JP2009225672A

JP2009225672A - GROWTH INHIBITION OF SCIRRHOUS GASTRIC CANCER CELL BY siRNA TO FGFR2

Info

Publication number: JP2009225672A
Application number: JP2008071510A
Authority: JP
Inventors: Isao Kaneko; 勲金子; Hiroaki Onda; 弘明恩田
Original assignee: HUMAN SCIENCE SHINKO ZAIDAN; NAT INST OF BIOMEDICAL INNOVAT; National Institute of Biomedical Innovation NIBIO
Current assignee: HUMAN SCIENCE SHINKO ZAIDAN; NAT INST OF BIOMEDICAL INNOVAT; National Institute of Biomedical Innovation NIBIO
Priority date: 2008-03-19
Filing date: 2008-03-19
Publication date: 2009-10-08

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid sequence of siRNA (small interfering RNA) having a strongly inhibitory action on growth of scirrhous gastric cancer cell by examining the nucleic acid sequence of siRNA to FGFR2 (Fibroblast Growth Factor Receptor 2), and to provide a pharmaceutical composition comprising the same as an active ingredient. <P>SOLUTION: The siRNA is an siRNA that inhibits a scirrhous gastric cancer cell growth by inhibiting expression of FGFR2 protein and is constituted of a double strand composed of a set of any RNA sequences represented by specific sequences. The pharmaceutical composition for treating scirrhous gastric cancer comprises the same. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

本発明は、スキルス胃がん細胞の増殖を阻害するＦＧＦＲ２に対するｓｉＲＮＡおよびこれらを含む医薬組成物に関する。 The present invention relates to siRNA against FGFR2 that inhibits the growth of Skills gastric cancer cells and pharmaceutical compositions containing these.

日本における胃がん死亡者数は年間約５万人と多いが、早期胃がんでは約９割、胃がんでも約６割と治癒率（治療後５年間生存している割合）は向上している。しかし、胃がん全体の約１０％を占めるスキルス胃がんは極めて悪性であり、ほとんど治療効果が認められない（非特許文献１）。その意味で、一刻も早くスキルスがんに有効な治療薬の開発が切望されている。そのために、これまで、インビトロ培養系で増殖可能な胃がん細胞の培養樹立細胞株を用いる研究が行われており、そのｍＲＮＡの発現解析から、ＦＧＦＲ２がスキルス胃がん細胞株であるＫＡＴＯ−ＩＩＩ細胞株に強発現していることが報告されている（非特許文献２）。 The number of deaths from gastric cancer in Japan is as high as about 50,000 per year, but the cure rate (rate of surviving for five years after treatment) has improved, with about 90% of early gastric cancer and about 60% of gastric cancer. However, Skills gastric cancer, which accounts for about 10% of all gastric cancer, is extremely malignant and hardly shows any therapeutic effect (Non-patent Document 1). In that sense, the development of an effective therapeutic agent for Skills cancer is eagerly desired. For this purpose, research has been conducted using established cell lines of gastric cancer cells that can be proliferated in an in vitro culture system. From the analysis of mRNA expression, FGFR2 has become a KATO-III cell line that is a Skills gastric cancer cell line. Strong expression has been reported (Non-patent Document 2).

さて、たんぱく質の機能を解析する手段の一つとして、標的のたんぱく質を特異的に発現している細胞に対し、低分子干渉性ＲＮＡ（以下、ｓｉＲＮＡという）をトランスフェクションして、ｍＲＮＡを特異的に分解することにより発現を阻害し、その機能を解析する手段がよく用いられている。実際に、ＦｉｂｒｏｂｌａｓｔＧｒｏｗｔｈＦａｃｔｏｒｒｅｃｅｐｔｏｒ２（以下、ＦＧＦＲ２という）に関しても、ＦＧＦＲ２に対するｓｉＲＮＡをＫＡＴＯ−ＩＩＩ細胞株にトランスフェクションし、ＫＡＴＯ−ＩＩＩ細胞株の増殖が阻害されることが報告されている（非特許文献３）。しかしながら、どの配列のｓｉＲＮＡが強い阻害作用を発揮するか、および他のスキルス胃がん細胞株への阻害作用については明らかにされていない。
Hansen, K.C. et al, Mol. Cell Proteomics, 2:299-314, 2003 Matunobu T., Ishiwata T. et al, Int. J. Onco, 2:307-314,2006 Masayuki T, et al., Clin Cancer Res, 13:3051-3057, 2007 金子勲：疾患関連たんぱく質解析研究平成18年度総括・分担研究報告書 p. 104-144 As a means of analyzing the function of a protein, a cell that specifically expresses a target protein is transfected with a small interfering RNA (hereinafter referred to as siRNA), and mRNA is specifically expressed. Means for inhibiting the expression by degrading the protein and analyzing its function are often used. In fact, it has been reported that the fibroblast growth factor receptor 2 (hereinafter referred to as FGFR2) is also transfected with siRNA against FGFR2 into the KATO-III cell line, and the growth of the KATO-III cell line is inhibited (non-native). Patent Document 3). However, it has not been clarified about which sequence of siRNA exerts a strong inhibitory action and the inhibitory action on other Skills gastric cancer cell lines.
Hansen, KC et al, Mol. Cell Proteomics, 2: 299-314, 2003 Matunobu T., Ishiwata T. et al, Int. J. Onco, 2: 307-314,2006 Masayuki T, et al., Clin Cancer Res, 13: 3051-3057, 2007 Isao Kaneko: Disease-related protein analysis research 2006 generalization / share research report p. 104-144

したがって、本発明の課題は、ＦＧＦＲ２に対するｓｉＲＮＡの核酸配列を検討し、スキルス胃がん細胞の増殖に対して阻害作用の強いｓｉＲＮＡの核酸配列およびそれを有効成分として含むスキルス胃がん治療用の医薬組成物を提供することである。 Accordingly, an object of the present invention is to examine the nucleic acid sequence of siRNA against FGFR2, and to provide a nucleic acid sequence of siRNA having a strong inhibitory effect on the growth of Skills gastric cancer cells and a pharmaceutical composition for treating Skills gastric cancer comprising the same as an active ingredient. Is to provide.

本発明者らは、上記課題を解決するために鋭意研究を行った。そして驚くべきことに、ＫＡＴＯ−ＩＩＩ細胞株にＦＧＦＲ２に対するｓｉＲＮＡ、特に配列番号５および６で表される核酸配列からなるｓｉＲＮＡをトランスフェクションすると細胞の増殖を著しく阻害することを見出した。 The inventors of the present invention have intensively studied to solve the above problems. Surprisingly, it has been found that KATO-III cell line significantly inhibits cell growth when transfected with siRNA against FGFR2, particularly siRNA consisting of nucleic acid sequences represented by SEQ ID NOs: 5 and 6.

すなわち、本発明は：
（１）ＦＧＦＲ２たんぱく質の発現を阻害することによりスキルス胃がん細胞株の増殖を阻害するｓｉＲＮＡであって、配列番号１および２、３および４、５および６、または７および８で表されるいずれかのＲＮＡ配列の組からなる二本鎖で構成されるｓｉＲＮＡ、
（２）ＦＧＦＲ２たんぱく質の発現を阻害することによりスキルス胃がん細胞株の増殖を阻害するｓｉＲＮＡであって、配列番号９および１０、１１および１２、１３および１４、または１５および１６で表されるいずれかのＲＮＡ配列の組からなる二本鎖で構成されるｓｉＲＮＡ、
（３）配列番号５および６で表されるＲＮＡ配列の組からなるｓｉＲＮＡであって、ＫＡＴＯ−ＩＩＩ細胞株およびＮＵＧＣ−４細胞株の増殖を阻害するｓｉＲＮＡ、
（４）有効成分として（１）〜（３）のいずれか１つに記載の１種またはそれ以上のｓｉＲＮＡを含む、スキルス胃がんの治療のための医薬組成物、
（５）有効成分として（３）に記載のｓｉＲＮＡを含む、スキルス胃がんの治療のための医薬組成物
を提供するものである。 That is, the present invention provides:
(1) An siRNA that inhibits the growth of Schirus gastric cancer cell line by inhibiting the expression of FGFR2 protein, which is represented by any of SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, or 7 and 8 SiRNA composed of a double strand consisting of a set of RNA sequences of
(2) An siRNA that inhibits the growth of Schirus gastric cancer cell line by inhibiting the expression of FGFR2 protein, which is represented by any of SEQ ID NOs: 9 and 10, 11 and 12, 13 and 14, or 15 and 16 SiRNA composed of a double strand consisting of a set of RNA sequences of
(3) siRNA consisting of a set of RNA sequences represented by SEQ ID NOs: 5 and 6, wherein the siRNA inhibits growth of KATO-III cell line and NUGC-4 cell line,
(4) A pharmaceutical composition for the treatment of Skills gastric cancer, comprising one or more siRNAs according to any one of (1) to (3) as an active ingredient,
(5) Provided is a pharmaceutical composition for the treatment of Skills gastric cancer comprising the siRNA described in (3) as an active ingredient.

本発明によれば、スキルス胃がん細胞に由来する細胞株、特にＫＡＴＯ−ＩＩＩ細胞株の細胞増殖を特異的に阻害することができる。これによりスキルス胃がんの細胞増殖に有効な成分を包含する医薬組成物を提供することも可能となり、スキルス胃がんを効果的に治療することができる。 According to the present invention, it is possible to specifically inhibit cell proliferation of a cell line derived from Skills gastric cancer cells, particularly a KATO-III cell line. Thereby, it is also possible to provide a pharmaceutical composition containing components effective for cell growth of Skills gastric cancer, and Skills gastric cancer can be effectively treated.

本発明は、スキルス胃がん細胞の増殖を阻害するｓｉＲＮＡに関するものである。本発明では、ＦＧＦＲ２に対するｓｉＲＮＡを提供する。ここで、ＦＧＦＲ２はＦｉｂｒｏｂｌａｓｔＧｒｏｗｔｈＦａｃｔｏｒｒｅｃｅｐｔｏｒ２の略称であり、線維芽増殖因子の膜受容体たんぱく質の一種であることを意味する。このたんぱく質はＦＧＦシグナルを介在して線維芽細胞をはじめとするさまざまな細胞に対して増殖活性や分化誘導など多彩な作用を示すことが知られているため、スキルス胃がんの細胞増殖にも関連することが示唆される。 The present invention relates to siRNA that inhibits the growth of Skills gastric cancer cells. In the present invention, siRNA against FGFR2 is provided. Here, FGFR2 is an abbreviation for Fibroblast Growth Factor receptor 2 and means a type of membrane receptor protein of fibroblast growth factor. Since this protein is known to exhibit various actions such as proliferation activity and differentiation induction on various cells including fibroblasts via FGF signal, it is also related to cell growth of Skills gastric cancer. It is suggested.

本発明のｓｉＲＮＡは、アンチセンス鎖とセンス鎖の１組からなる二本鎖で構成される。一般に、ｓｉＲＮＡは、ｄｓＲＮＡがＲＮａｓｅＩＩＩファミリーに属するＤｉｃｅｒと呼ばれる酵素によって分解された２１〜２５塩基の二本鎖ＲＮＡであり、複数のタンパク質で構成される複合体ＲＮＡＩｎｄｕｃｅｄＳｉｌｅｎｃｉｎｇＣｏｍｐｌｅｘ（ＲＩＳＣ）に取り込まれて活性型ＲＩＳＣを形成する。この活性型ＲＩＳＣは、取り込まれたｓｉＲＮＡのＲＮＡ配列を利用して標的ｍＲＮＡを特異的に認識・分解することにより遺伝子の発現を抑制し、たんぱく質の合成を阻害すること（この現象をＲＮＡｉという）が示されている。また、ｓｉＲＮＡのアンチセンス鎖は標的ｍＲＮＡと相補的である。本発明のｓｉＲＮＡは、Target siRNA-17、Target siRNA-18、Target siRNA-19およびTarget siRNA-20と名付けた以下の新規ＲＮＡ配列を含む。
Target siRNA-17のアンチセンス鎖（配列番号１）：
５’−ＧＡＵＵＧＡＵＧＧＡＣＣＣＧＵＡＵＵＣＵＵ−３’
Target siRNA-17のセンス鎖（配列番号２）：
５’−ＧＡＡＵＡＣＧＧＧＵＣＣＡＵＣＡＡＵＣＵＵ−３’
Target siRNA-18のアンチセンス鎖（配列番号３）：
５’−ＵＣＡＧＡＵＧＧＧＡＣＣＡＣＡＣＵＵＵＵＵ−３’
Target siRNA-18のセンス鎖（配列番号４）：
５’−ＡＡＡＧＵＧＵＧＧＵＣＣＣＡＵＣＵＧＡＵＵ−３’
Target siRNA-19のアンチセンス鎖（配列番号５）：
５’−ＵＧＵＵＧＧＵＣＣＡＧＵＡＵＧＧＵＧＣＵＵ−３’
Target siRNA-19のセンス鎖（配列番号６）：
５’−ＧＣＡＣＣＡＵＡＣＵＧＧＡＣＣＡＡＣＡＵＵ−３’
Target siRNA-20のアンチセンス鎖（配列番号７）：
５’−ＵＵＵＣＧＵＡＣＣＵＵＧＵＡＧＣＣＵＣＵＵ−３’
Target siRNA-20のセンス鎖（配列番号８）：
５’−ＧＡＧＧＣＵＡＣＡＡＧＧＵＡＣＧＡＡＡＵＵ−３’ The siRNA of the present invention is composed of a double strand consisting of a pair of an antisense strand and a sense strand. In general, siRNA is a 21 to 25 base double-stranded RNA in which dsRNA is degraded by an enzyme called Dicer belonging to the RNase III family, and is incorporated into a complex RNA Induced Silencing Complex (RISC) composed of a plurality of proteins. To form an active RISC. This active RISC specifically suppresses gene expression by specifically recognizing and degrading the target mRNA using the RNA sequence of the incorporated siRNA, and inhibits protein synthesis (this phenomenon is called RNAi). It is shown. The antisense strand of siRNA is complementary to the target mRNA. The siRNA of the present invention comprises the following novel RNA sequences named Target siRNA-17, Target siRNA-18, Target siRNA-19 and Target siRNA-20.
Antisense strand of Target siRNA-17 (SEQ ID NO: 1):
5'-GAUUGAUGGACCCCGUAUUCUU-3 '
Target siRNA-17 sense strand (SEQ ID NO: 2):
5'-GAAUACGGGUCCAUCAAUCUU-3 '
Antisense strand of Target siRNA-18 (SEQ ID NO: 3):
5'-UCAGAUGGGACCACACUUUUU-3 '
Target siRNA-18 sense strand (SEQ ID NO: 4):
5'-AAAGUGUGGUCCCAUCUGAUU-3 '
Antisense strand of Target siRNA-19 (SEQ ID NO: 5):
5'-UGUUGGUCCAGUAUGUGUGCUU-3 '
Target siRNA-19 sense strand (SEQ ID NO: 6):
5'-GCACCAAUACUGGACCAACAUU-3 '
Antisense strand of Target siRNA-20 (SEQ ID NO: 7):
5'-UUUCGUACCUUGUAGCCUCUU-3 '
Target siRNA-20 sense strand (SEQ ID NO: 8):
5'-GAGGCUACAGAGGUACGAAAUU-3 '

本発明のｓｉＲＮＡの構成ヌクレオチド配列は上記配列番号１〜８に示す配列であるが、これらの配列において、１個、２個、または３個の塩基が付加、欠失、置換されていてもよい。さらに本発明のｓｉＲＮＡには、安定性を高めるために、例えば、３’末端に２塩基、例えばＵＵなどのオーバーハングを付加しているが、それ以外の配列を付加してもよく、あるいは３’末端のＵＵを除いた配列であってもよい。従って、本発明のスキルス胃がん細胞株の増殖を阻害するｓｉＲＮＡは、下記に示す配列番号９および１０、１１および１２、１３および１４、または１５および１６で表されるいずれかのＲＮＡ配列の組からなる二本鎖で構成されるｓｉＲＮＡであってもよい。
Target siRNA-17のアンチセンス鎖から３’末端のＵＵを除いた配列（配列番号９）：
５’−ＧＡＵＵＧＡＵＧＧＡＣＣＣＧＵＡＵＵＣ−３’
Target siRNA-17のセンス鎖から３’末端のＵＵを除いた配列（配列番号１０）：
５’−ＧＡＡＵＡＣＧＧＧＵＣＣＡＵＣＡＡＵＣ−３’
Target siRNA-18のアンチセンス鎖から３’末端のＵＵを除いた配列（配列番号１１）：
５’−ＵＣＡＧＡＵＧＧＧＡＣＣＡＣＡＣＵＵＵ−３’
Target siRNA-18のセンス鎖から３’末端のＵＵを除いた配列（配列番号１２）：
５’−ＡＡＡＧＵＧＵＧＧＵＣＣＣＡＵＣＵＧＡ−３’
Target siRNA-19のアンチセンス鎖から３’末端のＵＵを除いた配列（配列番号１３）：
５’−ＵＧＵＵＧＧＵＣＣＡＧＵＡＵＧＧＵＧＣ−３’
Target siRNA-19のセンス鎖から３’末端のＵＵを除いた配列（配列番号１４）：
５’−ＧＣＡＣＣＡＵＡＣＵＧＧＡＣＣＡＡＣＡ−３’
Target siRNA-20のアンチセンス鎖から３’末端のＵＵを除いた配列（配列番号１５）：
５’−ＵＵＵＣＧＵＡＣＣＵＵＧＵＡＧＣＣＵＣ−３’
Target siRNA-20のセンス鎖から３’末端のＵＵを除いた配列（配列番号１６）：
５’−ＧＡＧＧＣＵＡＣＡＡＧＧＵＡＣＧＡＡＡ−３’ The nucleotide sequence of the siRNA of the present invention is the sequence shown in the above SEQ ID Nos. 1 to 8, but in these sequences, 1, 2 or 3 bases may be added, deleted or substituted. . Furthermore, in order to improve stability, the siRNA of the present invention has an overhang such as 2 bases, for example, UU, added to the 3 ′ end, but other sequences may be added, or 3 'It may be a sequence excluding the terminal UU. Therefore, the siRNA that inhibits the growth of the Skills gastric cancer cell line of the present invention is from any RNA sequence set represented by SEQ ID NOs: 9 and 10, 11 and 12, 13 and 14, or 15 and 16 shown below. The siRNA comprised by the double strand which consists of may be sufficient.
Sequence obtained by removing the UU at the 3 ′ end from the antisense strand of Target siRNA-17 (SEQ ID NO: 9):
5'-GAUUGAUGGACCCCGUAUUC-3 '
Sequence obtained by removing UU at the 3 ′ end from the sense strand of Target siRNA-17 (SEQ ID NO: 10):
5'-GAAUACGGGUCCAUCAAUC-3 '
Sequence obtained by removing the UU at the 3 ′ end from the antisense strand of Target siRNA-18 (SEQ ID NO: 11):
5'-UCAGAUGGGACCACACUUU-3 '
Sequence obtained by removing UU at the 3 ′ end from the sense strand of Target siRNA-18 (SEQ ID NO: 12):
5'-AAAGUGUGGUCCCAUCUGA-3 '
Sequence obtained by removing UU at the 3 ′ end from the antisense strand of Target siRNA-19 (SEQ ID NO: 13):
5'-UGUUGGUCCAGUAUGGUGC-3 '
Sequence obtained by removing UU at the 3 ′ end from the sense strand of Target siRNA-19 (SEQ ID NO: 14):
5'-GCACCAUAUCGUGACCAACA-3 '
Sequence obtained by removing the UU at the 3 ′ end from the antisense strand of Target siRNA-20 (SEQ ID NO: 15):
5'-UUCUCGUACCUUGAGCCUC-3 '
Sequence obtained by removing UU at the 3 ′ end from the sense strand of Target siRNA-20 (SEQ ID NO: 16):
5'-GAGGCUACAGAGGUACGAAA-3 '

本発明の上記ｓｉＲＮＡは当業者間で一般的な方法により作成することができる。本発明のｓｉＲＮＡを作成するのに最適な方法はｓｉＲＮＡを化学的に合成することであり、これ以外にも、ｓｉＲＮＡ発現ベクターに目的のｓｉＲＮＡヌクレオチド配列をクローン化して増幅すること、ｓｉＲＮＡを酵素を使用してインビトロ転写すること、ならびにｓｉＲＮＡ発現カセットをＰＣＲで合成することなどにより作成してもよいが、これらだけに限らない。 The above siRNA of the present invention can be prepared by a general method among those skilled in the art. The most suitable method for preparing the siRNA of the present invention is to chemically synthesize siRNA. In addition to this, the siRNA expression vector is cloned and amplified in an siRNA expression vector, and the siRNA is converted into an enzyme. It may be prepared by using in vitro transcription and synthesizing siRNA expression cassettes by PCR, but is not limited thereto.

本発明では、本発明のｓｉＲＮＡを有効成分とする医薬組成物を提供する。本発明の医薬組成物には、配列番号１〜１６で表される８種類のｓｉＲＮＡのうち１種以上を含んでよく、好ましくは配列番号５および６で表される組のｓｉＲＮＡを含むことである。さらに本発明の医薬組成物は、公知の抗がん剤を含んでいてもよい。 In this invention, the pharmaceutical composition which uses siRNA of this invention as an active ingredient is provided. The pharmaceutical composition of the present invention may contain one or more of 8 types of siRNAs represented by SEQ ID NOs: 1 to 16, and preferably contains siRNAs of a set represented by SEQ ID NOs: 5 and 6. is there. Furthermore, the pharmaceutical composition of the present invention may contain a known anticancer agent.

本発明の医薬組成物ではまた、有効成分としてのｓｉＲＮＡが製造業者から市販されているｓｉＲＮＡ発現ベクターなどの公知のｓｉＲＮＡ発現ベクターまたは使用の態様に合わせて適宜組み換えた公知のｓｉＲＮＡ発現ベクターにクローン化されていてもよい。 In the pharmaceutical composition of the present invention, siRNA as an active ingredient is also cloned into a known siRNA expression vector such as a siRNA expression vector commercially available from the manufacturer or a known siRNA expression vector appropriately recombined according to the mode of use. May be.

本発明の医薬組成物は、医薬上許容されるキャリアを含んでもよい。本発明で用いることができる医薬上許容されるキャリアは、生理食塩水、蒸留水、リンゲル液、緩衝生理食塩水、ブドウ糖溶液、マルトブドウ糖溶液、グリセロール、エタノールおよびリポソームからなる群から選択される１種またはそれ以上の成分であってよいが、これらだけに限らない。さらに本発明の医薬組成物に抗酸化剤、緩衝水溶液、静菌剤などのごとき他の一般的な添加物を添加してもよい。また、注射用溶液、ピル、カプセル、顆粒もしくはタブレット錠剤を製造するために、希釈剤、噴霧剤、界面活性剤、結合剤および潤滑剤をさらに添加してもよい。 The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier that can be used in the present invention is one selected from the group consisting of physiological saline, distilled water, Ringer's solution, buffered physiological saline, glucose solution, maltoglucose solution, glycerol, ethanol, and liposome. Or it may be a component more than it, but it is not restricted only to these. Furthermore, you may add other general additives, such as an antioxidant, buffered aqueous solution, a bacteriostatic agent, to the pharmaceutical composition of this invention. Diluents, sprays, surfactants, binders and lubricants may also be added to produce injectable solutions, pills, capsules, granules or tablet tablets.

本発明の医薬組成物を投与する形態は、経口投与または非経口投与（例えば、静脈内、皮下内、局所または腹膜の注射）であってもよく、患部である胃またはその近傍に効率的に有効成分を送達できればそれら以外の投与形態であってもよい。また、本発明の医薬組成物として投与される本発明のｓｉＲＮＡの有効な量を、患者の状態、例えば患者の重量、年齢、性別、および健康状態など、ならびに食事量、投与の頻度、投与の方法、排泄量および病気の重症度などによって決定することができる。本発明の医薬組成物として投与される本発明のｓｉＲＮＡの有効な量は、通常、１日あたり０．０１−１ｍｇ／ｋｇであり、好ましくは１日あたり０．０２−０．１ｍｇ／ｋｇである。 The pharmaceutical composition of the present invention may be administered orally or parenterally (for example, intravenous, subcutaneous, topical or peritoneal injection), and efficiently in or near the affected stomach. Other dosage forms may be used as long as the active ingredient can be delivered. In addition, the effective amount of the siRNA of the present invention administered as the pharmaceutical composition of the present invention is determined based on the patient's condition, such as the patient's weight, age, sex, and health status, and the amount of meal, frequency of administration, administration It can be determined by the method, the amount excreted and the severity of the disease. The effective amount of the siRNA of the present invention administered as the pharmaceutical composition of the present invention is usually 0.01-1 mg / kg per day, preferably 0.02-0.1 mg / kg per day. is there.

以下の実施例を示して本発明をさらに詳細かつ具体的に説明するが、実施例は本発明を限定するものではない。 The present invention will be described in more detail and specifically with reference to the following examples, but the examples are not intended to limit the present invention.

ｓｉＲＮＡによるヒト胃がん培養細胞株の増殖阻害
各種ヒト胃がん細胞株（スキルス胃がん株ＫＡＴＯ−ＩＩＩ、低分化充実型腺癌株のＭＫＮ−４５および中分化型腺癌株のＭＫＮ−７４）を用いて、それらの発現たんぱく質をcleavable ＩＣＡＴ（ｃＩＣＡＴ）法で解析した結果、スキルス胃がん細胞株のＫＡＴＯ−ＩＩＩ細胞株はＦＧＦＲ２を選択的に強発現していることを確認した（非特許文献４）。このことは、ＫＡＴＯ−ＩＩＩでのＦＧＦＲ２ｍＲＮＡの選択的強発現を、たんぱく質レベルでも確認したことを示す。そこで、ＦＧＦＲ２のｍＲＮＡレベルでその発現を阻害するｓｉＲＮＡを作成し、そのｓｉＲＮＡのＫＡＴＯ−ＩＩＩ以外のスキルス胃がん細胞株を含めた各種胃がん細胞の増殖に及ぼす効果を調べた。すなわち、ＦＧＦＲ２のｓｉＲＮＡ（DHARMACON）を、各胃がん細胞株（ＫＡＴＯ−ＩＩＩ、ＭＫＮ−４５、ＭＫＮ−７５、ＮＵＧＣ−４およびＯＣＵＭ−１）に、トランスフェクション試薬DharmaFECT 1（DHARMACON）を用いてトランスフェクションを行い、トランスフェクション後１−７日目の細胞増殖阻害効果（１０％牛胎児血清存在下）を、ＭＴＳ法（Promega）を用いて測定した。なお、ＦＧＦＲ２のｓｉＲＮＡとしては４種類（Target siRNA-17、Target siRNA-18、Target siRNA-19およびTarget siRNA-20（表１））を混合したもの（ON-TARGET plus SMART pool FGR2, DHARMACON）およびそれぞれ単独のTarget siRNAを、また、陰性コントロールとしてはNon-Target siRNAを使用し、各細胞株への最適濃度条件下でトランスフェクションを行った。
（表１）
ＦＧＦＲ２に対するｓｉＲＮＡの種類と配列

Inhibition of growth of cultured human gastric cancer cell lines by siRNA Using various human gastric cancer cell lines (Skills gastric cancer line KATO-III, MKN-45 of poorly differentiated solid adenocarcinoma line and MKN-74 of moderately differentiated adenocarcinoma line) As a result of analyzing the expressed proteins by the cleavable ICAT (cICAT) method, it was confirmed that the KATO-III cell line of the Skills gastric cancer cell line selectively and strongly expresses FGFR2 (Non-patent Document 4). This indicates that selective strong expression of FGFR2 mRNA in KATO-III was also confirmed at the protein level. Therefore, siRNA that inhibits its expression at the mRNA level of FGFR2 was prepared, and the effect of the siRNA on the growth of various gastric cancer cells including Skills gastric cancer cell lines other than KATO-III was examined. That is, FGFR2 siRNA (DHARMACON) was transfected into each gastric cancer cell line (KATO-III, MKN-45, MKN-75, NUGC-4 and OCUM-1) using the transfection reagent DharmaFECT 1 (DHARMACON). And the cell growth inhibitory effect (in the presence of 10% fetal bovine serum) 1-7 days after transfection was measured using the MTS method (Promega). FGFR2 siRNA is a mixture of four types (Target siRNA-17, Target siRNA-18, Target siRNA-19 and Target siRNA-20 (Table 1)) (ON-TARGET plus SMART pool FGR2, DHARMACON) Each target cell line was transfected using optimal Target siRNA and Non-Target siRNA as a negative control.
(Table 1)
SiRNA types and sequences for FGFR2

その結果、ｓｉＲＮＡ４種類の混合物（ON-TARGET plus SMART pool FGFR2, DHARMACON）をトランスフェクションさせた場合、ＫＡＴＯ−ＩＩＩ細胞株が最も強い阻害効果を受け、また、ＮＵＧＣ−４株も弱いながら阻害効果を受けた。一方、低分化非充実型腺がん細胞株のＭＫＮ−４５と中分化型腺がん細胞株のＭＫＮ−７４は、ほとんど阻害効果を受けなかった（図１）。また、スキルス胃がん細胞株のＯＣＵＭ−１は、ＦＧＦＲ２をＫＡＴＯ−ＩＩＩ株の５％程度発現しているにも関わらず、ほとんど阻害効果を受けなかった。なお、増殖阻害実験において、ＭＴＳ法の代わりに、ＢｒｄＵ法（Roche）で行ってもほぼ同様な結果が得られた（データ省略）。 As a result, when the siRNA four types of mixture (ON-TARGET plus SMART pool FGFR2, DHARMACON) were transfected, the KATO-III cell line received the strongest inhibitory effect, and the NUGC-4 strain also had a weak inhibitory effect. I received it. On the other hand, MKN-45, a poorly differentiated non-solid adenocarcinoma cell line, and MKN-74, a moderately differentiated adenocarcinoma cell line, received almost no inhibitory effect (FIG. 1). Skills gastric cancer cell line OCUM-1 was hardly affected even though FGFR2 was expressed in about 5% of KATO-III strain. In addition, in the growth inhibition experiment, almost the same results were obtained when the BrdU method (Roche) was used instead of the MTS method (data not shown).

次に、４種のｓｉＲＮＡのうち、どの配列を持つｓｉＲＮＡが最も阻害効果が強いかを、ＫＡＴＯ−ＩＩＩ、ＮＵＧＣ−４細胞株を用いて調べた。表１に各ｓｉＲＮＡのアンチセンス鎖のＲＮＡ配列とセンス鎖のＲＮＡ配列を示す。その結果、ＫＡＴＯ−ＩＩＩの場合、いずれのｓｉＲＮＡも陰性コントロールのNon-target siRNAより阻害効果は示したが、最も阻害効果の強いｓｉＲＮＡはTarget siRNA-19であり、ついでTarget siRNA-20、Target siRNA-18の順であった（図２）。また、ＮＵＧＣ−４の場合では、Target siRNA-19のみが阻害効果を示した。従って、Target siRNA-19が本発明のうち最も阻害効果の強いｓｉＲＮＡであると考えられる。 Next, it was investigated which siRNA having the strongest inhibitory effect among the four siRNAs using KATO-III and NUGC-4 cell lines. Table 1 shows the RNA sequence of the antisense strand and the RNA sequence of the sense strand of each siRNA. As a result, in the case of KATO-III, all siRNAs showed an inhibitory effect than the negative control Non-target siRNA, but the siRNA having the strongest inhibitory effect was Target siRNA-19, followed by Target siRNA-20, Target siRNA. -18 (Fig. 2). In the case of NUGC-4, only Target siRNA-19 showed an inhibitory effect. Therefore, it is considered that Target siRNA-19 is the most effective siRNA in the present invention.

以上のことより、ＦＧＦＲ２のｓｉＲＮＡは少なくともスキルス胃がん細胞株のある種のものに対しては増殖阻害効果を示し、その中でも最も強い阻害作用を示し、ＫＡＴＯ−ＩＩＩ細胞株およびＮＵＧＣ−４細胞株の両方に効果のあるｓｉＲＮＡはTarget siRNA-19であることが判明した。 From the above, siRNA of FGFR2 exhibits a growth inhibitory effect on at least certain types of Skills gastric cancer cell lines, and exhibits the strongest inhibitory action among them, KATO-III cell line and NUGC-4 cell line. An effective siRNA for both was found to be Target siRNA-19.

本発明は、がんの医薬分野およびがんの研究分野等において利用可能である。詳細には、スキルスがんの治療薬の製造分野およびスキルスがんの研究分野などにおいて利用可能である。 The present invention can be used in the medical field of cancer and the research field of cancer. Specifically, it can be used in the field of manufacturing a therapeutic drug for skill cancer and the field of research for skill cancer.

ＦＧＦＲ２のTarget siRNA４種混合物（ON-TARGET plus SMART pool FGFR2）による細胞増殖抑制実験の結果を示す。The result of the cell growth suppression experiment by Target siRNA 4 types mixture (ON-TARGET plus SMART pool FGFR2) of FGFR2 is shown. 各種ＦＧＦＲ２ｓｉＲＮＡ（Target siRNA-17、-18、-19および-20）による増殖阻害効果を示す。The growth inhibitory effect by various FGFR2 siRNA (Target siRNA-17, -18, -19, and -20) is shown.

SEQ ID NO:1:FGFR2 siRNA
SEQ ID NO:2:FGFR2 siRNA
SEQ ID NO:3:FGFR2 siRNA
SEQ ID NO:4:FGFR2 siRNA
SEQ ID NO:5:FGFR2 siRNA
SEQ ID NO:6:FGFR2 siRNA
SEQ ID NO:7:FGFR2 siRNA
SEQ ID NO:8:FGFR2 siRNA
SEQ ID NO:9:FGFR2 siRNA
SEQ ID NO:10:FGFR2 siRNA
SEQ ID NO:11:FGFR2 siRNA
SEQ ID NO:12:FGFR2 siRNA
SEQ ID NO:13:FGFR2 siRNA
SEQ ID NO:14:FGFR2 siRNA
SEQ ID NO:15:FGFR2 siRNA
SEQ ID NO:16:FGFR2 siRNA SEQ ID NO: 1: FGFR2 siRNA
SEQ ID NO: 2: FGFR2 siRNA
SEQ ID NO: 3: FGFR2 siRNA
SEQ ID NO: 4: FGFR2 siRNA
SEQ ID NO: 5: FGFR2 siRNA
SEQ ID NO: 6: FGFR2 siRNA
SEQ ID NO: 7: FGFR2 siRNA
SEQ ID NO: 8: FGFR2 siRNA
SEQ ID NO: 9: FGFR2 siRNA
SEQ ID NO: 10: FGFR2 siRNA
SEQ ID NO: 11: FGFR2 siRNA
SEQ ID NO: 12: FGFR2 siRNA
SEQ ID NO: 13: FGFR2 siRNA
SEQ ID NO: 14: FGFR2 siRNA
SEQ ID NO: 15: FGFR2 siRNA
SEQ ID NO: 16: FGFR2 siRNA

Claims

An siRNA that inhibits the growth of Schirus gastric cancer cell line by inhibiting the expression of FGFR2 protein, wherein any of the RNA sequences represented by SEQ ID NOs: 1 and 2, 3 and 4, 5 and 6, or 7 and 8 SiRNA comprised by the double strand which consists of these groups.

An siRNA that inhibits the growth of Schirus gastric cancer cell line by inhibiting the expression of FGFR2 protein, wherein any of the RNA sequences represented by SEQ ID NOs: 9 and 10, 11 and 12, 13 and 14, or 15 and 16 SiRNA comprised by the double strand which consists of these groups.

An siRNA consisting of a set of RNA sequences represented by SEQ ID NOs: 5 and 6, which inhibits the growth of KATO-III cell line and NUGC-4 cell line.

A pharmaceutical composition for the treatment of Skills gastric cancer, comprising one or more siRNAs according to any one of claims 1 to 3 as an active ingredient.

A pharmaceutical composition for the treatment of Skills gastric cancer comprising the siRNA according to claim 3 as an active ingredient.