JP7454207B2 - Pancreatic cancer cell invasion and metastasis inhibitor - Google Patents
Pancreatic cancer cell invasion and metastasis inhibitor Download PDFInfo
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Description
本発明は、膵癌細胞の浸潤や転移を有効に抑制できる膵癌細胞浸潤転移阻害剤に関するものである。 The present invention relates to an inhibitor of pancreatic cancer cell invasion and metastasis that can effectively suppress invasion and metastasis of pancreatic cancer cells.
「腫瘍」とは異常に増殖した細胞を指し、その異常増殖の原因が消失あるいは取り除かれても細胞の増殖が持続する状態をいう。腫瘍の中でも良性腫瘍は腫瘍の増殖が遅く、転移はしない。よって、一般的には切除すれば問題は無く、たとえ切除せずに放置しておいても命に別状はないといえる。一方、悪性腫瘍、即ちがんは、良性腫瘍とは異なり急速に増殖する上に、リンパ節や他の臓器に転移して増殖する。よって、例えば外科的手術により除去しても、僅かにでも残留したがん細胞や、既にリンパ節や他の臓器に転移していたがん細胞が再び増殖を開始することがある。よって、がんはいったん治療が終了した後の予後が悪く、各がんにおいては5年後生存率が調査されており、一般的に、治療によりがんが消失したとされてから5年経過後までに再発がない場合がようやく治癒と見なされる。 "Tumor" refers to abnormally proliferated cells, and refers to a state in which the cells continue to proliferate even after the cause of the abnormal proliferation has disappeared or been removed. Among tumors, benign tumors grow slowly and do not metastasize. Therefore, in general, there is no problem if it is removed, and even if it is left unresected, there is no danger to life. On the other hand, malignant tumors, that is, cancer, differ from benign tumors in that they proliferate rapidly and also metastasize and proliferate to lymph nodes and other organs. Therefore, even if cancer cells are removed by surgery, for example, even a small amount of cancer cells may remain, or cancer cells that have already metastasized to lymph nodes or other organs may start to proliferate again. Therefore, cancer has a poor prognosis once treatment is completed, and the survival rate of each cancer is investigated after 5 years. The disease is considered cured only when there is no recurrence.
膵癌は、がんの中で最も予後が悪いといわれている。その原因としては、膵臓が後腹膜臓器であるために早期発見が困難であることに加え、膵癌細胞の運動性が極めて高いため、例えば2cm以下の小さながんであっても、周囲の血管、胆管、神経などへすぐに浸潤し、また、近くのリンパ節に転移したり、肝臓などへ遠隔転移したりすることが挙げられる。 Pancreatic cancer is said to have the worst prognosis among cancers. This is because the pancreas is a retroperitoneal organ, which makes early detection difficult, and because pancreatic cancer cells have extremely high motility, even if the cancer is small, for example, 2 cm or less, surrounding blood vessels, bile ducts, etc. , it can quickly invade nerves, etc., and can also metastasize to nearby lymph nodes and distant metastases, such as to the liver.
上記のとおり、がんが悪性の腫瘍である所以は、特に他組織へ浸潤や転移することにあり、浸潤転移さえしなければ予後も良好なものになると考えられる。本発明者は、特に浸潤転移し易く予後の悪い膵癌の細胞の浸潤転移機構につき研究を進めてきた。 As mentioned above, cancer is a malignant tumor because it particularly invades and metastasizes to other tissues, and it is thought that the prognosis will be good if it does not invade and metastasize. The present inventors have been conducting research on the mechanism of invasion and metastasis of pancreatic cancer cells, which are particularly prone to invasion and metastasis and have a poor prognosis.
例えば本発明者は、ヒトインスリン様成長因子2mRNA結合タンパク質3(IGF2BP3)が膵癌細胞の浸潤転移に大きく関わっており、その部分ペプチドが膵癌細胞浸潤転移抑制ワクチンの有効成分として使用できること(特許文献1)や、膵癌細胞の細胞膜突起中のIGF2BP3に結合する特定のmRNAの翻訳を阻害するRNAが膵癌細胞浸潤転移阻害剤の有効成分として使用できること(特許文献2)を見出している。また、膵癌細胞の細胞膜突起に集積するRNAであって、エクソソームにより膵癌細胞外へ放出されるもの(特許文献3)や、膵癌細胞の細胞膜突起に集積する特定の糖タンパク質(特許文献4)が膵癌マーカーとして用いられ得ることを見出している。
For example, the present inventor has discovered that human insulin-
上述したように、本発明者は膵癌細胞の浸潤転移機構やその阻害方法などにつき研究を続けているが、ある統計では、膵癌はがんで死亡したケースの中で4番目に多いがんであり、有効な治療手段が依然として切望されている。
そこで本発明は、膵癌細胞の浸潤や転移を有効に抑制できる膵癌細胞浸潤転移阻害剤を提供することを目的とする。
As mentioned above, the present inventor continues to research the invasion and metastasis mechanism of pancreatic cancer cells and methods for inhibiting the same, but according to certain statistics, pancreatic cancer is the fourth most common cancer among cancer deaths. Effective therapeutic measures remain desperately needed.
Therefore, an object of the present invention is to provide an inhibitor of pancreatic cancer cell invasion and metastasis that can effectively suppress invasion and metastasis of pancreatic cancer cells.
本発明者は、上記課題を解決するために鋭意研究を重ねた。その結果、KH型スプライシング調節タンパク質が膵癌細胞の浸潤転移に関与しており、KH型スプライシング調節タンパク質RNAおよび/またはKH型スプライシング調節タンパク質に結合するsnoRNAをノックダウンすることにより膵癌細胞の浸潤転移を有効に抑制できることを見出して、本発明を完成した。
以下、本発明を示す。
The present inventor has conducted extensive research in order to solve the above problems. As a result, KH-type splicing regulatory protein is involved in the invasion and metastasis of pancreatic cancer cells, and knocking down KH-type splicing regulatory protein RNA and/or snoRNA that binds to KH-type splicing regulatory protein inhibits invasion and metastasis of pancreatic cancer cells. The present invention was completed by discovering that it can be effectively suppressed.
The present invention will be described below.
[1] KH型スプライシング調節タンパク質RNAおよび/またはKH型スプライシング調節タンパク質結合性snoRNAをノックダウンする化合物を有効成分として含むことを特徴とする膵癌細胞浸潤転移阻害剤。
[2] 上記化合物が、上記KH型スプライシング調節タンパク質結合性snoRNAに対するsiRNAである上記[1]に記載の膵癌細胞浸潤転移阻害剤。
[3] 上記化合物を含むナノ粒子を含む上記[1]または[2]に記載の膵癌細胞浸潤転移阻害剤。
[4] 上記KH型スプライシング調節タンパク質結合性snoRNAが、SNORA18およびSNORA22の少なくとも一方である上記[1]~[3]のいずれかに記載の膵癌細胞浸潤転移阻害剤。
[5] 上記KH型スプライシング調節タンパク質結合性snoRNAがSNORA18である上記[1]~[3]のいずれかに記載の膵癌細胞浸潤転移阻害剤。
[1] An inhibitor of pancreatic cancer cell invasion and metastasis, which comprises as an active ingredient a compound that knocks down KH-type splicing regulatory protein RNA and/or KH-type splicing regulatory protein-binding snoRNA.
[2] The pancreatic cancer cell invasion and metastasis inhibitor according to [1] above, wherein the compound is siRNA against the KH type splicing regulatory protein-binding snoRNA.
[3] The pancreatic cancer cell invasion and metastasis inhibitor according to [1] or [2] above, which contains nanoparticles containing the above compound.
[4] The pancreatic cancer cell invasion and metastasis inhibitor according to any one of [1] to [3] above, wherein the KH-type splicing regulatory protein-binding snoRNA is at least one of SNORA18 and SNORA22.
[5] The pancreatic cancer cell invasion and metastasis inhibitor according to any one of [1] to [3] above, wherein the KH type splicing regulatory protein-binding snoRNA is SNORA18.
本発明に係るKH型スプライシング調節タンパク質(KHSRP)は、従来、核内でのスプライシングや細胞質内でのmRNAの局在化と分解など、様々な細胞プロセスに関与するRNA結合性タンパク質であることが知られていたが、この度、本発明者は、KHSRPが膵癌細胞の細胞膜突起(葉状仮足)中にも局在し、膵癌細胞の運動性に関与していることを明らかにし、更にKHSRP自体のRNAまたはKHSRP結合性snoRNAをノックダウンすることにより膵癌細胞の浸潤転移を抑制できることを見出した。よって本発明は、膵癌細胞の浸潤転移を抑制できるものとして、非常に有用なものである。 The KH-type splicing regulatory protein (KHSRP) according to the present invention has conventionally been known to be an RNA-binding protein involved in various cellular processes such as splicing in the nucleus and localization and degradation of mRNA in the cytoplasm. However, the present inventors have now revealed that KHSRP is also localized in the cell membrane protrusions (lamellipodia) of pancreatic cancer cells and is involved in the motility of pancreatic cancer cells. It has been found that invasion and metastasis of pancreatic cancer cells can be suppressed by knocking down RNA or KHSRP-binding snoRNA. Therefore, the present invention is extremely useful as it can suppress invasion and metastasis of pancreatic cancer cells.
本発明に係る膵癌細胞浸潤転移阻害剤は、KH型スプライシング調節タンパク質RNAおよび/またはKH型スプライシング調節タンパク質結合性snoRNAをノックダウンする化合物を有効成分として含む。 The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention contains as an active ingredient a compound that knocks down KH-type splicing regulatory protein RNA and/or KH-type splicing regulatory protein-binding snoRNA.
KH型スプライシング調節タンパク質(KHSRP:KH-type Splicing Regulatory Protein)は、mRNAのAU-rich element(ARE)を認識する4つの隣接するK homology(KH)モチーフを含む一本鎖核酸結合性タンパク質であり、核内や細胞質顆粒中に存在し、mRNAのスプライシング、編集、局在、分解などに関与することが知られている。本発明者は、KHSRPが膵癌細胞の細胞膜突起中にも局在し、膵癌細胞の浸潤転移を促進することを見出し、更にKHSRP自体のRNAおよび/またはKHSRPに結合するsnoRNAをノックダウンすることにより膵癌細胞の浸潤転移を抑制できることを見出した。 KH-type splicing regulatory protein (KHSRP) is a single-stranded nucleic acid-binding protein containing four adjacent K homology (KH) motifs that recognize the AU-rich element (ARE) of mRNA. , is present in the nucleus and cytoplasmic granules, and is known to be involved in mRNA splicing, editing, localization, degradation, etc. The present inventors discovered that KHSRP is also localized in the cell membrane protrusions of pancreatic cancer cells and promotes invasion and metastasis of pancreatic cancer cells. It was found that invasion and metastasis of pancreatic cancer cells can be suppressed.
KHSRPのアミノ酸配列を配列番号1に、KHSRPの遺伝子の塩基配列を配列番号2に示す。配列番号2の塩基配列中、第111~第2246位がコーディング領域である。 The amino acid sequence of KHSRP is shown in SEQ ID NO: 1, and the nucleotide sequence of the KHSRP gene is shown in SEQ ID NO: 2. In the base sequence of SEQ ID NO: 2, positions 111 to 2246 are the coding region.
KHSRP RNAをノックダウンする化合物は、KHSRPをノックダウンできるものであれば特に制限されないが、例えば、KHSRP RNAの一部に結合するsiRNAおよびshRNAが挙げられる。KHSRP RNAに結合するsiRNAおよびshRNAは、KHSRP RNAの切断を誘導する。これらRNAは、細胞内に導入されても染色体内に組み込まれず変異を起こさないことから比較的安全である。また、siRNAは化学合成が比較的容易であり、二本鎖状態では安定である。 The compound that knocks down KHSRP RNA is not particularly limited as long as it can knock down KHSRP, and examples thereof include siRNA and shRNA that bind to a part of KHSRP RNA. siRNA and shRNA that bind to KHSRP RNA induce cleavage of KHSRP RNA. These RNAs are relatively safe because they are not integrated into chromosomes and do not cause mutations even when introduced into cells. In addition, siRNA is relatively easy to chemically synthesize and is stable in a double-stranded state.
siRNAは、標的RNAの部分塩基配列と相同なセンスRNAと、当該センスRNAとハイブリダイズ可能なアンチセンスRNAがハイブリダイズした21~23塩基対の二本鎖RNAであり、通常、3’末端はOHのままである一方で5’末端がリン酸化され、3’末端側は1塩基以上、4塩基以下突出していてもよい。siRNAには特異的なタンパク質が結合し、RISC(RNA-Induced-Silencing-Complex)と呼ばれる複合体が形成される。RISCはセンスRNAの塩基配列との相同配列を有するRNAを認識して結合し、RNaseIII様の酵素活性によりRNAを切断する。 siRNA is a double-stranded RNA of 21 to 23 base pairs, which is made by hybridizing a sense RNA that is homologous to a partial base sequence of the target RNA and an antisense RNA that can hybridize with the sense RNA. While remaining as OH, the 5' end may be phosphorylated, and the 3' end may protrude by at least 1 base and at most 4 bases. A specific protein binds to siRNA, forming a complex called RISC (RNA-Induced-Silencing-Complex). RISC recognizes and binds to RNA having a sequence homologous to the base sequence of sense RNA, and cleaves the RNA with RNase III-like enzymatic activity.
shRNAは一本鎖RNAからなり、標的RNAの部分塩基配列と相同なセンスRNA領域と、当該センスRNAとハイブリダイズ可能なアンチセンスRNA領域が環状構造を示すリンカーRNAにより結合されており、全体としてヘアピン状の構造を有する。shRNAは3’末端が1塩基以上、4塩基以下突出していてもよく、また、当該3’突出末端はDNAで構成されていてもよい。shRNAは、細胞内で分解され、siRNAと同様にRNA干渉を引き起こす。 shRNA consists of a single-stranded RNA, in which a sense RNA region homologous to a partial base sequence of the target RNA and an antisense RNA region capable of hybridizing with the sense RNA are connected by a linker RNA having a circular structure, and as a whole, It has a hairpin-like structure. The 3' end of shRNA may protrude by 1 or more bases and 4 or less bases, and the 3' protruding end may be composed of DNA. shRNA is degraded within cells and causes RNA interference similar to siRNA.
KHSRP RNAをノックダウンするための標的配列は、RNAiにより有効にノックダウンされるものであれば特に制限されないが、例えば、配列番号3~68の塩基配列のKHSRP RNAを挙げることができる。なお、配列番号3~68の塩基配列は、KHSRP遺伝子の21ntの塩基配列に加えて、2ntのoverhang配列を含んでいる。配列番号3~68の塩基配列は、特に制限されないが、KHSRP RNAをノックダウンするためのsiRNAのセンス鎖に含まれていることが好ましい。KHSRP RNAをノックダウンする化合物は、1種のみ用いてもよいが、2種以上を組み合わせて用いることが好ましい。 The target sequence for knocking down KHSRP RNA is not particularly limited as long as it can be effectively knocked down by RNAi, and examples thereof include KHSRP RNA having the base sequences of SEQ ID NOs: 3 to 68. Note that the base sequences of SEQ ID NOs: 3 to 68 include a 2nt overhang sequence in addition to the 21nt base sequence of the KHSRP gene. Although the base sequences of SEQ ID NOs: 3 to 68 are not particularly limited, they are preferably included in the sense strand of siRNA for knocking down KHSRP RNA. Although only one type of compound that knocks down KHSRP RNA may be used, it is preferable to use two or more types in combination.
KHSRP結合性RNAは、膵癌細胞の細胞質の顆粒、特にプロセッシングボディ(P-body)において、KHSRPに結合しているRNAをいう。P-bodyは細胞質に特徴的にみられる粒子様のRNA-タンパク質複合体で、多くのRNA代謝の場となっている。その構成タンパク質は細胞や種によっても大きく異なり、RNAの貯蔵や分解などいろいろな機能を果たす可能性がある。例えば,マイクロRNAによる翻訳抑制や,ポリAの短縮によるRNAの分化の場としても機能している。構成するタンパク質によって機能が変わりうる場所ともいえる(実験医学,2009年2月号,Vol.27,No.3)。 KHSRP-binding RNA refers to RNA that binds to KHSRP in cytoplasmic granules of pancreatic cancer cells, particularly in the processing body (P-body). P-bodies are particle-like RNA-protein complexes that are characteristically found in the cytoplasm, and are the site of many RNA metabolisms. Its constituent proteins vary greatly depending on the cell and species, and may perform a variety of functions such as RNA storage and degradation. For example, it functions as a site for translational suppression by microRNA and RNA differentiation by shortening polyA. It can also be said that the function can change depending on the constituent proteins (Jikken Igaku, February 2009 issue, Vol. 27, No. 3).
snoRNA(small nucleolar RNA,核小体低分子RNA)は、non-coding RNAの一種であり、タンパク質と複合体を形成してsnoRNP(small nucleolar ribonucleoprotein)として機能し、rRNAの修飾やプロセシングに関与する。KHSRP結合性RNAであるsnoRNAとしては、例えばSCARNA21、SNORA38、SNORA25、SNORA18、SNORA22、SNORA14B、SNORA71B、SNORA71D、SNORD97等が挙げられ、SNORA18およびSNORA22から選択される少なくとも一方が好ましく、SNORA18のノックダウンにより膵癌細胞の後腹膜浸潤が顕著に抑制されることからSNORA18が特に好ましい。 SnoRNA (small nucleolar RNA, nucleolar small RNA) is a type of non-coding RNA that forms a complex with proteins and functions as snoRNP (small nucleolar ribonucleoprotein), and is involved in rRNA modification and processing. . Examples of snoRNAs that are KHSRP-binding RNAs include SCARNA21, SNORA38, SNORA25, SNORA18, SNORA22, SNORA14B, SNORA71B, SNORA71D, and SNORD97, and at least one selected from SNORA18 and SNORA22 Preferably, by knockdown of SNORA18 SNORA18 is particularly preferred because retroperitoneal invasion of pancreatic cancer cells is significantly suppressed.
KHSRP結合性snoRNAをノックダウンする化合物は、KHSRP結合性snoRNAをノックダウンできるものであれば特に制限されないが、例えば、標的snoRNAに結合するsiRNAおよびshRNAが挙げられる。但し、miRNAは、標的mRNAの3’非翻訳領域に結合して翻訳を阻害するものであり、snoRNAはnon-coding RNAであるので、上記化合物はmiRNAでないことが好ましい。 The compound that knocks down KHSRP-binding snoRNA is not particularly limited as long as it can knock down KHSRP-binding snoRNA, and examples thereof include siRNA and shRNA that bind to target snoRNA. However, since miRNA inhibits translation by binding to the 3' untranslated region of target mRNA, and snoRNA is non-coding RNA, it is preferable that the above compound is not miRNA.
KHSRP結合性snoRNAの中で、例えばSNORA18は配列番号69の塩基配列を有し、SNORA22は配列番号70の塩基配列を有する。RNA干渉の標的となる塩基対数は21~23塩基対であることから、これら塩基配列の中から21~23塩基対を標的として選択すればよい。特に、SNORA18に対するsiRNAまたはshRNAとしては配列番号71~74を有するものが好ましく、SNORA22に対するsiRNAまたはshRNAとしては配列番号75~78を有するものが好ましい。これら好適塩基配列は、センス鎖の塩基配列でもアンチセンス鎖の塩基配列であってもよい。 Among the KHSRP-binding snoRNAs, for example, SNORA18 has the base sequence of SEQ ID NO: 69, and SNORA22 has the base sequence of SEQ ID NO: 70. Since the number of base pairs that can be a target for RNA interference is 21 to 23 base pairs, 21 to 23 base pairs may be selected as a target from among these base sequences. In particular, siRNAs or shRNAs against SNORA18 are preferably those having SEQ ID NOs: 71 to 74, and siRNAs or shRNAs against SNORA22 are preferably those having SEQ ID NOs: 75 to 78. These suitable base sequences may be the base sequences of the sense strand or the base sequences of the antisense strand.
(1)アンチセンス鎖の5’末端がAまたはUであり、(2)センス鎖の5’末端がGまたはCであり、且つ(3)アンチセンス鎖の5’末端部の7塩基中の4塩基以上はAまたはUであるRNAが、RNA干渉効果が高いものとして知られている。本発明で使用するsiRNAおよびshRNAは、かかる塩基配列を有するものであってもよいし、有さないものであってもよい。 (1) The 5' end of the antisense strand is A or U, (2) the 5' end of the sense strand is G or C, and (3) the 7 bases at the 5' end of the antisense strand are RNA in which four or more bases are A or U is known to have a high RNA interference effect. The siRNA and shRNA used in the present invention may or may not have such a base sequence.
siRNAおよびshRNAは、標的RNAをノックダウンすることができるものである限り、その一部または全部が天然のヌクレオチドではなく、天然のデオキシヌクレオチドや非天然ヌクレオチドで構成されているものであってもよい。かかるsiRNAおよびshRNAは、ヌクレアーゼ耐性が改善される他、ノックダウン活性自体が改善されることもある。非天然ヌクレオチドとしては、例えば、天然のヌクレオチドの2’位水酸基が、メトキシ基などのC1-6アルコキシ基、メトキシメトキシ基などのC1-6アルコキシ-C1-6アルコキシ基、フルオロ基などのハロゲノ基で置換されているものや、2’,4’-BNA、3’-アミノ-2’,4’-BNA、2’,4’-BNACOC、2’,4’-BNANC等、2’位と4’位が架橋されているもの等が挙げられる。これら変異は1種のみに限られず、例えば、置換ヌクレオチドと天然デオキシヌクレオチドを交互に配置する等、2種以上の変異を組み合わせてもよい。また、siRNAが有する特徴的な構造である3’末端突出部位(ダングリングエンド)のヌクレオシドを、同様に、天然のデオキシヌクレオチドであるチミジンに置換したり、塩基部欠損ヌクレオチドや、1,3-ベンゼンジメタノールおよび2,6-ピリジンジメタノール等の芳香族化合物で置換してもよい。 siRNA and shRNA may be partially or entirely composed of natural deoxynucleotides or non-natural nucleotides instead of natural nucleotides, as long as they can knock down the target RNA. . Such siRNA and shRNA not only have improved nuclease resistance but also may have improved knockdown activity itself. Examples of non-natural nucleotides include natural nucleotides in which the 2'-position hydroxyl group is a C 1-6 alkoxy group such as a methoxy group, a C 1-6 alkoxy-C 1-6 alkoxy group such as a methoxymethoxy group, a fluoro group, etc. those substituted with a halogeno group, 2',4'-BNA, 3'-amino-2',4'-BNA, 2',4'-BNA COC , 2',4'-BNA NC , etc. , those in which the 2' and 4' positions are crosslinked. These mutations are not limited to one type, and two or more types of mutations may be combined, for example, by alternately arranging substituted nucleotides and natural deoxynucleotides. In addition, the nucleoside at the 3'-end protruding site (dangling end), which is a characteristic structure of siRNA, may be substituted with thymidine, a natural deoxynucleotide, or a nucleoside with a missing base, or a 1,3- Aromatic compounds such as benzenedimethanol and 2,6-pyridine dimethanol may be substituted.
上記好適塩基配列を有するsiRNAおよびshRNAは、膵癌細胞においてRNA干渉作用によりKHSRP RNAまたはKHSRP結合性snoRNAの切断を促し、KHSRPとKHSRP結合性snoRNAとの結合を阻害し、延いては膵癌細胞の浸潤転移を抑制する。但し、上記好適塩基配列中に1塩基の欠失、置換もしくは付加を含む塩基配列を有するRNAであっても、同様にRNA干渉作用を示す場合がある。 siRNA and shRNA having the above-mentioned preferred base sequences promote the cleavage of KHSRP RNA or KHSRP-binding snoRNA in pancreatic cancer cells by RNA interference, inhibit the binding between KHSRP and KHSRP-binding snoRNA, and eventually lead to the invasion of pancreatic cancer cells. Suppress metastasis. However, even RNA having a base sequence that includes deletion, substitution, or addition of one base in the above-mentioned preferred base sequence may similarly exhibit an RNA interference effect.
本発明において「(塩基配列を)有する」とは、RNAが所定の塩基配列のみを有するか、或いは、所定の塩基配列に加え、それ以外の塩基配列を有してもよいとの意味であるが、上述したような5’末端の修飾や、3’末端における突出RNAまたは突出DNA以外、RNAの塩基配列は実質的に所定の塩基配列と同一であることが好ましい。 In the present invention, "having (a base sequence)" means that RNA has only a predetermined base sequence, or may have other base sequences in addition to the predetermined base sequence. However, it is preferable that the base sequence of the RNA is substantially the same as the predetermined base sequence, except for the modification of the 5' end as described above and the protruding RNA or DNA at the 3' end.
本発明に係る膵癌細胞浸潤転移阻害剤は、上記好適標的塩基配列を有するRNAに加え、上記好適標的塩基配列とストリンジェントな条件でハイブリダイズする塩基配列を有するRNAを含むことが好ましい。かかる二重鎖により、RNAをより安定化することができる。 The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention preferably contains, in addition to RNA having the above-mentioned preferred target base sequence, RNA having a base sequence that hybridizes with the above-mentioned preferred target base sequence under stringent conditions. Such double strands can make RNA more stable.
上記二重鎖は完全な相補鎖からなることが好ましいが、ストリンジェントな条件でハイブリダイズする範囲で完全に相補的でなくてもよいものとする。本発明において「ストリンジェントな条件」とは、150mM以上、900mM以下の塩化ナトリウム濃度、15mM以上、90mM以下のクエン酸ナトリウム濃度、即ち、1~6×SSC、0.1質量%以上、0.5質量%以下のSSDの水溶液中、42℃以上、55℃以下の温度で、5分間以上、15分間以下、1回または2回洗浄する条件をいう。また、センス鎖の塩基配列との完全相補配列に対するアンチセンス鎖の塩基配列の相同性としては、90%以上が好ましく、92%以上がより好ましく、95%以上がさらに好ましい。なお、塩基配列の相同性は、当業者であれば市販の配列解析ソフトウェアを用いて容易に決定することができる。 The double strands described above preferably consist of completely complementary strands, but do not need to be completely complementary as long as they hybridize under stringent conditions. In the present invention, "stringent conditions" refer to a sodium chloride concentration of 150 mM or more and 900 mM or less, a sodium citrate concentration of 15 mM or more and 90 mM or less, that is, 1 to 6 x SSC, 0.1% by mass or more, 0. It refers to the conditions of washing once or twice in an aqueous solution of SSD of 5% by mass or less at a temperature of 42° C. or higher and 55° C. or lower for 5 minutes or more and 15 minutes or less. Further, the homology of the base sequence of the antisense strand to a completely complementary sequence with the base sequence of the sense strand is preferably 90% or more, more preferably 92% or more, and still more preferably 95% or more. Note that the homology of base sequences can be easily determined by those skilled in the art using commercially available sequence analysis software.
本発明に係る膵癌細胞浸潤転移阻害剤の一つの態様では、上記センス鎖とアンチセンス鎖がそれぞれ独立に含まれており、これらが二本鎖RNA、即ちsiRNAを形成している。 In one embodiment of the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention, the sense strand and antisense strand are each independently included, and these form double-stranded RNA, ie, siRNA.
また、本発明に係る膵癌細胞浸潤転移阻害剤の別の態様では、上記センス鎖とアンチセンス鎖がリンカーRNAを介して結合した一本鎖RNAが有効成分として含まれており、当該一本鎖RNA、即ちshRNAはヘアピン構造を有しており、上記センス鎖とアンチセンス鎖がハイブリダイズしており、且つリンカーRNAが環状構造を有する。 Further, in another aspect of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention, a single-stranded RNA in which the above-mentioned sense strand and antisense strand are linked via a linker RNA is included as an active ingredient, and the single-stranded RNA is RNA, ie, shRNA, has a hairpin structure, the sense strand and antisense strand are hybridized, and the linker RNA has a circular structure.
上記センス鎖とアンチセンス鎖を含むsiRNA、並びに、上記センス鎖、リンカーRNAおよび上記アンチセンス鎖を含むshRNAは、化学合成してもよいし、T7RNAポリメラーゼを用いてインビトロ合成してもよい。 siRNA containing the sense strand and antisense strand, and shRNA containing the sense strand, linker RNA, and antisense strand may be chemically synthesized or may be synthesized in vitro using T7 RNA polymerase.
本発明に係る膵癌細胞浸潤転移阻害剤は、上記siRNAまたは上記shRNAを有効成分として1種のみ含むものであってもよいし、或いは2種以上含んでいてもよいものとする。2種以上含む場合の数としては、2以上、8以下が好ましく、3以上、5以下がより好ましい。 The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention may contain only one type of the above-mentioned siRNA or the above-mentioned shRNA as an active ingredient, or may contain two or more types of the above-mentioned siRNA or shRNA as an active ingredient. When two or more types are included, the number is preferably 2 or more and 8 or less, more preferably 3 or more and 5 or less.
本発明に係る膵癌細胞浸潤転移阻害剤は、KHSRP RNAまたはKHSRP結合性snoRNAをノックダウンするsiRNAまたはshRNAを有効成分として含むものであってもよいし、当該siRNAまたはshRNAを発現するベクターを有効成分として含むものであってもよい。 The pancreatic cancer cell invasion and metastasis inhibitor according to the present invention may contain as an active ingredient siRNA or shRNA that knocks down KHSRP RNA or KHSRP-binding snoRNA, or may contain a vector expressing the siRNA or shRNA as an active ingredient. It may also be included as
本発明に係る膵癌細胞浸潤転移阻害剤の剤形は、標的部位に有効成分が送達されるものであれば特に制限されず、例えば、注射剤、液剤、徐放剤とすることができる。これら製剤の溶媒としては水が好ましいが、生理食塩水、PBS、血清アルブミン溶液を用いるなどして製剤が最終的に等張液または略等張液となるようにすることが好ましい。その他、本発明に係るRNAを、直接的または間接的に葉酸へ結合させてもよい。膵癌細胞膜の表面には葉酸レセプターが高発現しているため、かかるRNA-葉酸複合体は、膵癌細胞へ選択的に送達および結合される可能性がある。また、本発明に係るRNAをキトサンナノ粒子に結合させてもよい。キトサンナノ粒子は、体内へ静注投与されたsiRNAの酵素分解を抑制する作用を有する。さらに、葉酸-キトサン複合ナノ粒子へ、本発明に係るRNAを結合させることが好ましい。その他、PEG化により血中滞留性の向上したリポソームや、DDS担体としてアテロコラーゲン等が知られている。 The dosage form of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention is not particularly limited as long as the active ingredient can be delivered to the target site, and may be, for example, an injection, a liquid, or a sustained release. Although water is preferred as a solvent for these preparations, it is preferable to use physiological saline, PBS, serum albumin solution, etc. so that the preparations are ultimately isotonic or approximately isotonic. In addition, the RNA according to the present invention may be directly or indirectly bound to folic acid. Since folate receptors are highly expressed on the surface of pancreatic cancer cell membranes, such RNA-folate complexes may be selectively delivered to and bound to pancreatic cancer cells. Furthermore, the RNA according to the present invention may be bound to chitosan nanoparticles. Chitosan nanoparticles have the effect of suppressing enzymatic degradation of siRNA intravenously administered into the body. Furthermore, it is preferable to bind the RNA according to the present invention to the folic acid-chitosan composite nanoparticles. In addition, liposomes with improved blood retention due to PEGylation and atelocollagen as DDS carriers are known.
本発明に係る膵癌細胞浸潤転移阻害剤の標的部位は、膵臓のみならず、膵癌細胞が転移したリンパ節や他の臓器であってもよい。また、標的部位へ有効成分をより確実に送達するために、剤形としては注射剤が好ましい。また、リポソームや高分子ミセルなど、公知の薬剤送達技術を用いてもよい。 The target site of the inhibitor of pancreatic cancer cell invasion and metastasis according to the present invention may be not only the pancreas but also lymph nodes and other organs to which pancreatic cancer cells have metastasized. Furthermore, in order to more reliably deliver the active ingredient to the target site, injections are preferred as the dosage form. Also, known drug delivery techniques such as liposomes and polymer micelles may be used.
本発明に係るDNAは、上記shRNAまたはsiRNAのセンス鎖および/もしくはアンチセンス鎖をコードする塩基配列を有することを特徴とする。更に、上記センス鎖とアンチセンス鎖を別の部位でコードするものであってもよいし、上記センス鎖、リンカーRNAおよび上記アンチセンス鎖を連続してコードするものであってもよい。 The DNA according to the present invention is characterized by having a base sequence encoding the sense strand and/or antisense strand of the shRNA or siRNA. Furthermore, the sense strand and the antisense strand may be encoded at separate sites, or the sense strand, linker RNA, and antisense strand may be encoded consecutively.
本発明に係るDNAは、上記shRNAまたはsiRNAのセンス鎖および/もしくはアンチセンス鎖をコードする塩基配列(遺伝子)が発現するよう、さらに、プロモーター、エンハンサー、サイレンサー、スプライシングドナー、アクセプターおよびポリAなどの調節領域を含んでいてもよい。特に、上記コード領域の5’末端側にプロモーターが存在し、3’末端側に転写を終結させるためのターミネーターが連結されていることが好ましい。 The DNA according to the present invention further includes a promoter, enhancer, silencer, splicing donor, acceptor, polyA, etc. so that the base sequence (gene) encoding the sense strand and/or antisense strand of the shRNA or siRNA is expressed. It may also include a regulatory region. In particular, it is preferable that a promoter be present at the 5' end of the coding region, and a terminator for terminating transcription be linked to the 3' end.
本発明に係るDNAは、上述したとおり上記shRNAまたはsiRNAのセンス鎖および/もしくはアンチセンス鎖をコードする塩基配列などを有するので、細胞内に導入されることにより、上記shRNAまたはsiRNAのセンス鎖および/もしくはアンチセンス鎖が細胞内で産生され、RNA干渉により標的RNAが分解される。よって、本発明に係るDNAが挿入されたベクターは、膵癌細胞浸潤転移阻害剤の有効成分となり得る。 As described above, the DNA according to the present invention has a base sequence encoding the sense strand and/or antisense strand of the shRNA or siRNA, and therefore, by being introduced into cells, the sense strand and the antisense strand of the shRNA or siRNA can be encoded. or the antisense strand is produced intracellularly and the target RNA is degraded by RNA interference. Therefore, a vector into which the DNA according to the present invention has been inserted can serve as an active ingredient of an inhibitor of pancreatic cancer cell invasion and metastasis.
本発明で用いるベクターは、プラスミドベクターや、無毒化したウィルスベクター、また、リポソームベクターなど公知のベクターから適宜選択すればよい。 Vectors used in the present invention may be appropriately selected from known vectors such as plasmid vectors, detoxified virus vectors, and liposome vectors.
本発明に係るベクターは、リポフェクトアミン法など脂質を媒体とする担体輸送法、リン酸カルシウムなどの化学物質を媒介する方法、マイクロインジェクション、遺伝子中による打ち込み法、電気穿孔法など、公知方法により標的細胞内へ送達することができる。 The vector according to the present invention can be delivered to target cells by known methods such as a carrier transport method using a lipid as a medium such as the lipofectamine method, a method using a chemical substance such as calcium phosphate, a microinjection method, a method of implantation into a gene, an electroporation method, etc. can be delivered within.
運動性、浸潤性および転移性の高い膵癌細胞において、細胞膜突起中にはKHSRPと結合して集積しているRNAが存在している。このRNAが膵癌細胞の運動性に関与することが、本発明者の実験により確認されている。本発明においては、膵癌細胞においてKHSRP RNAまたはKHSRP結合性snoRNAをノックダウンすることにより、膵癌細胞の浸潤転移を抑制することが可能となる。 In pancreatic cancer cells that are highly motile, invasive, and metastatic, RNA that binds to KHSRP and accumulates in cell membrane protrusions is present. Experiments conducted by the present inventors have confirmed that this RNA is involved in the motility of pancreatic cancer cells. In the present invention, by knocking down KHSRP RNA or KHSRP-binding snoRNA in pancreatic cancer cells, it is possible to suppress invasion and metastasis of pancreatic cancer cells.
本発明に係る膵癌細胞浸潤転移阻害剤の投与量や投与頻度は、それぞれの剤形や、患者の重篤度、年齢、性別、体重などにより適宜調整すればよい。 The dosage and frequency of administration of the pancreatic cancer cell invasion and metastasis inhibitor according to the present invention may be adjusted as appropriate depending on the respective dosage form, severity, age, sex, body weight, etc. of the patient.
以下、実施例を挙げて本発明をより具体的に説明するが、本発明はもとより下記実施例によって制限を受けるものではなく、前・後記の趣旨に適合し得る範囲で適当に変更を加えて実施することも勿論可能であり、それらはいずれも本発明の技術的範囲に包含される。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited by the Examples below, and modifications may be made as appropriate within the scope of the spirit of the preceding and following. Of course, other implementations are also possible, and all of them are included within the technical scope of the present invention.
実施例1: 膵癌細胞におけるKHSRPの局在確認
ヒト膵癌(PDAC)のS2-013細胞株はネブラスカ大学Dr.Michael Hollingsworthより寄贈され、同じくPDACのPANC-1細胞株はAmerican Type Culture Collectionから入手した。
免疫細胞化学的手法を用いて、2種類の膵癌細胞におけるKHSRPとアクチンの局在を確認した。結果を図1に示す。図1中の矢印は、細胞膜突起中に局在するKHSRPを示す。
図1に示される結果の通り、S2-013株でもPANC-1株でも、KHSRPは核内や細胞質のみならず細胞膜突起内に蓄積していることが認められた。また、細胞膜突起は重合したアクチン構造を有していた。
Example 1: Confirmation of localization of KHSRP in pancreatic cancer cells Human pancreatic cancer (PDAC) S2-013 cell line was obtained from University of Nebraska Dr. The PANC-1 cell line, also a PDAC, was a gift from Michael Hollingsworth and was obtained from the American Type Culture Collection.
Using immunocytochemical techniques, we confirmed the localization of KHSRP and actin in two types of pancreatic cancer cells. The results are shown in Figure 1. The arrow in FIG. 1 indicates KHSRP localized in cell membrane protrusions.
As shown in the results shown in FIG. 1, in both the S2-013 strain and the PANC-1 strain, KHSRP was found to accumulate not only in the nucleus and cytoplasm but also in cell membrane protrusions. Furthermore, the cell membrane protrusions had a polymerized actin structure.
実施例2: 膵癌細胞の運動性と浸潤性に対するKHSRPノックダウンの効果
(1)トランスウェル運動性アッセイとマトリゲル浸潤アッセイ
KHSRPが膵癌細胞の運動性と浸潤性に影響を及ぼすか否か調べた。具体的には、KHSRP siRNA(「TR311984」Origene Technologies社製)のベクターを用い、公知方法(TANIUCHI Kら,Oncotarget.,2014,5,6832-6845)に従って、KHSRPの発現が安定的に抑制されたS2-013株細胞を作製した。細胞は、KHSRP発現の抑制がウエスタンブロット分析によって確認されたもののみ使用した。KHSRPの発現が抑制されたS2-013株細胞を、上記文献に記載されたトランスウェル運動性アッセイとマトリゲル浸潤アッセイに付した。2種のスクランブルコントロールRNAi(Scr-1およびScr-2)を発現させたS2-013株細胞の結果と合わせて、それぞれの結果を図2に示す。図2中、「*」はt-テストにおいてコントロールに対してp<0.05で有意差があることを示す。
図2に示される結果の通り、KHSRPのmRNAを標的とするsiRNAを導入したKHSRP-RNAi S2-013株細胞(図2中、siKH-1とsiKH-2)の運動性と浸潤性は、スクランブルコントロール細胞に比べて有意に低いことが分かった。
また、KHSRPをコードするcDNAを増幅するために、S2-013株細胞のRNAを鋳型としてRT-PCRを行った。得られたPCR産物を、C-末端にmyc-DDKタグを生じるpCMV6-Entryベクター(Origene社製)に挿入した。以下、得られたベクターを「KHSRP-pCMV6」という。siKH-1およびsiKH-2クローン細胞に、KHSRP発現を回復させるため、トランスフェクション試薬(「X-tremeGENETM HP DNA Transfection Reagent」Roche社製)を用い、KHSRP-pCMV6を導入したsiKH-1およびsiKH-2クローン細胞を使って同様の実験を行ったところ、運動性と浸潤性が回復した(図示せず)。
Example 2: Effect of KHSRP knockdown on motility and invasiveness of pancreatic cancer cells (1) Transwell motility assay and Matrigel invasion assay It was investigated whether KHSRP affects the motility and invasiveness of pancreatic cancer cells. Specifically, the expression of KHSRP was stably suppressed using a vector of KHSRP siRNA (“TR311984” manufactured by Origene Technologies) according to a known method (TANIUCHI K et al., Oncotarget., 2014, 5, 6832-6845). S2-013 cell line was produced. Only cells whose suppression of KHSRP expression was confirmed by Western blot analysis were used. The S2-013 cell line in which KHSRP expression was suppressed was subjected to the transwell motility assay and Matrigel invasion assay described in the above-mentioned literature. The respective results are shown in FIG. 2 together with the results of S2-013 cell line expressing two types of scrambled control RNAi (Scr-1 and Scr-2). In FIG. 2, "*" indicates that there is a significant difference at p<0.05 from the control in the t-test.
As shown in Figure 2, the motility and invasiveness of KHSRP-RNAi S2-013 cell lines (siKH-1 and siKH-2 in Figure 2) into which siRNA targeting KHSRP mRNA was introduced were scrambled. It was found to be significantly lower than in control cells.
Furthermore, in order to amplify the cDNA encoding KHSRP, RT-PCR was performed using RNA from S2-013 cell line as a template. The obtained PCR product was inserted into pCMV6-Entry vector (manufactured by Origene) that generates a myc-DDK tag at the C-terminus. Hereinafter, the obtained vector will be referred to as "KHSRP-pCMV6". In order to restore KHSRP expression to siKH-1 and siKH-2 clone cells, KHSRP-pCMV6 was introduced into siKH-1 and siKH using a transfection reagent (“X-tremeGENE ™ HP DNA Transfection Reagent” manufactured by Roche). When similar experiments were performed using -2 clone cells, motility and invasiveness were restored (not shown).
(2)in vivo実験
6週齢の免疫適格性雌BALB/cSlc-nu/nuマウスを、日本SLC社から入手した。スクランブルコントロールRNAiクローン(Scr-1およびScr-2)と、KHSRPをノックダウンしたS2-013株細胞(siKH-1およびsiKH-2)8.0×105を10μLのPBSに懸濁させた。アベルチン(0.375g/kg体重)を腹腔内投与することによりマウスを麻酔し、各細胞懸濁液を膵臓の頭部にゆっくり注射した。移植から42日後にマウスを安楽死させた。後腹膜への腫瘍浸潤、および肺と肝臓への転移性病変の存在を、ヘマトキシリンおよびエオシン染色によって分析した。また、膵臓腫瘍を切除し、検査し、そして秤量した。結果を表1に示す。表中、「*」は、フィッシャーの正確確率検定において、Scr-1およびScr-2の結果に対してp<0.05で有意差があることを示す。
(2) In vivo experiment Six-week-old immunocompetent female BALB/cSlc-nu/nu mice were obtained from Japan SLC. 8.0×10 5 scrambled control RNAi clones (Scr-1 and Scr-2) and KHSRP-knocked-down S2-013 cell lines (siKH-1 and siKH-2) were suspended in 10 μL of PBS. Mice were anesthetized by intraperitoneal administration of avertin (0.375 g/kg body weight), and each cell suspension was slowly injected into the head of the pancreas. Mice were euthanized 42 days after transplantation. Tumor invasion into the retroperitoneum and the presence of metastatic lesions to the lungs and liver were analyzed by hematoxylin and eosin staining. A pancreatic tumor was also excised, examined, and weighed. The results are shown in Table 1. In the table, "*" indicates that there is a significant difference at p<0.05 between the results of Scr-1 and Scr-2 in Fisher's exact test.
表1に示される結果の通り、KHSRPがノックダウンされた膵癌細胞は、後腹膜への浸潤と肝臓および肺への転移のいずれも有意に抑制されていることが証明された。 As shown in Table 1, it was demonstrated that pancreatic cancer cells in which KHSRP was knocked down were significantly inhibited from infiltrating the retroperitoneum and metastasizing to the liver and lungs.
以上の結果より、KHSRPは膵癌細胞の運動性と浸潤性に重要な役割を果たしていることが示された。 The above results showed that KHSRP plays an important role in the motility and invasiveness of pancreatic cancer cells.
実施例3: KHSRPの局在位置の同定
ストレス顆粒とは、ストレス刺激に応答して一過性に形成される細胞内構造体であり、ストレスから細胞を防御する機構と考えられており、ストレス顆粒には、数種類のRNA結合タンパク質、40Sサブユニットリボソームタンパク質、および翻訳開始因子が存在する(Loreni Fら,Oncogene.,2014,33,2145-2156)。また、プロセシングボディ(P-body)は細胞質に特徴的にみられる粒子様の構造で、RNA-タンパク質の複合体であり、RNAの貯蔵や分解など、RNA代謝の場となっている。miRNAによるRNAサイレンシングの代替モデルとして、P-body内でmRNAがRISC(RNA-induced silencing complex)と相互作用するものが報告されている(Liu Jら,Nat.Cell.Biol.,2005,7,719-723)。これらは、非翻訳RNAを含み、翻訳機構を除外する細胞質病巣である(Teixeira Dら,Rna,2005,11,371-382)。
KHSRP含有顆粒がストレス顆粒(SG)かP-bodyかを調べるために、フィブロネクチン上で培養したS2-013株細胞を、抗KHSRP抗体、および抗G3BP抗体(SGマーカー)、または抗Ge-1/HEDLS(P-bodyマーカー)で二重標識した。結果を図3に示す。
図3に示される結果の通り、KHSRPは、細胞質顆粒においてG3BPと共存しない一方で、P-bodyと共局在していた。これらのデータは、P-bodyに局在するKHSRPが、膵癌細胞中の特定のmRNAのレベルを調節するように機能し得ることを示している。
Example 3: Identification of the localization location of KHSRP Stress granules are intracellular structures that are formed transiently in response to stress stimuli, and are thought to be a mechanism that protects cells from stress. Granules contain several types of RNA binding proteins, 40S subunit ribosomal proteins, and translation initiation factors (Loreni F et al., Oncogene., 2014, 33, 2145-2156). Furthermore, the processing body (P-body) is a particle-like structure characteristically found in the cytoplasm, is an RNA-protein complex, and serves as a site for RNA metabolism such as storage and degradation of RNA. As an alternative model of RNA silencing by miRNA, it has been reported that mRNA interacts with RISC (RNA-induced silencing complex) within the P-body (Liu J et al., Nat. Cell. Biol., 2005, 7). , 719-723). These are cytoplasmic foci that contain untranslated RNA and exclude the translation machinery (Teixeira D et al., RNA, 2005, 11, 371-382).
To examine whether KHSRP-containing granules are stress granules (SG) or P-bodies, S2-013 cell lines cultured on fibronectin were incubated with anti-KHSRP antibody, anti-G3BP antibody (SG marker), or anti-Ge-1/ Double labeling was performed with HEDLS (P-body marker). The results are shown in Figure 3.
As shown in FIG. 3, KHSRP did not colocalize with G3BP in cytoplasmic granules, but colocalized with P-body. These data indicate that P-body-localized KHSRP may function to regulate the levels of specific mRNAs in pancreatic cancer cells.
実施例4: KHSRPの標的転写産物の同定
KHSRP含有顆粒に局在するKHSRP結合転写物を同定するために、抗KHSRP抗体、およびフィブロネクチン上で増殖させたS2-013株細胞からの細胞抽出物を用いてRNA免疫沈降(RIP)を行い、続いて得られた免疫沈降物中のRNAを同定するための次世代配列決定を行った。RIPアッセイの結果、ウサギIgGアイソタイプコントロール免疫沈降物と比較して、抗KHSRP免疫沈降物中で有意に濃縮されている501個のRNAが同定された。501個のRNAから得られた完全な遺伝子リストを遺伝子発現オムニバスデータベース(www.ncbi.nlm.nih.gov/geo/)にアップロードした(GEO登録番号:GSE120853)。かなりの数のsnoRNAが、KHSRP結合性RNA候補のトップ10にリストされていた(表2)。
Example 4: Identification of target transcripts of KHSRP To identify KHSRP-bound transcripts localized to KHSRP-containing granules, anti-KHSRP antibodies and cell extracts from cell line S2-013 grown on fibronectin were used. RNA immunoprecipitation (RIP) was performed using the following method, followed by next-generation sequencing to identify the RNA in the obtained immunoprecipitate. The RIP assay identified 501 RNAs that were significantly enriched in the anti-KHSRP immunoprecipitates compared to the rabbit IgG isotype control immunoprecipitates. The complete gene list obtained from 501 RNAs was uploaded to the Gene Expression Omnibus database (www.ncbi.nlm.nih.gov/geo/) (GEO accession number: GSE120853). A significant number of snoRNAs were listed in the top 10 KHSRP binding RNA candidates (Table 2).
KHSRP結合性RNAの生物学的機能性についてさらに洞察を得るために、遺伝子オントロジー(GO)分析を行い、遺伝子リストと一致するGOタームを同定した。このGOセットは、アポトーシス、RNAスプライシング、翻訳、スプライセオソームを介した核内mRNAスプライシング、およびmRNAプロセシングを含んでいた。これらの機能に関連する任意のGOタームと一致したKHSRP結合性mRNAを表3に列挙する。 To gain further insight into the biological functionality of KHSRP-binding RNAs, we performed gene ontology (GO) analysis and identified GO terms that matched the gene list. This GO set included apoptosis, RNA splicing, translation, spliceosome-mediated nuclear mRNA splicing, and mRNA processing. KHSRP-binding mRNAs matched with any GO terms related to these functions are listed in Table 3.
本発明者は以前に、膵癌細胞組織におけるADP-リボシル化因子6(ARF6)およびRhoグアニンヌクレオチド交換因子4(ARHGEF4)の過剰発現が膵癌細胞の生存と相関し、それらが細胞膜突起を増加させることによって膵癌細胞の運動性および浸潤性を促進することを報告した。それに対して本研究では、KHSRPがsnoRNAと結合することが世界で初めて見出されたため、KHSRP結合性mRNAに焦点を合わせるのではなく、新規の所見であるKHSRP結合性snoRNAの機能を分析した。逆転写PCR(RT-PCR)を実施して、表2に記載のsnoRNAがKHSRPと免疫沈降することを確認した。その結果、表2に記載の全てのsnoRNAが抗KHSRP抗体で免疫沈降した一方で、アイソタイプ対照抗体で免疫沈降しなかった。
免疫細胞化学およびRNA蛍光in situハイブリダイゼーションを一緒に用い、フィブロネクチン上で培養されたS2-013株細胞の細胞質顆粒内でSNORA18およびSNORA22がKHSRPと共局在し、これらのsnoRNAがKHSRPと共局在することを示した(図4)。一方、対照ユビキチンC mRNAは、フィブロネクチン上で培養したS2-013株細胞においてKHSRPと共存しなかった(図4)。
The present inventors have previously shown that overexpression of ADP-ribosylation factor 6 (ARF6) and Rho guanine nucleotide exchange factor 4 (ARHGEF4) in pancreatic cancer cell tissues correlates with pancreatic cancer cell survival and that they increase cell membrane protrusions. reported that it promoted the motility and invasiveness of pancreatic cancer cells. In contrast, in this study, it was discovered for the first time in the world that KHSRP binds to snoRNA, so instead of focusing on KHSRP-binding mRNA, we analyzed the function of KHSRP-binding snoRNA, which is a novel finding. Reverse transcription PCR (RT-PCR) was performed to confirm that the snoRNAs listed in Table 2 were immunoprecipitated with KHSRP. As a result, all snoRNAs listed in Table 2 were immunoprecipitated with the anti-KHSRP antibody, but not with the isotype control antibody.
Using immunocytochemistry and RNA fluorescence in situ hybridization together, we showed that SNORA18 and SNORA22 colocalized with KHSRP in the cytoplasmic granules of S2-013 cell lines cultured on fibronectin, and that these snoRNAs colocalized with KHSRP. (Figure 4). On the other hand, control ubiquitin C mRNA did not coexist with KHSRP in S2-013 cell lines cultured on fibronectin (FIG. 4).
実施例5: 細胞膜突起におけるKHSRPの役割
フィブロネクチン上で培養したスクランブルコントロールsiRNAを恒常的に発現するS2-013株コントロールクローン(Scr-1)と、KHSRP特異的なsiRNAを恒常的に発現するKHSRP発現抑制S2-013株クローン(siKH-1)を共焦点顕微鏡で観察した。結果を図5に示す。図5中、矢印は、内部にアクチンが重合している細胞膜突起を示す。
図5に示される結果の通り、細胞膜突起におけるアクチン重合構造が、フィブロネクチン上で培養したScr-1 S2-013株コントロール細胞よりも、KHSRP発現が抑制されたsiKH-1 S2-013株細胞においてより少ないことが明らかにされた。逆に重合していないアクチン構造は、Scr-1 S2-013株コントロール細胞よりも、siKH-1 S2-013株細胞の細胞質においてより豊富であった。
モックコントロールベクター、または実施例2(1)で作製したKHSRP-pCMV6をコントロールRNAi S2-013株細胞またはKHSRP-RNAi S2-013株細胞に一時的に導入し、48時間後、共焦点顕微鏡で観察した。KHSRP-pCMV6の導入により、KHSRP-RNAi S2-013株細胞においても、細胞膜突起におけるアクチン重合構造が再現した(図示せず)。また、細胞膜突起を有する細胞を計数した。結果を図6に示す。
図6に示される結果の通り、細胞膜突起を有する細胞の数は、モックコントロールベクターを導入したKHSRP-RNAi S2-013株細胞よりも、KHSRP-pCMV6を導入したKHSRP-RNAi S2-013株細胞において有意に豊富であった。
これらの結果は、KHSRPがアクチンの重合を誘導して細胞膜突起の形成を増加させることを示している。
Example 5: Role of KHSRP in cell membrane protrusions S2-013 strain control clone (Scr-1) that constitutively expresses scrambled control siRNA cultured on fibronectin and KHSRP expression that constitutively expresses KHSRP-specific siRNA The suppressed S2-013 strain clone (siKH-1) was observed using a confocal microscope. The results are shown in Figure 5. In FIG. 5, arrows indicate cell membrane protrusions in which actin is polymerized.
As shown in Figure 5, the actin polymerization structure in cell membrane protrusions is more pronounced in siKH-1 S2-013 cell lines in which KHSRP expression is suppressed than in Scr-1 S2-013 control cells cultured on fibronectin. It was revealed that there were few. Conversely, unpolymerized actin structures were more abundant in the cytoplasm of siKH-1 S2-013 cells than in Scr-1 S2-013 control cells.
Mock control vector or KHSRP-pCMV6 prepared in Example 2 (1) was temporarily introduced into control RNAi S2-013 cell line or KHSRP-RNAi S2-013 cell line, and observed with a confocal microscope after 48 hours. did. By introducing KHSRP-pCMV6, actin polymerization structures in cell membrane protrusions were reproduced in KHSRP-RNAi S2-013 cells (not shown). In addition, cells having cell membrane protrusions were counted. The results are shown in FIG.
As shown in the results shown in Figure 6, the number of cells with membrane protrusions was higher in the KHSRP-RNAi S2-013 cell line introduced with KHSRP-pCMV6 than in the KHSRP-RNAi S2-013 cell line introduced with the mock control vector. It was significantly abundant.
These results indicate that KHSRP induces actin polymerization to increase the formation of plasma membrane protrusions.
実施例6: 細胞膜突起の形成におけるKHSRP結合性snoRNAの役割
本発明者は、そのmRNAがRNA結合タンパク質インスリン様成長因子2 mRNA結合タンパク質3(IGF2BP3)に結合しているARF6およびARHGEF4タンパク質が細胞膜突起に蓄積し、それによって膵癌細胞における細胞膜突起形成に寄与することを以前に報告している(Taniuchi Kら,Oncotarget.,2014,5,6832-6845;Taniuchi Kら,Int.J.Oncol.,2018,53,2224-2240)。
KHSRP結合性snoRNAであるSNORA18およびSNORA22が細胞膜突起の誘導に関与する否かを決定するために、スクランブルドコントロールsiRNA、SNORA18 siRNA、およびSNORA22 siRNAで一過性にトランスフェクトしたS2-013株細胞の細胞膜突起内のアクチン重合構造を分析した。
トランスフェクションの72時間後の半定量的RT-PCRデータに基づくと、SNORA18およびSNORA22の発現は、SNORA18 siRNAトランスフェクトまたはSNORA22 siRNAトランスフェクトS2-013株細胞よりも、スクランブルコントロールsiRNAトランスフェクトS2-013株細胞において著しく高いことが確認された。共焦点顕微鏡による観察では、フィブロネクチン上で培養したS2-013株細胞におけるSNORA18またはSNORA22のノックダウンが、アクチン重合構造を減少させることを明らかにした(SNORA18-ノックダウンについては図7、SNORA22-ノックダウンについては図8)。図7および図8中、矢印は、内部にアクチンが重合している細胞膜突起を示す。
更に、フィブロネクチン上で培養したS2-013株細胞におけるSNORA18およびSNORA22のノックダウンは、細胞膜突起の形成を有意に阻害した(図9)。
これらの結果は、ノンコーディングRNAであるSNORA18およびSNORA22が、膵癌細胞の細胞膜突起の形成において役割を果たすことを示している。
Example 6: Role of KHSRP-binding snoRNA in the formation of cell membrane protrusions The present inventors demonstrated that ARF6 and ARHGEF4 proteins, whose mRNAs bind to the RNA-binding protein insulin-
To determine whether the KHSRP-binding snoRNAs SNORA18 and SNORA22 are involved in the induction of cell membrane protrusions, S2-013 cell lines were transiently transfected with scrambled control siRNA, SNORA18 siRNA, and SNORA22 siRNA. We analyzed the actin polymerization structure within cell membrane protrusions.
Based on semi-quantitative RT-PCR data 72 hours after transfection, expression of SNORA18 and SNORA22 was higher in scrambled control siRNA-transfected S2-013 than in SNORA18 siRNA-transfected or SNORA22 siRNA-transfected S2-013 cells. It was confirmed that it was significantly high in cell lines. Confocal microscopy observation revealed that knockdown of SNORA18 or SNORA22 in S2-013 cells cultured on fibronectin reduced actin polymerized structures (Figure 7 for SNORA18-knockdown, SNORA22-knockdown For down, see Figure 8). In FIGS. 7 and 8, arrows indicate cell membrane protrusions in which actin is polymerized.
Furthermore, knockdown of SNORA18 and SNORA22 in S2-013 cells cultured on fibronectin significantly inhibited the formation of cell membrane protrusions (FIG. 9).
These results indicate that non-coding RNAs SNORA18 and SNORA22 play a role in the formation of cell membrane protrusions in pancreatic cancer cells.
実施例7: 膵癌細胞の運動性と浸潤性に対するKHSRP結合性snoRNAの役割
トランスウェル運動性アッセイにおいて、S2-013株細胞およびPANC-1株細胞の運動性は、スクランブルドコントロール細胞よりも、SNORA18-ノックダウン細胞およびSNORA22-ノックダウン細胞において有意に低かった(図10)。
マトリゲル浸潤アッセイにおいて、S2-013株細胞およびPANC-1株細胞の浸潤性は、コントロール細胞におけるよりもSNORA18-ノックダウン細胞およびSNORA22-ノックダウン細胞において有意に低かった(図11)。
これらの結果は、SNORA18およびSNORA22が膵癌細胞の運動性および浸潤性の促進と関連することを示している。
Example 7: Role of KHSRP-binding snoRNA on motility and invasiveness of pancreatic cancer cells In the transwell motility assay, the motility of S2-013 and PANC-1 cells was significantly higher than that of scrambled control cells. - significantly lower in knockdown cells and SNORA22-knockdown cells (Figure 10).
In the Matrigel invasion assay, the invasiveness of S2-013 and PANC-1 cells was significantly lower in SNORA18-knockdown and SNORA22-knockdown cells than in control cells (FIG. 11).
These results indicate that SNORA18 and SNORA22 are associated with promoting pancreatic cancer cell motility and invasiveness.
実施例8: 膵癌モデルマウスにおけるSNORA18とSNORA22のノックダウンによる膵癌細胞の浸潤と転移に対する効果
インビボイメージング研究によれば、膵癌(PDAC)の同所性マウスモデル(マウス膵臓にヒト膵癌株細胞を移植したヒト膵癌浸潤・転移マウスモデル)に静脈内注射された葉酸(FA)修飾ポリエチレングリコール(PEG)-キトサンオリゴ糖乳酸(COL)ナノ粒子を付加したsiRNAは、マウス膵臓のS2-013株由来PDAC腫瘍におけるPDAC細胞に取り込まれる(Taniuchi Kら,Oncotarget.,2019,10,2869-2886)。そこで、インビボでの浸潤性および転移に対するSNORA18およびSNORA22の効果を研究するために、SNORA18およびSNORA22に対するsiRNA-FA-PEG-COLナノ粒子をPDAC同所性マウスモデルに静脈内注射した。SNORA18 siRNAとしては、配列番号4の塩基配列を有するセンス鎖が相補的なアンチセンス鎖と結合した21塩基の二本鎖siRNAを用い、SNORA22 siRNAとしては、配列番号8の塩基配列を有するセンス鎖が相補的なアンチセンス鎖と結合した21塩基の二本鎖siRNAを用いた。
6週齢のヌードマウスの膵臓にヒト膵癌細胞株S2-013を移植した膵癌浸潤転移モデルを用いた。具体的には、第1日目にイソフルランを用いた吸入麻酔により48匹のヌードマウスを麻酔し、開腹して膵臓を露出した。100万個のヒト膵癌細胞株S2-013を0.1mLのPBSに懸濁し、各マウスの膵臓内に移植した。siRNAはSNORA18とSNORA22に対するものと、コントロールとしてスクランブルsiRNAを用いた。合成した各siRNAを葉酸キトサンナノ粒子に付加した。ヒト膵癌細胞を移植した各群のマウスにそれぞれの葉酸キトサンナノ粒子付加siRNAを1回/週の頻度で尾静脈へ投与した。マウスに全身投与されたsiRNAは、キトサンナノ粒子によりヒト膵癌組織まで受動的に送達され、葉酸が膵癌細胞上の葉酸受容体を介して効率的なエンドサイトーシスを促すことを期待できる。合計5回のsiRNA静注投与を行った。S2-013株細胞の移植から6週間後、マウスを安楽死させ、膵癌組織、肺、および肝臓の切片をヘマトキシリン-エオシン染色し、後腹膜浸潤、腹膜播種、ならびに肝臓および肺への転移の存在を判定した。各マウスの開腹写真を図12に、コントロールマウスの後腹膜、肺および肝臓のマトキシリン-エオシン染色写真を図13に、また、結果を表4にまとめる。なお、表4中、「*」は、フィッシャー直接検定において、3種類のコントロール群れであるPBS投与コントロールマウス、スクランブルコントロールsiRNA-COLナノ粒子投与コントロールマウス、およびスクランブルコントロールsiRNA-FA-PEG-COLナノ粒子投与コントロールマウスに対してp<0.05で有意差があることを示す。
Example 8: Effect of knockdown of SNORA18 and SNORA22 on pancreatic cancer cell invasion and metastasis in pancreatic cancer model mouse siRNA attached with folic acid (FA)-modified polyethylene glycol (PEG)-chitosan oligosaccharide lactate (COL) nanoparticles was intravenously injected into a mouse model of human pancreatic cancer invasion and metastasis (human pancreatic cancer invasion/metastasis mouse model). It is taken up by PDAC cells in tumors (Taniuchi K et al., Oncotarget., 2019, 10, 2869-2886). Therefore, to study the effects of SNORA18 and SNORA22 on invasiveness and metastasis in vivo, siRNA-FA-PEG-COL nanoparticles against SNORA18 and SNORA22 were intravenously injected into a PDAC orthotopic mouse model. As SNORA18 siRNA, a 21-base double-stranded siRNA in which a sense strand having the base sequence of SEQ ID NO: 4 was combined with a complementary antisense strand was used, and as SNORA22 siRNA, a sense strand having the base sequence of SEQ ID NO: 8 was used. A 21-base double-stranded siRNA was used, which was bound to a complementary antisense strand.
A pancreatic cancer invasion/metastasis model was used in which the human pancreatic cancer cell line S2-013 was transplanted into the pancreas of a 6-week-old nude mouse. Specifically, on the first day, 48 nude mice were anesthetized by inhalation anesthesia using isoflurane, and the abdomen was opened to expose the pancreas. One million human pancreatic cancer cell lines S2-013 were suspended in 0.1 mL of PBS and transplanted into the pancreas of each mouse. siRNA was used against SNORA18 and SNORA22, and scrambled siRNA was used as a control. Each synthesized siRNA was added to folic acid chitosan nanoparticles. Each group of mice transplanted with human pancreatic cancer cells received each folic acid chitosan nanoparticle-added siRNA once a week into the tail vein. siRNA systemically administered to mice is passively delivered to human pancreatic cancer tissues by chitosan nanoparticles, and it can be expected that folic acid will promote efficient endocytosis via folate receptors on pancreatic cancer cells. siRNA was administered intravenously a total of 5 times. Six weeks after transplantation of S2-013 cell lines, mice were euthanized and sections of pancreatic cancer tissue, lungs, and liver were stained with hematoxylin-eosin to determine the presence of retroperitoneal invasion, peritoneal dissemination, and metastasis to the liver and lungs. was determined. A laparotomy photograph of each mouse is shown in FIG. 12, a matoxylin-eosin stained photograph of the retroperitoneum, lungs and liver of a control mouse is shown in FIG. 13, and the results are summarized in Table 4. In Table 4, "*" indicates three types of control groups, PBS-administered control mice, scrambled control siRNA-COL nanoparticle-administered control mice, and scrambled control siRNA-FA-PEG-COL nanoparticles, in Fisher's exact test. Significant difference is indicated at p<0.05 compared to particle-administered control mice.
3種類のコントロール群の膵癌モデルマウスには、広範囲の腹膜癌腫症が70%に見られ(図12)、後腹膜の局所浸潤と、肺および肝臓への転移が高率で認められた(図13)。
一方、SNORA18およびSNORA22に対するsiRNA-FA-PEG-COLナノ粒子を投与した膵癌モデルマウスでは、対照群と比較して、後腹膜浸潤および肺転移が有意に阻害されており、特にSNORA18に対するsiRNA-FA-PEG-COLナノ粒子は、後腹膜浸潤を強く阻害した(表4)。
In the pancreatic cancer model mice of the three control groups, extensive peritoneal carcinomatosis was observed in 70% (Figure 12), and local invasion of the retroperitoneum and metastasis to the lungs and liver were observed at a high rate (Figure 12). 13).
On the other hand, in pancreatic cancer model mice administered with siRNA-FA-PEG-COL nanoparticles directed against SNORA18 and SNORA22, retroperitoneal invasion and lung metastasis were significantly inhibited compared to the control group. -PEG-COL nanoparticles strongly inhibited retroperitoneal invasion (Table 4).
Claims (4)
上記KH型スプライシング調節タンパク質RNAをノックダウンする化合物が、上記KH型スプライシング調節タンパク質RNAに結合するsiRNAまたはshRNAであり、
上記KH型スプライシング調節タンパク質結合性snoRNAをノックダウンする化合物が、上記KH型スプライシング調節タンパク質結合性snoRNAである、SNORA18またはSNORA22に結合するsiRNAまたはshRNAであることを特徴とする膵癌細胞浸潤転移阻害剤。 Containing as an active ingredient a compound that knocks down KH type splicing regulatory protein RNA and/or KH type splicing regulatory protein binding snoRNA,
The compound that knocks down the KH type splicing regulatory protein RNA is siRNA or shRNA that binds to the KH type splicing regulatory protein RNA,
Inhibition of invasion and metastasis of pancreatic cancer cells, characterized in that the compound that knocks down the KH-type splicing regulatory protein-binding snoRNA is siRNA or shRNA that binds to SNORA18 or SNORA2 2 , which is the KH-type splicing regulatory protein-binding snoRNA. agent.
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JP2015227292A (en) | 2014-05-30 | 2015-12-17 | 国立大学法人高知大学 | Pancreatic cancer cell invasion metastasis inhibition vaccine |
JP2016214239A (en) | 2015-05-15 | 2016-12-22 | 国立大学法人高知大学 | Pancreatic cancer marker |
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JP2015227292A (en) | 2014-05-30 | 2015-12-17 | 国立大学法人高知大学 | Pancreatic cancer cell invasion metastasis inhibition vaccine |
JP2016214239A (en) | 2015-05-15 | 2016-12-22 | 国立大学法人高知大学 | Pancreatic cancer marker |
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Mol. Oncol.,2018年11月15日,13, [2],p.212-227 |
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