド配列からなり、且つmiR-1290と同等の機能を有するヌクレオチドを含む核酸
を含む、剤（以下、「本発明の剤」ともいう。）を提供する。上記(a)及び(b)の核酸を包括して「本発明の核酸」ともいう。
1. 1. Agent of the present invention The present invention is a prophylactic and / or therapeutic agent for mesothelioma.
(a) Nucleic acid containing the nucleotide sequence of miR-1290 represented by SEQ ID NO: 1 or
(b) An agent comprising a nucleic acid containing a nucleotide having 70% or more identity with the nucleotide sequence of (a) above and having a function equivalent to that of miR-1290 (hereinafter, "the agent of the present invention"). Also called.). The nucleic acids of (a) and (b) above are also collectively referred to as "nucleic acid of the present invention".
本発明の核酸は、RNA、RNAとDNAのキメラ核酸（以下、キメラ核酸と称する）またはハイブリッド核酸である。ここにおいて、キメラ核酸とは、１本鎖又は２本鎖の核酸において一本の核酸の中にRNAとDNAを含むことをいい、ハイブリッド核酸とは、二本鎖において、一方の鎖がRNAまたはキメラ核酸でもう一方の鎖がDNAまたはキメラ核酸である核酸をいう。 The nucleic acid of the present invention is RNA, a chimeric nucleic acid of RNA, RNA and DNA (hereinafter referred to as chimeric nucleic acid) or a hybrid nucleic acid. Here, the chimeric nucleic acid means that RNA and DNA are contained in one nucleic acid in a single-stranded or double-stranded nucleic acid, and a hybrid nucleic acid means that in a double-stranded nucleic acid, one strand is RNA or A nucleic acid in which the other strand is DNA or a chimeric nucleic acid.
本発明の核酸は、１本鎖または２本鎖である。２本鎖の態様には、２本鎖RNA、２本鎖キメラ核酸、RNA／DNAハイブリッド、RNA／キメラ核酸ハイブリッド、キメラ核酸／キメラ核酸ハイブリッドおよびキメラ核酸／DNAハイブリッドが含まれる。本発明の核酸は、好ましくは１本鎖RNA、１本鎖キメラ核酸、２本鎖RNA、２本鎖キメラ核酸、RNA／DNAハイブリッド、RNA／キメラ核酸ハイブリッド、キメラ核酸／キメラ核酸ハイブリッドまたはキメラ核酸／DNAハイブリッドであり、より好ましくは１本鎖RNA、１本鎖キメラ核酸、２本鎖RNA、２本鎖キメラ核酸、RNA／DNAハイブリッド、キメラ核酸／キメラ核酸ハイブリッドまたはRNA／キメラ核酸ハイブリッドである。 The nucleic acid of the present invention is single-stranded or double-stranded. Double-stranded embodiments include double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid and chimeric nucleic acid / DNA hybrid. The nucleic acid of the present invention is preferably single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, RNA / chimeric nucleic acid hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid or chimeric nucleic acid. / DNA hybrid, more preferably single-stranded RNA, single-stranded chimeric nucleic acid, double-stranded RNA, double-stranded chimeric nucleic acid, RNA / DNA hybrid, chimeric nucleic acid / chimeric nucleic acid hybrid or RNA / chimeric nucleic acid hybrid. ..
本発明の核酸の長さは、哺乳動物（好ましくはヒト）の中皮腫細胞、好ましくはMPM細胞の増殖を抑制する活性を有する限り、その長さに上限はない。しかし、合成の容易さや抗原性の問題等を考慮すると、本発明の核酸の長さは、例えば約200塩基以下、好ましくは約130塩基以下、より好ましくは約50塩基以下であり、最も好ましくは30塩基以下である。下限としては、例えば15塩基以上、好ましくは、17塩基以上である。なお、核酸がステム-ループ型の２次構造をとることによりハイブリッドを形成する場合の核酸の長さは、該ハイブリッドの片側鎖の長さとして考えるものとする。 There is no upper limit to the length of the nucleic acid of the present invention as long as it has an activity of suppressing the growth of mammalian (preferably human) mesothelioma cells, preferably MPM cells. However, considering the ease of synthesis, the problem of antigenicity, etc., the length of the nucleic acid of the present invention is, for example, about 200 bases or less, preferably about 130 bases or less, more preferably about 50 bases or less, and most preferably. It is 30 bases or less. The lower limit is, for example, 15 bases or more, preferably 17 bases or more. The length of the nucleic acid when the nucleic acid forms a hybrid by adopting a stem-loop type secondary structure is considered as the length of one side strand of the hybrid.
miR-1290は既知の分子であり、miRBaseにはヒト（hsa-miR-1290; Accession No. MI0006352）、チンパンジー（ptr-miR-1290; Accession No. MI0008491）、ボルネオオランウータン（ppy-miR-1290; Accession No. MI0015246）のpre-miRNA配列がエントリーされており、いずれも成熟型miRNAは、配列番号１（ヒト成熟型miR-1290のAccession No.はMIMAT0005880)で表されるヌクレオチド配列からなる。即ち、成熟型miR-1290とは、配列番号１で表されるヌクレオチド配列からなる１本鎖または２本鎖の核酸分子を意味する。 miR-1290 is a known molecule, and miRBase includes humans (hsa-miR-1290; Accession No. MI0006352), chimpanzees (ptr-miR-1290; Accession No. MI0008491), and Bornean orangutans (ppy-miR-1290; The pre-miRNA sequence of Accession No. MI0015246) has been entered, and the mature miRNA consists of the nucleotide sequence represented by SEQ ID NO: 1 (Accession No. of human mature miR-1290 is MIMAT0005880). That is, the mature miR-1290 means a single-stranded or double-stranded nucleic acid molecule consisting of the nucleotide sequence represented by SEQ ID NO: 1.
miR-1290は、中皮腫細胞内へ取り込まれると、該細胞の増殖を抑制する活性を有する。本発明において、「miR-1290と同等の機能を有する」とは、中皮腫細胞の増殖を抑制する活性を有することを意味する。当該活性を有する限り、その程度は問わない。マイクロRNAは標的mRNAとハイブリッドを形成し、タンパク質への翻訳を抑制したり、標的mRNAを切断したりして、その機能を抑制する。従って、miR-1290と同等の機能を有するヌクレオチドとしては、miR-1290の標的mRNAと、生理的な条件（例えば0.1M リン酸緩衝液 (pH7.0) 25℃）下でハイブリッドを形成するヌクレオチドが挙げられる。miR-1290の標的mRNAは、例えば、TagetScan、TargetMiner、miRDB等の各種アルゴリズムを用いて検索することができる。 When miR-1290 is taken up into mesothelioma cells, it has an activity of suppressing the proliferation of the cells. In the present invention, "having a function equivalent to that of miR-1290" means having an activity of suppressing the proliferation of mesothelioma cells. As long as it has the activity, the degree does not matter. MicroRNA hybridizes with the target mRNA and suppresses its function by suppressing translation into protein or cleaving the target mRNA. Therefore, as a nucleotide having the same function as miR-1290, a nucleotide that hybridizes with the target mRNA of miR-1290 under physiological conditions (for example, 0.1 M phosphate buffer (pH 7.0) 25 ° C). Can be mentioned. The target mRNA of miR-1290 can be searched by using various algorithms such as TagetScan, TargetMiner, and miRDB.
本発明において用いられる「miR-1290と同等の機能を有するヌクレオチド」のヌクレオチド配列は、配列番号１で表されるヌクレオチド配列と70%以上、好ましくは80%以上、より好ましくは90%以上、さらに好ましくは95%以上の同一性を有する。 The nucleotide sequence of the "nucleotide having the same function as miR-1290" used in the present invention is 70% or more, preferably 80% or more, more preferably 90% or more, and further, the nucleotide sequence represented by SEQ ID NO: 1. It preferably has 95% or more identity.
「同一性」とは、当該技術分野において公知の数学的アルゴリズムを用いて２つのヌクレオチド配列をアラインさせた場合の、最適なアラインメント（好ましくは、該アルゴリズムは最適なアラインメントのために配列の一方もしくは両方へのギャップの導入を考慮し得るものである）における、オーバーラップする全ヌクレオチド残基に対する、同一ヌクレオチド残基の割合（％）を意味する。 "Identity" is the optimal alignment when two nucleotide sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm is one of the sequences for optimal alignment or one of the sequences. It means the ratio (%) of the same nucleotide residue to the total number of overlapping nucleotide residues in which the introduction of a gap in both can be considered).
本明細書において、ヌクレオチド配列における同一性は、例えば相同性計算アルゴリズムNCBI BLAST-2（National Center for Biotechnology Information Basic Local Alignment Search Tool）を用い、以下の条件（ギャップオープン＝5ペナルティ；ギャップエクステンション＝2ペナルティ；ｘ＿ドロップオフ＝50；期待値＝10；フィルタリング＝ON）にて２つのヌクレオチド配列をアラインすることにより、計算することができる。 In the present specification, for the identity in the nucleotide sequence, for example, the homology calculation algorithm NCBI BLAST-2 (National Center for Biotechnology Information Basic Local Alignment Search Tool) is used, and the following conditions (gap open = 5 penalty; gap extension = 2) are used. It can be calculated by aligning the two nucleotide sequences with a penalty; x_dropoff = 50; expected value = 10; filtering = ON).
配列番号１で表されるヌクレオチド配列と70%以上の同一性を有するヌクレオチド配列としては、配列番号１で表されるヌクレオチド配列において１もしくは複数のヌクレオチドが欠失、置換、挿入または付加されたヌクレオチド配列、例えば、(1)配列番号１で表されるヌクレオチド配列中の１〜６個（好ましくは１〜４個、より好ましくは１または２個、さらに好ましくは１個）のヌクレオチドが欠失したヌクレオチド配列、(2)配列番号１で表されるヌクレオチド配列に１〜６個（好ましくは１〜４個、より好ましくは１または２個、さらに好ましくは１個）のヌクレオチドが付加されたヌクレオチド配列、(3)配列番号１で表されるヌクレオチド配列に１〜６個（好ましくは１〜４個、より好ましくは１または２個、さらに好ましくは１個）のヌクレオチドが挿入されたアミノ酸配列、(4)配列番号１で表されるヌクレオチド配列中の１〜６個（好ましくは１〜４個、より好ましくは１または２個、さらに好ましくは１個）のヌクレオチドが他のヌクレオチドで置換されたヌクレオチド配列、または(5)上記(1)〜(4)の変異が組み合わせれたヌクレオチド配列（この場合、変異したヌクレオチドの総和が、１〜６個、好ましくは１〜４個、より好ましくは１または２個、さらに好ましくは１個）である。 As a nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, one or more nucleotides are deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 1. The sequence, for example, (1) 1 to 6 (preferably 1 to 4, more preferably 1 or 2, more preferably 1) nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 was deleted. Nucleotide sequence, (2) Nucleotide sequence in which 1 to 6 (preferably 1 to 4, more preferably 1 or 2, still more preferably 1) nucleotides are added to the nucleotide sequence represented by SEQ ID NO: 1. , (3) An amino acid sequence in which 1 to 6 (preferably 1 to 4, more preferably 1 or 2, more preferably 1) nucleotides are inserted into the nucleotide sequence represented by SEQ ID NO: 1. 4) A nucleotide in which 1 to 6 (preferably 1 to 4, more preferably 1 or 2, still more preferably 1) nucleotides in the nucleotide sequence represented by SEQ ID NO: 1 are replaced with other nucleotides. Sequence, or (5) Nucleotide sequence in which the mutations of (1) to (4) above are combined (in this case, the total number of mutated nucleotides is 1 to 6, preferably 1 to 4, more preferably 1 or Two, more preferably one).
マイクロRNAは5’末端側2〜8番目のヌクレオチド配列（シード領域）が標的mRNAとのハイブリッド形成・翻訳抑制に主に寄与するので（Current Biology, 15, R458-R460 (2005)）、配列番号１で表されるヌクレオチド配列中2〜8番目のヌクレオチド配列は保存されることが望ましい。 Since the 2nd to 8th nucleotide sequences (seed regions) on the 5'-terminal side of microRNA mainly contribute to the suppression of hybrid formation and translation with the target mRNA (Current Biology, 15, R458-R460 (2005)), SEQ ID NO: It is desirable that the 2nd to 8th nucleotide sequences in the nucleotide sequence represented by 1 are preserved.
一実施態様において、「配列番号１で表されるヌクレオチド配列において１もしくは複数のヌクレオチドが欠失、置換、挿入または付加されたヌクレオチド配列」は、成熟型miR-1290のアイソフォーム（isomiR）であり得る。miR-1290のisomiRとしては、中国科学技術大学から提供されるIsomiR Bank（Bioinformatics, 32(13): 2069-2071 (2016)）にエントリーされているヌクレオチド配列からなるヌクレオチドが挙げられる。 In one embodiment, "a nucleotide sequence in which one or more nucleotides are deleted, substituted, inserted or added in the nucleotide sequence represented by SEQ ID NO: 1" is an isoform (isomiR) of mature miR-1290. obtain. The isomiR of miR-1290 includes nucleotides consisting of nucleotide sequences entered in IsomiR Bank (Bioinformatics, 32 (13): 2069-2071 (2016)) provided by the University of Science and Technology of China.
好ましい一実施態様において、配列番号１で表されるヌクレオチド配列と70%以上の同一性を有するヌクレオチド配列は、配列番号１で表されるヌクレオチド配列に含まれる連続する15塩基以上（好ましくは16塩基以上、より好ましくは17塩基以上、さらに好ましくは18塩基）の部分配列である。 In a preferred embodiment, the nucleotide sequence having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 is 15 or more consecutive bases (preferably 16 bases) contained in the nucleotide sequence represented by SEQ ID NO: 1. The above is a partial sequence of 17 bases or more, more preferably 18 bases).
好ましい実施態様において、本発明の核酸は、RISC複合体に取り込まれて標的mRNAに作用する。本発明の核酸が効率よくRISC複合体に取り込まれて成熟miR-1290にプロセッシングされるためには、該核酸は2本鎖核酸（ステム-ループ構造をとることで2重鎖を形成する1本鎖核酸を含む）であることが好ましい。 In a preferred embodiment, the nucleic acids of the invention are incorporated into the RISC complex and act on the target mRNA. In order for the nucleic acid of the present invention to be efficiently incorporated into the RISC complex and processed into mature miR-1290, the nucleic acid is a double-stranded nucleic acid (one that forms a double strand by adopting a stem-loop structure). (Contains chain nucleic acid) is preferable.
本発明の核酸が2本鎖核酸（ステム-ループ構造をとることで2重鎖を形成する1本鎖核酸を含む）である場合、相補鎖（パッセンジャー鎖）配列は、配列番号１で表されるヌクレオチド配列又はそれと70%以上の同一性を有するヌクレオチド配列に対して、完全相補的であってもよいし、ミスマッチを含んでもよい。ミスマッチの数としては、例えば、１〜１０個、好ましくは１〜８個、より好ましくは１〜６個が挙げられる。但し、効率的な標的mRNAの機能抑制のためには、標的nRNAとのハイブリッド形成の安定性だけでなく、2本鎖miRNAの5’末端側の5ヌクレオチドの対合安定性が寄与するとされているので、当該領域中にはミスマッチを含まないことが望ましい。 When the nucleic acid of the present invention is a double-stranded nucleic acid (including a single-stranded nucleic acid that forms a double strand by adopting a stem-loop structure), the complementary strand (passenger strand) sequence is represented by SEQ ID NO: 1. It may be completely complementary to a nucleotide sequence or a nucleotide sequence having 70% or more identity with the same, or may contain a mismatch. Examples of the number of mismatches include 1 to 10, preferably 1 to 8, and more preferably 1 to 6. However, it is said that not only the stability of hybrid formation with the target nRNA but also the pairing stability of the 5 nucleotides on the 5'end side of the double-stranded miRNA contribute to the efficient suppression of the function of the target mRNA. Therefore, it is desirable that the region does not include a mismatch.
本明細書において、核酸が「miR-1290」や「miR-1290と同等の機能を有するヌクレオチド」を含むとは、該核酸のヌクレオチド配列中に「miR-1290」や「miR-1290と同等の機能を有するヌクレオチド」のヌクレオチド配列が含まれることを意味する。 In the present specification, the inclusion of "miR-1290" or "nucleotide having a function equivalent to miR-1290" in a nucleic acid is equivalent to "miR-1290" or "miR-1290" in the nucleotide sequence of the nucleic acid. It means that the nucleotide sequence of "functional nucleotide" is included.
天然型の核酸は、細胞中に存在する核酸分解酵素によって容易に分解されるので、本発明の核酸は、各種分解酵素に対して抵抗性となるように修飾し、修飾体としてもよい。本発明の修飾体には、配列番号１で表されるヌクレオチド配列と70%以上の同一性を有し、且つ、miR-1290と同等の機能を有するヌクレオチドの範囲で、配列の修飾も含めた各種修飾をされた修飾体が含まれる。修飾体における修飾の例としては、例えば、糖鎖部分が修飾されているもの（例えば、2’-Oメチル化）、塩基部分が修飾されているもの、リン酸部分やヒドロキシル部分が修飾されているもの（例えば、ビオチン、アミノ基、低級アルキルアミン基、アセチル基等）を挙げることができるが、これに限定されない。 Since the natural nucleic acid is easily degraded by the nucleic acid degrading enzyme present in the cell, the nucleic acid of the present invention may be modified so as to be resistant to various degrading enzymes to be a modified product. The modified product of the present invention includes modification of the sequence within the range of nucleotides having 70% or more identity with the nucleotide sequence represented by SEQ ID NO: 1 and having the same function as miR-1290. Various modified modified products are included. Examples of modifications in the modified product include those in which the sugar chain moiety is modified (for example, 2'-O methylation), those in which the base moiety is modified, and those in which the phosphoric acid moiety and the hydroxyl moiety are modified. Examples include, but are not limited to, biotin, amino groups, lower alkylamine groups, acetyl groups, etc.
本発明の核酸は、5’または3’末端に、付加的な塩基を有していてもよい。該付加的塩基の長さは、通常5塩基以下である。該付加的塩基は、DNAでもRNAでもよいが、DNAを用いると核酸の安定性を向上させることができる場合がある。このような付加的塩基の配列としては、例えばug-3’、uu-3’、tg-3’、tt-3’、ggg-3’、guuu-3’、gttt-3’、ttttt-3’、uuuuu-3’などの配列が挙げられるが、これらに限定されるものではない。 The nucleic acid of the present invention may have an additional base at the 5'or 3'end. The length of the additional base is usually 5 bases or less. The additional base may be DNA or RNA, but DNA may be used to improve the stability of the nucleic acid. Examples of such additional base sequences include ug-3', uu-3', tg-3', tt-3', ggg-3', guuu-3', gttt-3', and ttttt-3. Sequences such as', uuuuu-3'can be mentioned, but are not limited to these.
本発明の核酸の好ましい態様としては、成熟型miR-1290、その前駆体等の核酸を挙げることができる。本発明の核酸の好ましい別の態様としては、成熟型miRNAと同様の活性を有するヌクレオチドを含む核酸、例えば、内在性の成熟型miR-1290を模倣するように合成された、miR-1290 mimicなどを使用することができる。市販のものを利用することもできる。例えば、AmbionTM Pre-miRTM miRNA precursor moleculeが例示できる。 Preferred embodiments of the nucleic acid of the present invention include nucleic acids such as mature miR-1290 and its precursors. Another preferred embodiment of the nucleic acid of the invention is a nucleic acid containing a nucleotide having activity similar to that of a mature miRNA, such as miR-1290 mimic synthesized to mimic the endogenous mature miR-1290. Can be used. Commercially available products can also be used. For example, Ambion TM Pre-miR TM miRNA precursor molecule can be exemplified.
miR-1290の前駆体とは、細胞内のプロセシングや、２本鎖核酸の開裂の結果、細胞内において成熟型miR-1290を生じ得る核酸を意味する。該前駆体としては、miR-1290のpri-miRNAやpre-miRNA等を挙げることができる。pri-miRNAはmiRNA遺伝子の一次転写産物（1本鎖RNA）であり、通常数百〜数千塩基程度の長さを有する。pre-miRNAは、pri-miRNAが細胞内プロセシングを受けることにより生じるヘアピン構造を有する1本鎖RNAであり、通常70〜110塩基の長さを有する。
miR-1290のpri-miRNAやpre-miRNAは公知の分子であり、例えばサンガー研究所が作成しているmiRBaseデータベース等に開示されている。miR-1290の好適なpre-miRNAとしては、配列番号２（Accession No. MI0006352）で表されるヒトpre-miR-1290（hsa-miR-1290）のヌクレオチド配列からなる1本鎖RNAを挙げることが出来る。
The precursor of miR-1290 means a nucleic acid that can give rise to mature miR-1290 in the cell as a result of intracellular processing or cleavage of the double-stranded nucleic acid. Examples of the precursor include miR-1290 pri-miRNA and pre-miRNA. pri-miRNA is a primary transcript (single-strand RNA) of the miRNA gene and usually has a length of several hundred to several thousand bases. Pre-miRNA is a single-stranded RNA having a hairpin structure generated by subjecting pri-miRNA to intracellular processing, and usually has a length of 70 to 110 bases.
The pri-miRNA and pre-miRNA of miR-1290 are known molecules and are disclosed in, for example, the miRBase database created by the Sanger Institute. Suitable pre-miRNAs of miR-1290 include single-stranded RNA consisting of the nucleotide sequence of human pre-miR-1290 (hsa-miR-1290) represented by SEQ ID NO: 2 (Accession No. MI0006352). Can be done.
また、ループ部分を介して、例えば、配列番号１で表されるヌクレオチド配列（第１の配列）と、その相補配列（第２の配列）とが連結された一本鎖核酸であって、ステム-ループ型の構造をとることにより、第１の配列が第２の配列と２本鎖構造状の形状を有する人工核酸も本発明の核酸の好ましい態様の１つである。この場合、ループ部分は、3〜10ヌクレオチド程度の任意のヌクレオチド配列であり得る。 Further, it is a single-stranded nucleic acid in which, for example, the nucleotide sequence represented by SEQ ID NO: 1 (first sequence) and its complementary sequence (second sequence) are linked via a loop portion, and is a stem. -An artificial nucleic acid having a loop-type structure in which the first sequence has a double-stranded structure with the second sequence is also one of the preferred embodiments of the nucleic acid of the present invention. In this case, the loop moiety can be any nucleotide sequence of about 3-10 nucleotides.
本発明の核酸は、従来公知の手法を用いて、哺乳動物細胞（ヒト細胞等）から単離することにより、または化学的に合成することにより、または遺伝子組み換え技術を用いて産生することにより得ることができる。また、適宜市販されている核酸を用いることも可能である。 The nucleic acid of the present invention is obtained by isolating it from mammalian cells (human cells, etc.) using a conventionally known method, by chemically synthesizing it, or by producing it using a genetic recombination technique. be able to. It is also possible to use commercially available nucleic acids as appropriate.
本発明の核酸は、適切な発現ベクターのプロモーターの下流に挿入することにより、細胞内に導入して転写されることにより生合成される形態として提供することもできる。発現ベクターとしては、宿主細胞において機能的なプロモーターを含有しているものが用いられる。プロモーターとしては、例えば、RNA polymerase II (pol II)系プロモーターやRNA polymerase III (pol III)系プロモーター等をあげることができる。pol II系プロモーターとしては、例えば、サイトメガロウイルス（ヒトCMV）のIE(immediate early)遺伝子のプロモーター、SV40の初期プロモーター等をあげることができる。それらを用いた発現ベクターとして、例えば、pCDNA6.2-GW/miR（Invitrogen社製）、pSilencer 4.1-CMV（Ambion社製）等を例示することができる。pol III系プロモーターとしては、例えば、U6 RNAやH1 RNAあるいはtRNAのプロモーターをあげることができる。それらを用いた発現ベクターとして、例えば、pSINsi-hH1 DNA（タカラバイオ社製）、pSINsi-hU6 DNA（タカラバイオ社製）、pENTR/U6（Invitrogen社製）等をあげることができる。 The nucleic acid of the present invention can also be provided as a form that is biosynthesized by being introduced into cells and transcribed by inserting it downstream of the promoter of an appropriate expression vector. As the expression vector, a vector containing a functional promoter in the host cell is used. Examples of the promoter include RNA polymerase II (pol II) -based promoters and RNA polymerase III (pol III) -based promoters. Examples of the pol II promoter include the promoter of the IE (immediate early) gene of cytomegalovirus (human CMV) and the early promoter of SV40. Examples of expression vectors using them include pCDNA6.2-GW / miR (manufactured by Invitrogen) and pSilencer 4.1-CMV (manufactured by Ambion). Examples of the pol III promoter include U6 RNA, H1 RNA, and tRNA promoters. Examples of expression vectors using them include pSINsi-hH1 DNA (manufactured by Takara Bio Inc.), pSINsi-hU6 DNA (manufactured by Takara Bio Inc.), pENTR / U6 (manufactured by Invitrogen), and the like.
あるいは、ウイルスベクター内のプロモーター下流に本発明の核酸を挿入して組換えウイルスベクターを作製し、該ベクターをパッケージング細胞に導入して組換えウイルスを産生し、該ウイルスを宿主細胞に感染させることにより、該宿主細胞内に本発明の核酸を導入・発現させることもできる。
パッケージング細胞はウイルスのパッケジ−ングに必要なタンパク質をコードする遺伝子のいずれかを欠損している組換えウイルスベクターの、該欠損するタンパク質を補給できる細胞であればいずれの細胞でもよく、例えばヒト腎臓由来のHEK293細胞、マウス繊維芽細胞NIH3T3などを用いることができる。パッケージング細胞で補給するタンパク質としては、レトロウイルスベクターの場合はマウスレトロウイルス由来のgag、pol、envなどのタンパク質、レンチウイルスベクターの場合はHIVウイルス由来のgag、pol、env、vpr、vpu、vif、tat、rev、nefなどのタンパク質、アデノウイルスベクターの場合はアデノウイルス由来のE1A,E1Bなどのタンパク質、また、アデノ随伴ウイルスベクターの場合はRep(p5、p19、p40)、Vp(Cap)などのタンパク質を用いることができる。
Alternatively, the nucleic acid of the present invention is inserted downstream of the promoter in the viral vector to prepare a recombinant viral vector, and the vector is introduced into packaging cells to produce a recombinant virus, which infects host cells. Thereby, the nucleic acid of the present invention can be introduced and expressed in the host cell.
The packaging cell may be any cell of the recombinant viral vector lacking any of the genes encoding the protein required for virus packaging, as long as it can supplement the defective protein, for example, human. HEK293 cells derived from the kidney, mouse fibroblasts NIH3T3, etc. can be used. Proteins supplemented by packaging cells include proteins such as mouse retrovirus-derived gag, pol, and env in the case of retroviral vectors, and HIV virus-derived gag, pol, env, vpr, vpu, in the case of lentiviral vectors. Proteins such as vif, tat, rev, nef, proteins such as E1A, E1B derived from adenovirus in the case of adenovirus vectors, and Rep (p5, p19, p40), Vp (Cap) in the case of adeno-related viral vectors. And other proteins can be used.
本発明の剤は、有効量の本発明の核酸に加え、任意の担体、例えば医薬上許容される担体を含むことができ、医薬組成物の形態で医薬として適用される。 In addition to an effective amount of the nucleic acid of the present invention, the agent of the present invention can contain any carrier, for example, a pharmaceutically acceptable carrier, and is applied pharmaceutically in the form of a pharmaceutical composition.
医薬上許容される担体としては、例えば、ショ糖、デンプン等の賦形剤、セルロース、メチルセルロース等の結合剤、デンプン、カルボキシメチルセルロース等の崩壊剤、ステアリン酸マグネシウム、エアロジル等の滑剤、クエン酸、メントール等の芳香剤、安息香酸ナトリウム、亜硫酸水素ナトリウム等の保存剤、クエン酸、クエン酸ナトリウム等の安定剤、メチルセルロース、ポリビニルピロリド等の懸濁剤、界面活性剤等の分散剤、水、生理食塩水等の希釈剤、ベースワックス等が挙げられるが、それらに限定されるものではない。 Pharmaceutically acceptable carriers include, for example, excipients such as sucrose and starch, binders such as cellulose and methyl cellulose, disintegrants such as starch and carboxymethyl cellulose, lubricants such as magnesium stearate and aerodyl, citric acid, etc. Fragrances such as menthol, preservatives such as sodium benzoate and sodium hydrogen sulfite, stabilizers such as citric acid and sodium citrate, suspending agents such as methylcellulose and polyvinylpyrrolid, dispersants such as surfactants, water, Diluting agents such as physiological saline, base wax and the like can be mentioned, but the present invention is not limited thereto.
本発明の核酸の中皮腫細胞内への導入を促進するために、本発明の剤は更に核酸導入用試薬を含むことができる。該核酸導入用試薬としては、アテロコラーゲン；リポソーム；ナノパーティクル；リポフェクチン、リプフェクタミン（lipofectamine）、DOGS（トランスフェクタム）、DOPE、DOTAP、DDAB、DHDEAB、HDEAB、ポリブレン、あるいはポリ（エチレンイミン）(PEI)等の陽イオン性脂質等を用いることが出来る。 In order to promote the introduction of the nucleic acid of the present invention into mesothelioma cells, the agent of the present invention may further contain a reagent for introducing nucleic acid. Reagents for introducing nucleic acids include atelocollagen; liposomes; nanoparticles; lipofectin, lipofectamine, DOGS (transfectum), DOPE, DOTAP, DDAB, DHDEAB, HDEAB, polybrene, or poly (ethyleneimine) (PEI). And the like, cationic lipids and the like can be used.
本発明の剤は、経口的にまたは非経口的に、哺乳動物に対して投与することが可能であるが、本発明の剤は、非経口的に投与するのが望ましい。 Although the agent of the present invention can be administered orally or parenterally to mammals, it is desirable that the agent of the present invention be administered parenterally.
非経口的な投与（例えば、皮下注射、筋肉注射、局所注入、腹腔内投与など）に好適な製剤としては、水性および非水性の等張な無菌の注射液剤があり、これには抗酸化剤、緩衝液、制菌剤、等張化剤等が含まれていてもよい。また、水性および非水性の無菌の懸濁液剤が挙げられ、これには懸濁剤、可溶化剤、増粘剤、安定化剤、防腐剤等が含まれていてもよい。当該製剤は、アンプルやバイアルのように単位投与量あるいは複数回投与量ずつ容器に封入することができる。また、有効成分および医薬上許容される担体を凍結乾燥し、使用直前に適当な無菌のビヒクルに溶解または懸濁すればよい状態で保存することもできる。 Suitable formulations for parenteral administration (eg, subcutaneous, intramuscular, topical, intraperitoneal, etc.) include aqueous and non-aqueous isotonic sterile injections, which are antioxidants. , Buffer solution, antibacterial agent, isotonic agent and the like may be contained. Also included are aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizing agents, thickeners, stabilizers, preservatives and the like. The pharmaceutical product can be encapsulated in a container in a unit dose or a plurality of doses like an ampoule or a vial. In addition, the active ingredient and a pharmaceutically acceptable carrier can be freeze-dried and stored in a state where it can be dissolved or suspended in a suitable sterile vehicle immediately before use.
医薬組成物中の本発明の核酸の含有量は、例えば、医薬組成物全体の約0.1ないし100重量%である。 The content of the nucleic acid of the present invention in the pharmaceutical composition is, for example, about 0.1 to 100% by weight of the entire pharmaceutical composition.
本発明の剤の投与量は、投与の目的、投与方法、中皮腫の種類、腫瘍の大きさ、投与対象者の状態（性別、年齢、体重など）によって異なるが、成人に注射により局所投与する場合、通常、本発明の核酸の量として1 pmol/kg以上10 nmol/kg以下、全身投与では2 nmol/kg以上50 nmol/kg以下が望ましい。かかる投与量を１〜１０回、より好ましくは５〜１０回投与することが望ましい。 The dose of the agent of the present invention varies depending on the purpose of administration, the administration method, the type of mesothelioma, the size of the tumor, and the condition of the subject (gender, age, body weight, etc.), but is locally administered to adults by injection. In general, the amount of the nucleic acid of the present invention is preferably 1 pmol / kg or more and 10 nmol / kg or less, and 2 nmol / kg or more and 50 nmol / kg or less for systemic administration. It is desirable to administer such a dose 1 to 10 times, more preferably 5 to 10 times.
本発明の剤は、投与対象に対して好ましくない影響を及ぼさない範囲で、他の抗中皮腫薬と併用することができる。そのような抗中皮腫薬としては、例えば、シスプラチン、ペメトレキセド、カルボプラチン、ゲムシタビン、ビノレルビン等の化学療法剤が挙げられる。好ましくは、ペメトレキセド、シスプラチン、カルボプラチン及びそれらの組み合せが挙げられる。化学療法剤以外の抗がん剤としては、例えば、ベバシズマブ等の抗VEGF抗体薬、免疫チェックポイント阻害薬等を挙げることができる。これらの抗中皮腫薬の投与方法・投与量は、臨床上で通常使用されている用法・用量に従うことができる。 The agent of the present invention can be used in combination with other anti-mesothelioma agents as long as it does not have an unfavorable effect on the administration subject. Examples of such anti-mesothelioma agents include chemotherapeutic agents such as cisplatin, pemetrexed, carboplatin, gemcitabine and vinorelbine. Preferred include pemetrexed, cisplatin, carboplatin and combinations thereof. Examples of anticancer agents other than chemotherapeutic agents include anti-VEGF antibody agents such as bevacizumab and immune checkpoint inhibitors. The administration method / dose of these anti-mesothelioma drugs can follow the dosage / administration usually used clinically.
本発明の核酸と他の抗中皮腫薬との併用に際しては、それらの投与形態は特に限定されず、投与時に、両者が組み合わされていればよい。このような投与形態としては、例えば、（１）本発明の核酸と他の抗中皮腫薬とを同時に製剤化して得られる単一の製剤の投与、（２）本発明の核酸と他の抗中皮腫薬とを別々に製剤化して得られる2種の製剤の同一投与経路での同時投与、（３）本発明の核酸と他の抗中皮腫薬とを別々に製剤化して得られる2種の製剤の同一投与経路での時間差をおいての投与、（４）本発明の核酸と他の抗中皮腫薬とを別々に製剤化して得られる2種の製剤の異なる投与経路での同時投与、（５）本発明の核酸と他の抗中皮腫薬とを別々に製剤化して得られる2種の製剤の異なる投与経路での時間差をおいての投与（例えば、本発明の核酸→他の抗中皮腫薬の順序での投与、あるいは逆の順序での投与）等が挙げられる。 When the nucleic acid of the present invention is used in combination with other anti-mesothelioma drugs, the administration form thereof is not particularly limited, and both may be combined at the time of administration. Such administration forms include, for example, (1) administration of a single preparation obtained by simultaneously formulating the nucleic acid of the present invention and another anti-mesothelioma drug, and (2) the nucleic acid of the present invention and other administration forms. Simultaneous administration of two preparations obtained by separately formulating an anti-mesothelioma drug by the same route of administration, (3) Obtaining by separately formulating the nucleic acid of the present invention and another anti-mesothelioma drug Administration of the two preparations with a time lag in the same administration route, (4) Different administration routes of the two preparations obtained by separately formulating the nucleic acid of the present invention and another anti-mesothelioma drug. (5) Administration of two preparations obtained by separately formulating the nucleic acid of the present invention and another anti-mesothelioma drug by different routes of administration (for example, the present invention). Nucleic acid → administration of other anti-mesothelioma drugs in the order, or in the reverse order) and the like.
本発明の剤は、その有効成分である本発明の核酸が中皮腫組織（中皮腫細胞）に送達されるように、哺乳動物（例えば、ラット、マウス、モルモット、ウサギ、ヒツジ、ウマ、ブタ、ウシ、サル、ヒト、好ましくはヒト）に対して安全に投与される。 The agent of the present invention comprises mammals (eg, rats, mice, guinea pigs, rabbits, sheep, horses, etc.) so that the nucleic acid of the present invention, which is an active ingredient thereof, is delivered to mesothelioma tissue (mesothelioma cells). It is safely administered to pigs, cows, monkeys, humans, preferably humans).
本発明の核酸は、中皮腫細胞の増殖を抑制する活性を有するので、本発明の剤を中皮腫患者等に対して投与することにより、中皮腫を治療及び／又は予防することが出来る。 Since the nucleic acid of the present invention has an activity of suppressing the proliferation of mesothelioma cells, it is possible to treat and / or prevent mesothelioma by administering the agent of the present invention to a mesothelioma patient or the like. You can.
本発明の剤が適用できる中皮腫としては、例えば、胸膜中皮腫（MPM）、腹膜中皮腫、心膜中皮腫及び精巣鞘膜中皮腫などが挙げられる。好ましくは、MPM及び腹膜中皮腫、より好ましくはMPMである。
本発明の剤は、中皮腫細胞においてmiR-1290の発現が低下している患者に投与するのが望ましい。miR-1290の発現レベルは、例えば、後述の本発明の診断剤を用いて測定することができる。
Examples of mesothelioma to which the agent of the present invention can be applied include pleural mesothelioma (MPM), peritoneal mesothelioma, pericardial mesothelioma and testicular mesothelioma. MPM and peritoneal mesothelioma, more preferably MPM.
The agent of the present invention is preferably administered to patients with reduced expression of miR-1290 in mesothelioma cells. The expression level of miR-1290 can be measured, for example, using the diagnostic agent of the present invention described below.
２．中皮腫の診断剤・判定方法
本発明は、被験者から採取した生体試料中の、miR-1290、miR-345-5p及びmiR-4732-5pからなる群より選択される1以上のmiRNAのレベルを測定することを含む、中皮腫の判定方法（以下、「本発明の判定方法」ともいう。）を提供するものである。
2. Diagnostic Agent / Judgment Method for Mesothelioma The present invention presents the level of one or more miRNAs selected from the group consisting of miR-1290, miR-345-5p and miR-4732-5p in a biological sample collected from a subject. Provided is a method for determining mesothelioma (hereinafter, also referred to as “determination method for the present invention”), which comprises measuring.
本発明の判定方法において、中皮腫は、悪性の中皮腫を意味し、胸膜中皮腫（MPM）、腹膜中皮腫、心膜中皮腫及び精巣鞘膜腫を含む。患者数の多さという点から、中皮腫はMPM及び腹膜中皮腫が好ましく、MPMがより好ましい。 In the determination method of the present invention, mesothelioma means malignant mesothelioma and includes pleural mesothelioma (MPM), peritoneal mesothelioma, pericardial mesothelioma and testicular mesothelioma. From the viewpoint of the large number of patients, MPM and peritoneal mesothelioma are preferable as mesothelioma, and MPM is more preferable.
本発明の判定方法において、被験者は、哺乳動物（例えば、ラット、マウス、モルモット、ウサギ、ヒツジ、ウマ、ブタ、ウシ、サル、ヒト）であれば特に限定されないが、好ましくはヒトである。被験者としては、例えば、中皮腫に罹患していることが疑われる者（例、アスベストへの曝露経験がある者、胸水や腹水の貯留が認められる者等）などが挙げられる。 In the determination method of the present invention, the subject is not particularly limited as long as it is a mammal (for example, rat, mouse, guinea pig, rabbit, sheep, horse, pig, cow, monkey, human), but is preferably a human. Subjects include, for example, those suspected of having mesothelioma (eg, those who have been exposed to asbestos, those who have pleural effusion or ascites), and the like.
本発明の判定方法において用いられ得る生体試料としては、中皮腫が発生し得る部位（例、胸膜、腹膜、心膜、精巣鞘膜、肺及び横隔膜等）の中皮組織の細胞、あるいは当該細胞から分泌されるエクソソームが含まれる限り特に限定されないが、例えば、胸膜、腹膜、心膜、精巣鞘膜、肺及び横隔膜等の中皮組織の生検サンプルやそれらの培養上清、あるいは、胸水、腹水、心嚢液、血液、血漿、血清、リンパ液、尿及び唾液等の体液が挙げられる。 As a biological sample that can be used in the determination method of the present invention, cells in the mesothelial tissue of a site where mesothelium may occur (eg, pleura, peritoneum, peritoneum, testis sheath, lung, diaphragm, etc.), or the said It is not particularly limited as long as it contains exosomes secreted from cells, but for example, biopsy samples of mesothelial tissues such as pleura, peritoneum, peritoneum, testis sheath, lung and diaphragm, their culture supernatants, or pleural fluid. , Peritoneum, pleura, blood, plasma, serum, lymph, urine, saliva and other body fluids.
本発明の判定方法において発現レベルが測定されるmiR-1290、miR-345-5p及びmiR-4732-5pには、それぞれ成熟型、pri-miRNA及びpre-miRNAが含まれるが、好ましくは、これら全ての型の発現レベルの合計または成熟型の発現レベル、より好ましくは成熟型の発現レベルが測定される。
miR-1290の成熟型及びその前駆体については、上述のとおりである。miR-345-5p及びmiR-4732-5pも既知の分子であり、ヒト成熟型及びpre-miR-345-5pは、miRBaseにそれぞれAccession No. MIMAT0000772（配列番号３）及びMI0000825（配列番号４）として、ヒト成熟型及びpre- miR-4732-5pは、それぞれAccession No. MIMAT0019855（配列番号５）及びMI0017369（配列番号６）としてエントリーされている。
本発明の判定方法における測定対象としてのmiR-1290、miR-345-5p及びmiR-4732-5pには、各miRNAのアイソフォーム（isomiR）及びそれらの前駆体（pri-miRNA及びpre-miRNA）も含まれる。これらのisomiRとしては、上述のIsomiR Bankにエントリーされているヌクレオチド配列からなるヌクレオチドが挙げられる。
The miR-1290, miR-345-5p and miR-4732-5p whose expression levels are measured in the determination method of the present invention include mature forms, pri-miRNA and pre-miRNA, respectively, but preferably these. The sum of the expression levels of all types or the expression level of the mature form, more preferably the expression level of the mature form, is measured.
The mature form of miR-1290 and its precursors are as described above. miR-345-5p and miR-4732-5p are also known molecules, and human mature and pre-miR-345-5p have Accession No. MIMAT0000772 (SEQ ID NO: 3) and MI0000825 (SEQ ID NO: 4) in miRBase, respectively. The mature human form and pre-miR-4732-5p have been entered as Accession No. MIMAT0019855 (SEQ ID NO: 5) and MI0017369 (SEQ ID NO: 6), respectively.
The miR-1290, miR-345-5p and miR-4732-5p as measurement targets in the determination method of the present invention include isoforms (isomiR) of each miRNA and their precursors (pri-miRNA and pre-miRNA). Is also included. Examples of these isomiRs include nucleotides consisting of the nucleotide sequences entered in the above-mentioned IsomiR Bank.
例えば、miR-1290、miR-345-5p及びmiR-4732-5pの発現レベルは、各miRNAを特異的に検出し得る核酸プローブ及び／又は核酸プライマーを用いて、自体公知の方法により測定することが出来る。該測定方法としては、例えば、RT-PCR、ノザンブロッティング、in situ ハイブリダイゼーション、核酸アレイ等を挙げることができる。または、市販のキット（例えば、TaqMan（登録商標）MicroRNA Cells-to-CTTM Kit）によっても測定できる。 For example, the expression levels of miR-1290, miR-345-5p and miR-4732-5p should be measured by methods known per se using a nucleic acid probe and / or nucleic acid primer capable of specifically detecting each miRNA. Can be done. Examples of the measurement method include RT-PCR, Northern blotting, in situ hybridization, nucleic acid array and the like. Alternatively, it can be measured with a commercially available kit (eg, TaqMan® MicroRNA Cells-to-CT TM Kit).
miR-1290、miR-345-5p又はmiR-4732-5pを特異的に検出し得る核酸プローブとしては、配列番号１、３又は５で表されるヌクレオチド配列に含まれる、15塩基以上、好ましくは16塩基以上、より好ましくは17塩基以上の連続したヌクレオチド配列またはその相補配列を含むポリヌクレオチドを挙げることが出来る。
miR-1290、miR-345-5p又はmiR-4732-5pを特異的に検出し得る核酸プライマーは、当該miRNA又はその相補的核酸のヌクレオチド配列の一部又は全部の領域を特異的に増幅し得るように設計されたものであればいかなるものであってもよい。そのような核酸プライマーは、例えば、配列番号２、４又は６で表されるヌクレオチド配列の配列情報に基づいて、適宜設計することができる。
As a nucleic acid probe capable of specifically detecting miR-1290, miR-345-5p or miR-4732-5p, 15 bases or more, preferably 15 bases or more, contained in the nucleotide sequence represented by SEQ ID NO: 1, 3 or 5. Examples thereof include polynucleotides containing a continuous nucleotide sequence of 16 bases or more, more preferably 17 bases or more, or a complementary sequence thereof.
A nucleic acid primer capable of specifically detecting miR-1290, miR-345-5p or miR-4732-5p can specifically amplify a part or all of the nucleotide sequence region of the miRNA or its complementary nucleic acid. Any can be used as long as it is designed as such. Such nucleic acid primers can be appropriately designed based on, for example, the sequence information of the nucleotide sequence represented by SEQ ID NO: 2, 4 or 6.
核酸プローブ及び核酸プライマーは、特異的検出に支障を生じない範囲で付加的配列（検出対象のポリヌクレオチドと相補的でないヌクレオチド配列）を含んでいてもよい。
また、核酸プローブ及び核酸プライマーは、適当な標識剤、例えば、放射性同位元素（例：125I、131I、3H、14C等）、酵素（例：β-ガラクトシダーゼ、β-グルコシダーゼ、アルカリフォスファターゼ、パーオキシダーゼ、リンゴ酸脱水素酵素等）、蛍光物質（例：フルオレスカミン、フルオレッセンイソチオシアネート等）、発光物質（例：ルミノール、ルミノール誘導体、ルシフェリン、ルシゲニン等）などで標識されていてもよい。あるいは、蛍光物質（例：FAM、VIC等）の近傍に該蛍光物質の発する蛍光エネルギーを吸収するクエンチャー（消光物質）がさらに結合されていてもよい。かかる実施態様においては、検出反応の際に蛍光物質とクエンチャーとが分離して蛍光が検出される。
Nucleic acid probes and nucleic acid primers may contain additional sequences (nucleotide sequences that are not complementary to the polynucleotide to be detected) as long as they do not interfere with specific detection.
In addition, the nucleic acid probe and the nucleic acid primer are suitable labeling agents, for example, radioisotopes (eg 125 I, 131 I, 3 H, 14 C, etc.), enzymes (eg β-galactosidase, β-glucosidase, alkaline phosphatase). , Peroxidase, malic acid dehydrogenase, etc.), fluorescent substances (eg, fluorescamine, fluoressen isothiocyanate, etc.), luminescent substances (eg, luminol, luminol derivatives, luciferin, lucigenin, etc.) Good. Alternatively, a quencher (quenching substance) that absorbs the fluorescence energy emitted by the fluorescent substance may be further bonded in the vicinity of the fluorescent substance (eg, FAM, VIC, etc.). In such an embodiment, the fluorescent substance and the quencher are separated during the detection reaction and fluorescence is detected.
核酸プローブ及び核酸プライマーは、DNA、RNA、キメラ核酸のいずれであってもよく、また、1本鎖であっても2本鎖であってもよい。核酸プローブまたはプライマーは、例えば、miR-1290については配列番号１または２、miR-345-5pについては配列番号３または４、miR-4732-5pについては配列番号５または６で、それぞれ表されるヌクレオチド配列の情報に基づいて、DNA/RNA自動合成機を用いて常法に従って合成することができる。 The nucleic acid probe and nucleic acid primer may be any of DNA, RNA, and chimeric nucleic acid, and may be single-stranded or double-stranded. Nucleic acid probes or primers are represented, for example, by SEQ ID NO: 1 or 2 for miR-1290, SEQ ID NO: 3 or 4 for miR-345-5p, and SEQ ID NO: 5 or 6 for miR-4732-5p, respectively. Based on the nucleotide sequence information, it can be synthesized according to a conventional method using an automatic DNA / RNA synthesizer.
測定対象のmiRNA又はその相補的核酸を特異的に検出し得る核酸プローブを、適切な支持体の上に結合して、核酸アレイとして提供してもよい。支持体としては、当該分野で通常用いられている支持体であれば特に限定されず、例えば、メンブレン（例えば、ナイロン膜）、ビーズ、ガラス、プラスチック、金属等が挙げられる。 A nucleic acid probe capable of specifically detecting the miRNA to be measured or its complementary nucleic acid may be bound on an appropriate support and provided as a nucleic acid array. The support is not particularly limited as long as it is a support usually used in the art, and examples thereof include a membrane (for example, a nylon membrane), beads, glass, plastic, and metal.
次に、測定されたmiR-1290、miR-345-5p及びmiR-4732-5pの発現レベルに基づいて、被験者が中皮腫に罹患している又は将来罹患する可能性が高いか否かが判定される。後述の実施例に示すように、正常細胞に比べて、中皮腫細胞胞ではmiR-1290、miR-345-5p及びmiR-4732-5pの発現レベルが低い。上記判定は、miR-1290の発現レベルと中皮腫との間の負の相関に基づき行われる。 Then, based on the measured expression levels of miR-1290, miR-345-5p and miR-4732-5p, whether the subject has or is likely to have mesothelioma. It is judged. As shown in the examples below, the expression levels of miR-1290, miR-345-5p and miR-4732-5p are lower in mesothelioma cells than in normal cells. The above determination is based on a negative correlation between miR-1290 expression levels and mesothelioma.
例えば、健常者（ネガティブコントロール）、必要に応じてさらに中皮腫患者（ポジティブコントロール）から採取した生体試料中の、miR-1290、miR-345-5p及びmiR-4732-5pのうちの1以上のmiRNAの発現レベルを測定し、被験者から採取した生体試料中の該miRNAの発現レベルを、ネガティブコントロール及びポジティブコントロールのそれと比較する。発現レベルの比較は、好ましくは、有意差の有無に基づいて行われる。
その結果、被験者におけるこれらのmiRNAの発現レベルがネガティブコントロールと比べて低かった場合には、該被験者は、中皮腫に罹患している又は将来罹患する可能性が高いと判定することができる。
For example, one or more of miR-1290, miR-345-5p and miR-4732-5p in biological samples taken from healthy individuals (negative control) and, if necessary, mesothelioma patients (positive control). The expression level of the miRNA is measured, and the expression level of the miRNA in the biological sample collected from the subject is compared with that of the negative control and the positive control. The comparison of expression levels is preferably based on the presence or absence of significant differences.
As a result, if the expression level of these miRNAs in a subject is lower than that of the negative control, it can be determined that the subject has or is likely to suffer from mesothelioma.
本発明の中皮腫の予防及び／又は治療剤（本発明の剤）の投与対象としては、中皮腫細胞におけるmiR-1290の発現レベルが低下している患者が望ましいため、本発明の判定方法は、本発明の剤の投与対象の選別に有用である。 The determination of the present invention is made because it is desirable that the subject of administration of the preventive and / or therapeutic agent for mesothelioma of the present invention (the agent of the present invention) is a patient in which the expression level of miR-1290 in mesothelioma cells is decreased. The method is useful for selecting the administration target of the agent of the present invention.
本発明はまた、miR-1290を特異的に検出し得る核酸プローブ及び／又は核酸プライマー、miR-345-5pを特異的に検出し得る核酸プローブ及び／又は核酸プライマー、並びにmiR-4732-5pを特異的に検出し得る核酸プローブ及び／又は核酸プライマーからなる群より選択される1以上の核酸プローブ及び／又は核酸プライマーを含む、中皮腫の診断剤（以下、「本発明の剤（II）」ともいう。）を提供する。本発明の剤（II）に含まれる核酸プローブ及び核酸プライマーは、上記「本発明の判定方法」において使用されるものと同様である。 The present invention also provides a nucleic acid probe and / or a nucleic acid primer capable of specifically detecting miR-1290, a nucleic acid probe and / or a nucleic acid primer capable of specifically detecting miR-345-5p, and miR-4732-5p. A diagnostic agent for mesenteric tumors, which comprises one or more nucleic acid probes and / or nucleic acid primers selected from the group consisting of nucleic acid probes and / or nucleic acid primers that can be specifically detected (hereinafter, "the agent (II) of the present invention). Also called.). The nucleic acid probe and nucleic acid primer contained in the agent (II) of the present invention are the same as those used in the above-mentioned "determination method of the present invention".
核酸プローブ及び核酸プライマーは、通常、水もしくは適当な緩衝液（例：TEバッファー、PBSなど）中に適当な濃度となるように溶解されるか、凍結乾燥された状態で、適切な容器中に収容される。或いは、該核酸プローブが固相担体上に固定された核酸アレイの態様で、本発明の剤（II）に含まれ得る。 Nucleic acid probes and nucleic acid primers are usually dissolved in water or a suitable buffer (eg, TE buffer, PBS, etc.) to a suitable concentration, or lyophilized in a suitable container. Be housed. Alternatively, the nucleic acid probe may be included in the agent (II) of the present invention in the form of a nucleic acid array immobilized on a solid phase carrier.
本発明の剤（II）は、対象miRNAの測定方法に応じて、当該方法の実施に必要な他の成分を構成としてさらに含んでいてもよい。例えば、ノザンブロッティングや核酸アレイを測定に用いる場合には、本発明の剤（II）は、ブロッティング緩衝液、標識化試薬、ブロッティング膜等をさらに含むことができる。in situ ハイブリダイゼーションを測定に用いる場合には、本発明の剤（II）は、標識化試薬、発色基質等をさらに含むことができる。RT-PCRを測定に用いる場合には、本発明の剤（II）は、10×PCR反応緩衝液、10×MgCl2水溶液、10×dNTPs水溶液、Taq DNAポリメラーゼ、逆転写酵素等を更に含むことができる。 The agent (II) of the present invention may further contain other components necessary for carrying out the method, depending on the method for measuring the target miRNA. For example, when Northern blotting or a nucleic acid array is used for measurement, the agent (II) of the present invention can further include a blotting buffer, a labeling reagent, a blotting membrane and the like. When in situ hybridization is used for measurement, the agent (II) of the present invention can further contain a labeling reagent, a color-developing substrate, and the like. When RT-PCR is used for measurement, the agent (II) of the present invention further contains 10 × PCR reaction buffer, 10 × MgCl 2 aqueous solution, 10 × dNTPs aqueous solution, Taq DNA polymerase, reverse transcriptase and the like. Can be done.
３．抗中皮腫活性を有する物質のスクリーニング方法
本発明はまた、被検物質が中皮腫細胞におけるmiR-1290の発現を増強するか否かを評価することを含む、抗中皮腫活性を有する物質のスクリーニング方法（以下、「本発明のスクリーニング方法」ともいう。）を提供する。
3. 3. Screening Methods for Substances with Anti-Mesothelioma Activity The present invention also has anti-mesothelioma activity, including assessing whether the test substance enhances the expression of miR-1290 in mesothelioma cells. A substance screening method (hereinafter, also referred to as “the screening method of the present invention”) is provided.
本発明のスクリーニング方法に供される被検物質は、いかなる公知化合物および新規化合物であってもよく、例えば、核酸、糖質、脂質、蛋白質、ペプチド、有機低分子化合物、コンビナトリアルケミストリー技術を用いて作製された化合物ライブラリー、ランダムペプチドライブラリー、あるいは微生物、動植物、海洋生物等由来の天然成分等が挙げられる。 The test substance used in the screening method of the present invention may be any known compound or a novel compound, for example, using a nucleic acid, a sugar, a lipid, a protein, a peptide, an organic low molecular weight compound, or a combinatorial chemistry technique. Examples thereof include a prepared compound library, a random peptide library, and natural components derived from microorganisms, animals and plants, marine organisms, and the like.
本発明の探索方法は、以下の工程を含む：
（１）被検物質と中皮腫細胞とを接触させること；
（２）被検物質を接触させた細胞におけるmiR-1290の発現レベルを測定し、該発現レベルを、被検物質を接触させない対照細胞におけるmiR-1290の発現レベルと比較すること；並びに
（３）上記（２）の比較結果に基づいて、miR-1290の発現レベルを増大させた被検物質を、抗中皮腫活性を有する物質として選択すること。
The search method of the present invention includes the following steps:
(1) Contacting the test substance with mesothelioma cells;
(2) Measure the expression level of miR-1290 in cells contacted with the test substance and compare the expression level with the expression level of miR-1290 in the control cells not contacted with the test substance; and (3). ) Based on the comparison result of (2) above, a test substance having an increased expression level of miR-1290 should be selected as a substance having anti-mesothelioma activity.
本発明のスクリーニング方法において、発現レベルが測定されるmiR-1290には、成熟型、pri-miRNAおよびpre-miRNAが含まれるが、好ましくは、これら全ての型の発現レベルの合計または成熟型の発現レベル、より好ましくは成熟型の発現レベルが測定される。 In the screening method of the present invention, miR-1290 whose expression level is measured includes mature, pri-miRNA and pre-miRNA, preferably the sum of the expression levels of all these types or the mature type. Expression levels, more preferably mature expression levels, are measured.
中皮腫細胞としては、胸膜中皮腫（MPM）、腹膜中皮腫、心膜中皮腫、精巣鞘膜腫等の各種中皮腫患者から切除した中皮腫、好ましくはMPM又は腹膜中皮腫、より好ましくはMPMの腫瘍組織から単離した中皮腫細胞などが挙げられる。中皮腫細胞は、初代培養細胞でもよいし、そこから樹立された細胞株であってもよい。 The mesothelioma cells include mesothelioma resected from various mesothelioma patients such as pleural mesothelioma (MPM), pericardial mesothelioma, pericardial mesothelioma, and testis sheath mesothelioma, preferably MPM or in the peritoneum. Examples include mesothelioma, more preferably mesothelioma cells isolated from MPM tumor tissue. The mesothelioma cell may be a primary cultured cell or a cell line established from the primary cultured cell.
被検物質と中皮腫細胞との接触は培地中で行われる。培地は、例えば、約5〜20%のウシ胎仔血清を含む最少必須培地（MEM）、ダルベッコ改変イーグル培地（DMEM）などである。培養条件も同様に適宜決定されるが、例えば、培地のpHは約6〜約8であり、培養温度は通常約30〜約40℃であり、培養時間は約12〜約72時間である。 Contact between the test substance and the mesothelioma cells takes place in the medium. The medium is, for example, the minimum essential medium (MEM) containing about 5 to 20% fetal bovine serum, Dulbecco's modified Eagle's medium (DMEM), and the like. The culturing conditions are similarly determined as appropriate, but for example, the pH of the medium is about 6 to about 8, the culturing temperature is usually about 30 to about 40 ° C., and the culturing time is about 12 to about 72 hours.
miR-1290の発現レベルの測定は、上記「本発明の判定方法」の項で述べた方法に従って行うことができる。 The expression level of miR-1290 can be measured according to the method described in the above section "Determination method of the present invention".
発現レベルの比較は、好ましくは、有意差の有無に基づいて行なわれ得る。なお、被検物質を接触させない対照細胞におけるmiR-1290の発現レベルは、被検物質を接触させた細胞におけるmiR-1290の発現レベルの測定に対し、事前に測定した発現レベルであっても、同時に測定した発現レベルであってもよいが、実験の精度、再現性の観点から同時に測定した発現レベルであることが好ましい。 Comparison of expression levels can preferably be based on the presence or absence of significant differences. The expression level of miR-1290 in the control cells not contacted with the test substance may be the expression level measured in advance with respect to the measurement of the expression level of miR-1290 in the cells contacted with the test substance. The expression level may be measured at the same time, but it is preferable that the expression level is measured at the same time from the viewpoint of accuracy and reproducibility of the experiment.
比較の結果、miR-1290の発現レベルを増大させた物質が、抗中皮腫活性を有する物質として選択される。 As a result of comparison, a substance having an increased expression level of miR-1290 is selected as a substance having anti-mesothelioma activity.
本発明のスクリーニング方法で得られる化合物は、新たな中皮腫の予防及び／又は治療剤の開発のための候補物質として有用である。 The compounds obtained by the screening method of the present invention are useful as candidate substances for the development of new preventive and / or therapeutic agents for mesothelioma.
以下に、実施例を挙げて本発明をより具体的に説明するが、本発明はそれらの実施例等により何ら限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to those examples.
細胞培養
理研細胞バンク（つくば、日本）より、悪性胸膜中皮腫（MPM）細胞株であるACC-MESO1及びACC-MESO4を入手した。ATCC（American Type Culture Collection、マナッサス、バージニア、米国）より、MPM細胞株であるMSTO-211H及び非悪性中皮細胞株であるMeT-5A細胞を購入した。MPM細胞株であるL324、N407及びK921は、産業医科大学 第2外科で樹立されたものを用いた。GlutaMAXサプリメント（Invitrogen、カールスバッド、カリフォルニア、米国）、10％ 非働化ウシ胎児血清（FBS）及び1％（v / w）ペニシリン/ストレプトマイシンを添加したRPMI-1640培地、199培地及びDMEMを用いて、MPM及びMeT-5A細胞をそれぞれ5％ CO2雰囲気中、37℃で維持した。
Cell Culture We obtained malignant pleural mesothelioma (MPM) cell lines ACC-MESO1 and ACC-MESO4 from RIKEN Cell Bank (Tsukuba, Japan). We purchased the MPM cell line MSTO-211H and the non-malignant mesenteric cell line MeT-5A cells from ATCC (American Type Culture Collection, Manassas, Virginia, USA). The MPM cell lines L324, N407 and K921 used were those established in the Second Department of Surgery, University of Occupational and Environmental Health. Using GlutaMAX supplements (Invitrogen, Carlsbad, CA, USA), RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% (v / w) penicillin / streptomycin, 199 medium and DMEM. MPM and MeT-5A cells were maintained at 37 ° C. in a 5% CO 2 atmosphere, respectively.
実施例１ MPM細胞で発現レベルが低下するmiRNAの同定
6種類のヒトMPM細胞株（MESO1, MESO4, K921, L324, N407, 211H）とヒト不死化胸膜中皮細胞株（MeT-5A）の細胞から、miRNeasy Mini Kit（Qiagen、Hilden、ドイツ）を用いて全RNAで抽出した。得られた全RNAについて、microRNAの発現量を3D-Geneのチップ（東レ社）を用いて網羅的に解析した。2,565種類のmicroRNAから、MeT-5A細胞での値を、6種類のMPM細胞の中で最も高い値で除した値を求め、この値が2以上になる3種類のmicroRNA（hsa-miR-1290、hsa-miR-345-5p及びhsa-miR-4732-5p）を同定した（表１）。
Example 1 Identification of miRNAs whose expression levels are reduced in MPM cells
Using miRNeasy Mini Kit (Qiagen, Hilden, Germany) from cells of 6 types of human MPM cell lines (MESO1, MESO4, K921, L324, N407, 211H) and human immortalized pleural mesothelial cell line (MeT-5A) Was extracted with total RNA. For all the obtained RNA, the expression level of microRNA was comprehensively analyzed using a 3D-Gene chip (Toray Industries, Inc.). From 2,565 types of microRNAs, the value in MeT-5A cells was divided by the highest value among the 6 types of MPM cells, and 3 types of microRNAs (hsa-miR-1290) with this value of 2 or more were obtained. , Hsa-miR-345-5p and hsa-miR-4732-5p) were identified (Table 1).
実施例２ リアルタイムPCRによる検証
実施例１にて同定した3種のmiRNAについて、それらの発現レベルをリアルタイムPCRにより検証した。TaqMan MicroRNA Reverse Transcription Kit（米国マサチューセッツ州ウォルサム）を用いて、StepOne PlusリアルタイムPCRシステム（米国マサチューセッツ州ウォルサム）でマイクロRNAのcDNAを合成し、特別に設計されたTaqManプローブを用いてqRT-PCRを行った。各miRNAの増幅に用いたプライマー配列は、ThermoFisher Scientificより購入した。PCR反応は、95℃で20秒間保持した後、40サイクルの増幅（95℃で30秒間の変性、60℃で30秒間のアニーリング及び伸長）にて行った。各細胞のmiRNAとU6（内部標準）のCT値を求め、MeT-5Aが1になるようにΔΔCT法で比を求めた。
その結果、3種類のmiRNAはいずれも、MeT-5A細胞に比べて、MPM細胞での発現が20%以下に低下していた（図１）。
Example 2 Verification by real-time PCR The expression levels of the three miRNAs identified in Example 1 were verified by real-time PCR. Using the TaqMan MicroRNA Reverse Transcription Kit (Waltham, Mass., USA), microRNA cDNA was synthesized with the StepOne Plus real-time PCR system (Waltham, Mass., USA), and qRT-PCR was performed using a specially designed TaqMan probe. It was. Primer sequences used for amplification of each miRNA were purchased from Thermo Fisher Scientific. The PCR reaction was carried out by holding at 95 ° C. for 20 seconds and then performing 40 cycles of amplification (denaturation at 95 ° C. for 30 seconds, annealing and elongation at 60 ° C. for 30 seconds). The CT values of miRNA and U6 (internal standard) of each cell were determined, and the ratio was determined by the ΔΔCT method so that MeT-5A became 1.
As a result, the expression of all three types of miRNA was reduced to 20% or less in MPM cells as compared with MeT-5A cells (Fig. 1).
実施例３ miR-1290過剰発現によるMPM細胞の細胞増殖抑制
miRNA発現プラスミドの構築のために、hsa-miR-1290（配列番号２）、hsa-miR-345-5p（配列番号４）及びhsa-miR-4732-5p（配列番号６）の5’末端にBamH1リンカー（GGATCC）、3’末端に（dT）5及びEcoRIリンカー（GAATTC）を付加した２本鎖オリゴDNAを合成し、pSIH1-H1-GFP-T2A-Puroベクター（System Biosciences、Palo Alto、California、United States）のBamHI-EcoRI部位に連結した。製造業者のプロトコルに従って、System Biosciencesのレンチウイルスパッケージングシステムから、各miRNAの発現カセットを含むレンチウイルスを入手した。 MESO1細胞をこれらのウイルスに感染させた後、5 μg/mLのピューロマイシン存在下で各miRNAを発現する細胞（MESO1-1290、MESO1-345-5p、MESO1-4732-5p）を選択した。miRNAを含まないベクターを導入したMESO1細胞（MESO1-ctrl）をコントロール細胞として用いた。
実施例２と同様にして、導入された各miRNAの発現レベルをリアルタイムPCRにより測定し、U6（内部標準）で補正した後、コントール細胞の値が1になるようにΔΔCT法で比を求めた。その結果、各miRNAは、MESO1-ctrlに比べ、いずれも100倍以上に高発現していることが確認された（図２）。
Example 3 Cell proliferation suppression of MPM cells by overexpression of miR-1290
At the 5'end of hsa-miR-1290 (SEQ ID NO: 2), hsa-miR-345-5p (SEQ ID NO: 4) and hsa-miR-4732-5p (SEQ ID NO: 6) for the construction of miRNA expression plasmids. Double-stranded oligo DNA with BamH1 linker (GGATCC), 3'end (dT) 5 and EcoRI linker (GAATTC) was synthesized and pSIH1-H1-GFP-T2A-Puro vector (System Biosciences, Palo Alto, California). , United States) was ligated to the BamHI-EcoRI site. Lentiviruses containing expression cassettes for each miRNA were obtained from System Biosciences' lentivirus packaging system according to the manufacturer's protocol. After infecting MESO1 cells with these viruses, cells expressing each miRNA in the presence of 5 μg / mL puromycin (MESO1-1290, MESO1-345-5p, MESO1-4732-5p) were selected. MESO1 cells (MESO1-ctrl) into which a miRNA-free vector was introduced were used as control cells.
In the same manner as in Example 2, the expression level of each introduced miRNA was measured by real-time PCR, corrected by U6 (internal standard), and then the ratio was determined by the ΔΔCT method so that the control cell value became 1. .. As a result, it was confirmed that each miRNA was 100 times more highly expressed than MESO1-ctrl (Fig. 2).
次に、作製されたmiRNA過剰発現細胞を、5％ CO2雰囲気中、37℃で4日間培養した。１日毎に細胞数を測定し、培養開始1日後の細胞数を1とした相対値で、細胞数の経日変化を図３に示した。また、各細胞の倍加時間を表２に示す。 Next, the prepared miRNA overexpressing cells were cultured at 37 ° C. for 4 days in a 5% CO 2 atmosphere. The number of cells was measured every day, and the diurnal change in the number of cells was shown in FIG. 3 as a relative value with the number of cells 1 day after the start of culturing as 1. Table 2 shows the doubling time of each cell.
hsa-miR-1290過剰発現細胞（MESO1-1290）のみが、コントロール細胞（MESO1-ctrl）と比べて、顕著に細胞増殖能が低下していた。 Only the hsa-miR-1290 overexpressing cells (MESO1-1290) had significantly reduced cell proliferation ability as compared with the control cells (MESO1-ctrl).
本発明の核酸は、中皮腫細胞の増殖を顕著に抑制することができ、かつ正常細胞に取り込まれても細胞障害が生じにくいと考えられるので、副作用が少ない中皮腫の治療薬として大いに有用である。 Since the nucleic acid of the present invention can remarkably suppress the proliferation of mesothelioma cells and is considered to be less likely to cause cell damage even when taken up by normal cells, it is highly useful as a therapeutic agent for mesothelioma with few side effects. It is useful.
Claims (10)
(a) 配列番号１で表されるmiR-1290のヌクレオチド配列を含む核酸、又は
(b) 上記(a)のヌクレオチド配列と70%以上、好ましくは80%以上、より好ましくは90%以上の同一性を有するヌクレオチド配列からなり、且つmiR-1290と同等の機能を有するヌクレオチドを含む核酸
を含有する、剤。 A prophylactic and / or therapeutic agent for mesothelioma
(a) Nucleic acid containing the nucleotide sequence of miR-1290 represented by SEQ ID NO: 1 or
(b) Consists of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more identity with the nucleotide sequence of (a) above, and contains a nucleotide having a function equivalent to that of miR-1290. An agent containing a nucleic acid.
(a) 配列番号１で表されるmiR-1290のヌクレオチド配列を含む核酸、又は
(b) 上記(a)のヌクレオチド配列と70%以上、好ましくは80%以上、より好ましくは90%以上の同一性を有するヌクレオチド配列からなり、且つmiR-1290と同等の機能を有するヌクレオチドを含む核酸
の有効量を、対象に投与することを含む、方法。 A method for preventing and / or treating mesothelioma.
(a) Nucleic acid containing the nucleotide sequence of miR-1290 represented by SEQ ID NO: 1 or
(b) Consists of a nucleotide sequence having 70% or more, preferably 80% or more, more preferably 90% or more identity with the nucleotide sequence of (a) above, and contains a nucleotide having a function equivalent to that of miR-1290. A method comprising administering to a subject an effective amount of nucleic acid.
（１）被検物質と中皮腫細胞とを接触させること；
（２）被検物質を接触させた細胞におけるmiR-1290の発現レベルを測定し、該発現レベルを、被検物質を接触させない対照細胞におけるmiR-1290の発現レベルと比較すること；並びに
（３）上記（２）の比較結果に基づいて、miR-1290の発現レベルを増大させた被検物質を、抗中皮腫活性を有する物質として選択すること。 Screening method for substances with anti-mesothelioma activity, including the following steps:
(1) Contacting the test substance with mesothelioma cells;
(2) Measure the expression level of miR-1290 in cells contacted with the test substance and compare the expression level with the expression level of miR-1290 in the control cells not contacted with the test substance; and (3). ) Based on the comparison result of (2) above, a test substance having an increased expression level of miR-1290 should be selected as a substance having anti-mesothelioma activity.
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