JP7385847B2 - Cancer growth inhibitor containing a snoRNA expression inhibitor as an active ingredient - Google Patents
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Description
本発明は、核小体低分子ＲＮＡ（ｓｍａｌｌ ｎｕｃｌｅｏｌａｒ ＲＮＡ：ｓｎｏＲＮＡ）の発現抑制作用を有する化合物を有効成分とする新規ながん増殖抑制剤、更にはがんの治療剤に関する。 TECHNICAL FIELD The present invention relates to a novel cancer growth inhibitor, which contains as an active ingredient a compound that suppresses the expression of small nucleolar RNA (snoRNA), and furthermore, to a therapeutic agent for cancer.
ｓｎｏＲＮＡは、核小体（ｎｕｃｌｅｏｌｕｓ）に存在するＲＮＡのことであり、図１に示すように、ノンコーディングＲＮＡ（ｎｏｎ－ｃｏｒｄｉｎｇ ＲＮＡ）の１つである。リボゾームＲＮＡ（ｒＲＮＡ）及びその他のＲＮＡ遺伝子のメチル化やシュードウリジン化の化学修飾を導く小さなＲＮＡ分子の一群である（非特許文献１）。
ｓｎｏＲＮＡは、リボゾーム遺伝子のイントロンの中にコードされていて、ＲＮＡポリメラーゼＩＩによって合成される。そして、ｓｎｏＲＮＡは、蛋白質と結合して核小体低分子リボ核酸蛋白質（ｓｎｏＲＮＰ）に変化する。ｓｎｏＲＮＰ複合体は、ｒＲＮＡ等の化学修飾の標的部位に相補的に結合し、ｒＲＮＡ等の化学修飾を触媒する。
ｒＲＮＡ等の結合を誘導するのは、ｓｎｏＲＮＡの塩基配列であり、その塩基配列に対して相補的なｒＲＮＡの塩基配列部分に結合する。
ｓｎｏＲＮＡはその配列によって主にｂｏｘＣ／ｄとｂｏｘＨ／ＡＣＡの二種に分けられており、その相補的塩基配列により、ＲＮＡ修飾酵素を正しい位置に並べる機能を有しているとされている。SnoRNA is RNA present in the nucleolus, and as shown in FIG. 1, it is a type of non-coding RNA. It is a group of small RNA molecules that lead to chemical modifications such as methylation and pseudouridination of ribosomal RNA (rRNA) and other RNA genes (Non-Patent Document 1).
SnoRNAs are encoded in introns of ribosomal genes and are synthesized by RNA polymerase II. Then, snoRNA binds to protein and changes into nucleolar small ribonucleic acid protein (snoRNP). The snoRNP complex binds complementary to a target site for chemical modification of rRNA, etc., and catalyzes chemical modification of rRNA, etc.
It is the base sequence of snoRNA that induces the binding of rRNA, etc., and binds to the base sequence portion of rRNA that is complementary to the base sequence.
SnoRNAs are mainly divided into two types, boxC/d and boxH/ACA, depending on their sequences, and their complementary base sequences are said to have the function of arranging RNA modifying enzymes in the correct position.
従って、ｓｎｏＲＮＡが変異を起こす場合や、異常に高発現する場合には、核小体の中でリボソームを適切に構築することが出来難くなる。即ち、リボソームが異常を起こすので、タンパク質の合成が正しく行われなくなり、そのため細胞は正常に機能できなくなり、異常に増殖したり、死滅したりするようになる。ｓｎｏＲＮＡ等の異常により、リボソーム異常を起こし、その結果引き起こされる疾患群は「リボソーム病」と呼ばれている。代表的な疾患は、ダイアモンド・ブラックファン貧血（ＤＢＡ）という先天性の赤芽球形成不全症であり、リボソームタンパク質（ＲＰ）をコードする遺伝子の異常と深く関連している。造血異常の他にも、角化不全、骨格異常、脾臓や肝臓の異常を示す疾患もリボソーム病に含まれている。
現在では、ｓｎｏＲＮＡが、疾患のバイオマーカーとして使用される多いが（特許文献１）、ｓｎｏＲＮＡを疾患の治療剤として使用されることは未だ知られていない。ｓｎｏＲＮＡの用途としては、老化の進行を阻止するために、ある特定のｓｎｏＲＮＡを補充療法として投与することが知られている状況である（特許文献２）。Therefore, when snoRNA mutates or is abnormally highly expressed, it becomes difficult to appropriately construct ribosomes within the nucleolus. That is, as ribosomes become abnormal, protein synthesis is not performed properly, and cells are therefore unable to function normally, leading to abnormal proliferation or death. Abnormalities in snoRNA and the like cause ribosomal abnormalities, and a group of diseases caused as a result are called "ribosomal diseases." A typical disease is Diamond-Blackfan anemia (DBA), a congenital erythroblast aplasia that is closely related to abnormalities in the gene encoding ribosomal protein (RP). In addition to hematopoietic abnormalities, ribosomal diseases also include diseases showing hypokeratosis, skeletal abnormalities, and abnormalities of the spleen and liver.
Currently, snoRNAs are often used as disease biomarkers (Patent Document 1), but it is not yet known that snoRNAs can be used as therapeutic agents for diseases. As for the use of snoRNA, it is known that a certain snoRNA is administered as a replacement therapy in order to prevent the progression of aging (Patent Document 2).
本発明は、がん細胞の生体内での定着、増殖を抑制する、新たな作用機序のがんの治療剤を提供することを目的とする。 An object of the present invention is to provide a therapeutic agent for cancer with a new mechanism of action that suppresses the colonization and proliferation of cancer cells in vivo.
本発明者は、小胞体ストレス応答に関与するｎｏｎ－ｃｏｒｄｉｎｇ ＲＮＡの研究を進める中で、タンパク合成に係る核小体のストレス応答に着目し、ストレス応答を制御するｓｎｏＲＮＡとがんとの関係を検討した。
がん細胞では、増殖が盛んなために、タンパク合成が異常に亢進しており、がん細胞自体は常に飢餓状態にある。そのため、がん細胞では、増殖に必要なタンパクの合成に特化したリボソーム構築が行われている。それをコントロールしているのが図１に示すようにｓｎｏＲＮＡと考えられる。即ち、がん細胞に特有の高発現ｓｎｏＲＮＡは、がんの生存と増殖に必要なタンパクを供給するためのリボソーム構築に大きく寄与していると考えられた。
そこで、本発明者は、がん細胞に特異的高発現のｓｎｏＲＮＡをピックアップし、そのｓｎｏＲＮＡの発現を抑制すれば、がんの生存と増殖に必要なタンパクを供給するリボソーム構築が困難になると考えた。リボソーム構築が困難になれば、がんの生存と増殖に必要なタンパクが供給できず、がん細胞は生存と増殖が困難になり、死滅して行くと考えた。
本発明者は、上記のコンセプトに基づいて、多くのがん細胞で特異的に高発現のｓｎｏＲＮＡを選択した。そして、選択した高発現のｓｎｏＲＮＡの発現を抑制することによって、がんの増殖が抑制できることを見出した。即ち、がんの増殖抑制は、がんのアポトーシスと生体の免疫反応により、がんの死滅を誘導することになる。このように、本発明者は、ｓｎｏＲＮＡの発現抑制に基づく新たな作用機序のがんの増殖抑制剤、あるいはがんの治療剤を見出すことができた。
また、本発明者は、がん細胞に特異的に高発現するｓｎｏＲＮＡとして、ＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂ、ＳＮＯＲＡ６９、ＳＮＯＲＤ１１１、ＳＮＯＲＤ９９、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ１０５Ｂ、ＳＮＯＲＤ３７、ＳＮＯＲＤ５４を特定することができ、これらのｓｎｏＲＮＡのいずれか一つ以上を発現抑制することによって、がん細胞を増殖抑制できることを見出した。
本発明者は、以上の知見に基づいて本発明を完成した。While conducting research on non-coding RNAs involved in endoplasmic reticulum stress responses, the present inventors focused on the stress response of the nucleolus involved in protein synthesis, and investigated the relationship between snoRNAs that control stress responses and cancer. investigated.
Because cancer cells proliferate actively, protein synthesis is abnormally accelerated, and the cancer cells themselves are constantly in a state of starvation. Therefore, cancer cells construct ribosomes specialized for synthesizing proteins necessary for proliferation. It is thought that snoRNA controls this, as shown in Figure 1. That is, highly expressed snoRNAs unique to cancer cells were thought to greatly contribute to ribosome construction to supply proteins necessary for cancer survival and proliferation.
Therefore, the present inventor believes that if we pick up highly expressed snoRNAs specific to cancer cells and suppress the expression of these snoRNAs, it will become difficult to construct ribosomes that supply proteins necessary for cancer survival and proliferation. Ta. They believed that if ribosome assembly becomes difficult, the proteins necessary for cancer survival and proliferation will not be supplied, and cancer cells will have difficulty surviving and proliferating and will die.
Based on the above concept, the present inventors selected snoRNAs that are specifically highly expressed in many cancer cells. They have also discovered that cancer growth can be suppressed by suppressing the expression of selected highly expressed snoRNAs. In other words, suppression of cancer growth induces death of the cancer through apoptosis of the cancer and immune response of the body. Thus, the present inventors were able to discover a cancer growth inhibitor or a cancer therapeutic agent with a new mechanism of action based on suppression of snoRNA expression.
In addition, the present inventor was able to identify SNORA50C, SNORA56, SNORD42B, SNORA69, SNORD111, SNORD99, SNORA74A, SNORD105B, SNORD37, and SNORD54 as snoRNAs that are highly expressed specifically in cancer cells, and these snoRNAs of We have discovered that by suppressing the expression of one or more of these, it is possible to suppress the proliferation of cancer cells.
The present inventor completed the present invention based on the above knowledge.
即ち、本発明の要旨は以下の通りである。
（１）がん細胞で特異的に高発現するｓｎｏＲＮＡの発現抑制剤を有効成分とする、がんの増殖抑制剤。
（２）上記がん細胞が、乳癌、結腸癌、腎癌、肝癌、肺癌（肺腺癌、肺扁平上皮癌）、胃癌のいずれかである、上記（１）に記載のがんの増殖抑制剤。
（３）上記ｓｎｏＲＮＡが、ＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂ、ＳＮＯＲＡ６９、ＳＮＯＲＤ１１１、ＳＮＯＲＤ９９、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ１０５Ｂ、ＳＮＯＲＤ３７、ＳＮＯＲＤ５４の中から一つ以上が選択されるものである、上記（１）又は（２）に記載のがんの増殖抑制剤。
（４）上記がん細胞が、乳癌（例えば、浸潤性乳癌）の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂの中から一つ以上選択されるものである、上記（１）又は（２）に記載の乳癌（例えば、浸潤性乳癌）の増殖抑制剤。
（５）上記がん細胞が、結腸癌の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＡ５６及び／又はＳＮＯＲＡ６９である、上記（１）又は（２）に記載の結腸癌の増殖抑制剤。
（６）上記がん細胞が、腎癌（例えば、腎淡明細胞癌）の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＤ１１１及び／又はＳＮＯＲＤ９９である、上記（１）又は（２）に記載の腎癌（例えば、腎淡明細胞癌）の増殖抑制剤。
（７）上記がん細胞が、肝癌（例えば、肝細胞癌）の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＡ７４Ａ及び／又はＳＮＯＲＤ９９である、上記（１）又は（２）に記載の肝癌（例えば、肝細胞癌）の増殖抑制剤。
（８）上記がん細胞が、肺癌（例えば、肺腺癌）の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＤ１０５Ｂ及び／又はＳＮＯＲＤ３７である、上記（１）又は（２）に記載の肺癌（例えば、肺腺癌）の増殖抑制剤。
（９）上記がん細胞が、肺癌（例えば、肺扁平上皮癌）の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＡ７４Ａ及び／又はＳＮＯＲＤ５４である、上記（１）又は（２）に記載の肺癌（例えば、肺扁平上皮癌）癌の増殖抑制剤。
（１０）上記がん細胞が、胃癌の細胞であり、上記ｓｎｏＲＮＡがＳＮＯＲＡ６９である、上記（１）又は（２）に記載の胃癌の増殖抑制剤。That is, the gist of the present invention is as follows.
(1) A cancer growth inhibitor whose active ingredient is an expression inhibitor of snoRNA that is specifically highly expressed in cancer cells.
(2) Suppression of cancer growth according to (1) above, wherein the cancer cells are any of breast cancer, colon cancer, kidney cancer, liver cancer, lung cancer (lung adenocarcinoma, lung squamous cell carcinoma), and gastric cancer. agent.
(3) The snoRNA described in (1) or (2) above is one or more selected from among SNORA50C, SNORA56, SNORD42B, SNORA69, SNORD111, SNORD99, SNORA74A, SNORD105B, SNORD37, and SNORD54. cancer growth inhibitor.
(4) The cancer cell is a breast cancer cell (for example, invasive breast cancer), and the snoRNA is selected from one or more of SNORA50C, SNORA56, and SNORD42B, (1) or (2) above. ) The agent for inhibiting the growth of breast cancer (eg, invasive breast cancer).
(5) The colon cancer growth inhibitor according to (1) or (2) above, wherein the cancer cells are colon cancer cells and the snoRNA is SNORA56 and/or SNORA69.
(6) The renal cancer according to (1) or (2) above, wherein the cancer cell is a renal cancer cell (for example, renal clear cell carcinoma), and the snoRNA is SNORD111 and/or SNORD99. For example, a growth inhibitor for renal clear cell carcinoma).
(7) The liver cancer cell according to (1) or (2) above, wherein the cancer cell is a liver cancer (e.g., hepatocellular carcinoma) cell, and the snoRNA is SNORA74A and/or SNORD99. cancer) growth inhibitor.
(8) The lung cancer cell according to (1) or (2) above, wherein the cancer cell is a lung cancer (e.g., lung adenocarcinoma) cell, and the snoRNA is SNORD105B and/or SNORD37. cancer) growth inhibitor.
(9) The lung cancer cell according to (1) or (2) above, wherein the cancer cell is a lung cancer cell (e.g., lung squamous cell carcinoma), and the snoRNA is SNORA74A and/or SNORD54. Squamous cell carcinoma) cancer growth inhibitor.
(10) The growth inhibitor for gastric cancer according to (1) or (2) above, wherein the cancer cells are gastric cancer cells and the snoRNA is SNORA69.
（１１）ｓｎｏＲＮＡの発現抑制剤が、配列番号１のｓｉＲＮＡ、配列番号１のｓｉＲＮＡ、配列番号２のｓｉＲＮＡ、配列番号３のｓｉＲＮＡ、配列番号４のｓｉＲＮＡ、配列番号５のｓｉＲＮＡ、配列番号６のｓｉＲＮＡ、配列番号７のｓｉＲＮＡ、配列番号８のｓｉＲＮＡ、配列番号９のｓｉＲＮＡ、配列番号１０のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）に記載のがんの増殖抑制剤。
（１２）ｓｎｏＲＮＡの発現抑制剤が、配列番号１のｓｉＲＮＡ、配列番号１のｓｉＲＮＡ、配列番号２のｓｉＲＮＡ、配列番号３のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の乳癌（例えば、浸潤性乳癌）の増殖抑制剤。
（１３）ｓｎｏＲＮＡの発現抑制剤が、配列番号２のｓｉＲＮＡ、配列番号４のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の結腸癌の増殖抑制剤。
（１４）ｓｎｏＲＮＡの発現抑制剤が、配列番号５のｓｉＲＮＡ、配列番号１０のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の腎癌（例えば、腎淡明細胞癌）の増殖抑制剤。
（１５）ｓｎｏＲＮＡの発現抑制剤が、配列番号６のｓｉＲＮＡ、配列番号１０のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の肝癌（例えば、肝細胞癌）の増殖抑制剤。
（１６）ｓｎｏＲＮＡの発現抑制剤が、配列番号７のｓｉＲＮＡ、配列番号８のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の肺癌（例えば、肺腺癌）の増殖抑制剤。
（１７）ｓｎｏＲＮＡの発現抑制剤が、配列番号６のｓｉＲＮＡ、配列番号９のｓｉＲＮＡの中から、一つ以上が選択されるものである、上記（１）又は（２）に記載の肺癌（例えば、肺扁平上皮癌）の増殖抑制剤。
（１８）ｓｎｏＲＮＡの発現抑制剤が、配列番号４のｓｉＲＮＡである、上記（１）又は（２）に記載の胃癌の増殖抑制剤。
（１９）上記（１）～（１８）のいずれかに記載のがんの増殖抑制剤を有効成分とする、がん治療剤。
（２０） 配列番号１～１０の何れかの塩基配列からなるＲＮＡ。
（２１） ＲＮＡがｓｉＲＮＡである（２０）に記載のＲＮＡ。(11) The snoRNA expression inhibitor is siRNA of SEQ ID NO: 1, siRNA of SEQ ID NO: 1, siRNA of SEQ ID NO: 2, siRNA of SEQ ID NO: 3, siRNA of SEQ ID NO: 4, siRNA of SEQ ID NO: 5, siRNA of SEQ ID NO: 6. siRNA, siRNA of SEQ ID NO. 7, siRNA of SEQ ID NO. 8, siRNA of SEQ ID NO. 9, and siRNA of SEQ ID NO. Antiproliferative agents.
(12) The above (1) where the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 1, siRNA of SEQ ID NO: 1, siRNA of SEQ ID NO: 2, and siRNA of SEQ ID NO: 3. The agent for inhibiting the growth of breast cancer (eg, invasive breast cancer) according to 1) or (2).
(13) Colon cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 2 and siRNA of SEQ ID NO: 4. Antiproliferative agents.
(14) The renal cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 5 and siRNA of SEQ ID NO: 10. For example, a growth inhibitor for renal clear cell carcinoma).
(15) The liver cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 6 and siRNA of SEQ ID NO: 10 (e.g. , hepatocellular carcinoma) growth inhibitor.
(16) The lung cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 7 and siRNA of SEQ ID NO: 8 (e.g. , lung adenocarcinoma) growth inhibitor.
(17) The lung cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is one or more selected from siRNA of SEQ ID NO: 6 and siRNA of SEQ ID NO: 9 (e.g. , lung squamous cell carcinoma) growth inhibitor.
(18) The growth inhibitor for gastric cancer according to (1) or (2) above, wherein the snoRNA expression inhibitor is siRNA of SEQ ID NO: 4.
(19) A cancer therapeutic agent containing the cancer growth inhibitor according to any one of (1) to (18) above as an active ingredient.
(20) RNA consisting of any base sequence of SEQ ID NOs: 1 to 10.
(21) The RNA according to (20), wherein the RNA is siRNA.
本発明のがん治療剤は、がん細胞に特異的に高発現するｓｎｏＲＮＡを抑制又は発現させないようにすることによって、がん細胞の増殖を抑制し、がんの治療を行うものである。がん細胞に高発現するｓｎｏＲＮＡを抑制又は発現させないために使用される発現抑制剤としては、ｓｉＲＮＡ、ｓｈＲＮＡ、ｄｓＲＮＡのようなＲＮＡ干渉を利用するもの、アンチセンスオリゴヌクレオチド、デコイ等の公知の手段を使用することが出来る。
がん細胞に特異的に高発現するｓｎｏＲＮＡとしては、ＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂ、ＳＮＯＲＡ６９、ＳＮＯＲＤ１１１、ＳＮＯＲＤ９９、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ１０５Ｂ、ＳＮＯＲＤ３７、ＳＮＯＲＤ５４を特定することができ、これらの中から一つ以上のｓｎｏＲＮＡを発現抑制できる剤を使用することによって、乳癌、結腸癌、腎癌、肝癌、肺腺癌、肺扁平上皮癌、胃癌の増殖を抑制できることを見出している。それ故、本発明のがん増殖抑制剤は、これらのがんの有効な治療薬となる。The cancer therapeutic agent of the present invention suppresses the proliferation of cancer cells and treats cancer by suppressing or preventing the expression of snoRNA that is highly expressed specifically in cancer cells. Expression inhibitors used to suppress or prevent the expression of snoRNAs that are highly expressed in cancer cells include those that utilize RNA interference such as siRNA, shRNA, and dsRNA, and known means such as antisense oligonucleotides and decoys. can be used.
Snorna, which is specifically expressed in cancer cells, can identify Snora50C, Snora56, Snord42B, Snora69, Snord111, Snord99, Snod99, Snord105B, Snord37, Snord54. , One or more Snorna from these It has been found that the growth of breast cancer, colon cancer, kidney cancer, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, and gastric cancer can be suppressed by using an agent capable of suppressing expression. Therefore, the cancer growth inhibitor of the present invention is an effective therapeutic agent for these cancers.
本発明のがんの増殖抑制剤は、がん細胞で特異的に高発現するｓｎｏＲＮＡの発現抑制剤を含む剤であり、特に、この発現抑制剤を有効成分として含む剤である。
本発明の「がん細胞」とは、特に限定されるものではないが、ｓｎｏＲＮＡを高発現するがん細胞として、乳癌（浸潤性乳癌、非浸潤性乳癌）、結腸癌、腎癌（淡明細胞癌、乳頭状腎癌、嫌色素性腎癌、多房嚢胞性腎癌、紡錘細胞癌、集合管癌）、肝癌（肝細胞癌、肝内胆管癌）、肺癌（小細胞肺癌（肺腺癌、肺扁平上皮癌、肺大細胞癌がん）、非小細胞肺癌）、胃癌のいずれかのがん細胞を挙げることができる。The cancer growth inhibitor of the present invention is an agent containing an expression inhibitor of snoRNA that is specifically highly expressed in cancer cells, and particularly an agent containing this expression inhibitor as an active ingredient.
The "cancer cells" of the present invention are not particularly limited, but include cancer cells that highly express snoRNA, including breast cancer (invasive breast cancer, non-invasive breast cancer), colon cancer, and renal cancer (clear cancer). cell carcinoma, papillary renal carcinoma, chromophobe renal carcinoma, multilocular cystic renal carcinoma, spindle cell carcinoma, collecting duct carcinoma), liver cancer (hepatocellular carcinoma, intrahepatic cholangiocarcinoma), lung cancer (small cell lung cancer Cancer, squamous cell lung cancer, large cell lung cancer), non-small cell lung cancer), and gastric cancer can be mentioned.
本発明の「特異的に高発現するｓｎｏＲＮＡ」とは、健常人と比較して、がん細胞において顕著に又は有意に発現しているｓｎｏＲＮＡのことを言う。好ましくは、健常人の発現量の約４倍以上のｓｎｏＲＮＡの発現量を示すがん細胞のことを言う。より好ましくは約５倍以上のｓｎｏＲＮＡの発現量を持つがん細胞を挙げることができる。
がん特有の高発現ｓｎｏＲＮＡとしては、例えばＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂ、ＳＮＯＲＡ６９、ＳＮＯＲＤ１１１、ＳＮＯＲＤ９９、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ１０５Ｂ、ＳＮＯＲＤ３７、ＳＮＯＲＤ５４を挙げることができる。
このうち、乳癌（例えば、浸潤性乳癌）細胞特有の高発現ｓｎｏＲＮＡとして、ｓｎｏＲＮＡがＳＮＯＲＡ５０Ｃ、ＳＮＯＲＡ５６、ＳＮＯＲＤ４２Ｂを挙げることができ、結腸癌細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＡ５６、ＳＮＯＲＡ６９を挙げることができ、腎癌（例えば、腎淡明細胞癌）細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＤ１１１、ＳＮＯＲＤ９９を挙げることができ、肝癌（例えば、肝細胞癌）細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ９９を挙げることができ、肺癌細胞、例えば、肺腺癌細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＤ１０５Ｂ、ＳＮＯＲＤ３７を挙げることができ、また例えば、肺扁平上皮癌細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＡ７４Ａ、ＳＮＯＲＤ５４を挙げることができ、胃癌細胞特有の高発現ｓｎｏＲＮＡとして、ＳＮＯＲＡ６９を挙げることができる。The term "specifically highly expressed snoRNA" in the present invention refers to snoRNA that is significantly or significantly expressed in cancer cells compared to healthy individuals. Preferably, it refers to cancer cells that exhibit an expression level of snoRNA that is about four times or more than that of a healthy person. More preferred are cancer cells that express about 5 times or more snoRNA.
Examples of highly expressed snoRNAs specific to cancer include SNORA50C, SNORA56, SNORD42B, SNORA69, SNORD111, SNORD99, SNORA74A, SNORD105B, SNORD37, and SNORD54.
Among these, highly expressed snoRNAs specific to breast cancer (e.g., invasive breast cancer) cells include SNORA50C, SNORA56, and SNORD42B, and highly expressed snoRNAs specific to colon cancer cells include SNORA56 and SNORA69. , SNORD111 and SNORD99 are highly expressed snoRNAs specific to renal cancer (e.g., renal clear cell carcinoma) cells, and SNORA74A and SNORD99 are highly expressed snoRNAs specific to liver cancer (e.g., hepatocellular carcinoma) cells. Examples of highly expressed snoRNAs specific to lung cancer cells, for example, lung adenocarcinoma cells, include SNORD105B and SNORD37. For example, examples of highly expressed snoRNAs specific to lung squamous cell carcinoma cells include SNORA74A and SNORD54. SNORA69 can be mentioned as a highly expressed snoRNA unique to gastric cancer cells.
本発明の「発現抑制剤」とは、特異的に高発現するｓｎｏＲＮＡの発現を抑制する剤であれば特に限定はされない。標的となるｓｎｏＲＮＡの発現を抑制する剤は、標的ｓｎｏＲＮＡをコードしている遺伝子の転写、転写後調節等のいずれかの段階で標的ｓｎｏＲＮＡの発現に対する抑制作用を発揮するものであってよい。また、標的ｓｎｏＲＮＡを分解するもの、標的ｓｎｏＲＮＡの機能発現を抑制するものであってもよい。
標的ｓｎｏＲＮＡの発現を抑制する剤として、具体的には、標的ｓｎｏＲＮＡをコードする遺伝子の転写を抑制する核酸分子としてデコイ核酸等が挙げられ、標的ｓｎｏＲＮＡをＲＮＡ干渉作用により分解するＲＮＡ分子又はその前駆体としてｓｉＲＮＡ、ｓｈＲＮＡ、ｄｓＲＮＡ等が挙げられ、標的ｓｎｏＲＮＡにハイブリダイズしてその機能の発現を抑制したり、分解したりする核酸分子としてアンチセンス核酸等が挙げられる。これらの核酸医薬は、１種単独で使用してもよく、また２種以上を組み合わせて使用してもよい。また、これらの核酸医薬の塩基配列は、標的分子をコードする遺伝子の塩基配列の情報に基づいて、当業者が公知の手法により適宜設計することができる。これらの核酸医薬の中でも、臨床応用への容易性等の観点から、好ましくは、ｓｉＲＮＡ、ｓｈＲＮＡ、ｄｓＲＮＡが挙げられ、更に好ましくはｓｉＲＮＡが挙げられる。
好ましいｓｉＲＮＡとして、表２、表３に記載の配列番号１～１０の何れかの塩基配列を含むｓｉＲＮＡを挙げることができる。これらのｓｉＲＮＡの長さの上限は、３０塩基程度であり、特に２７塩基程度とすることができる。中でも、表２、表３に記載の配列番号１～１０の何れかの塩基配列からなるｓｉＲＮＡが好ましい。The "expression inhibitor" of the present invention is not particularly limited as long as it is an agent that inhibits the expression of snoRNA that is specifically highly expressed. The agent that suppresses the expression of the target snoRNA may exert a suppressive effect on the expression of the target snoRNA at any stage such as transcription of the gene encoding the target snoRNA or post-transcriptional regulation. Furthermore, it may be one that degrades the target snoRNA or one that suppresses the functional expression of the target snoRNA.
Examples of agents that suppress the expression of target snoRNAs include decoy nucleic acids and the like as nucleic acid molecules that suppress the transcription of genes encoding target snoRNAs, and RNA molecules or their precursors that degrade target snoRNAs by RNA interference. Examples of the snoRNA include siRNA, shRNA, dsRNA, etc., and examples of the nucleic acid molecule that hybridizes to the target snoRNA to suppress the expression of its function or degrade it include antisense nucleic acids. These nucleic acid drugs may be used alone or in combination of two or more. Furthermore, the base sequences of these nucleic acid drugs can be appropriately designed by those skilled in the art using known techniques based on information on the base sequence of the gene encoding the target molecule. Among these nucleic acid medicines, from the viewpoint of ease of clinical application, siRNA, shRNA, and dsRNA are preferred, and siRNA is more preferred.
Preferred siRNAs include siRNAs containing any of the base sequences of SEQ ID NOs: 1 to 10 listed in Tables 2 and 3. The upper limit of the length of these siRNAs is about 30 bases, particularly about 27 bases. Among these, siRNA consisting of any of the base sequences of SEQ ID NOs: 1 to 10 listed in Tables 2 and 3 is preferred.
また、前記発現抑制剤がＲＮＡ分子である場合は、生体内で生成し得るようにデザインされたものであってもよい。具体的には、当該ＲＮＡ分子をコードしているＤＮＡを哺乳動物細胞用の発現ベクターに挿入したものであってもよい。このような発現ベクターとしては、例えば、レトロウイルス、レンチウイルス、アデノウイルス、アデノ随伴ウイルス、ヘルペスウイルス、センダイウイルス等のウイルスベクターや、動物細胞発現プラスミド等が挙げられる。
また、前記発現抑制剤がＲＮＡ分子である場合は、安定性改善のために化学修飾が施されたものであってもよい。化学修飾ＲＮＡとして、例えば、ホスホロチオエート、モルフォリノホスホロジアミデート、ボラノホスフェート、ＬＮＡ（Ｌｏｃｋｅｄ Ｎｕｃｌｅｉｃ Ａｃｉｄ）のような核酸アナログを含むＲＮＡや、２’－Ｏ－メチル化ＲＮＡ、２’－Ｏ－メトキシエチル化ＲＮＡ等が挙げられる。Furthermore, when the expression inhibitor is an RNA molecule, it may be designed to be produced in vivo. Specifically, the DNA encoding the RNA molecule may be inserted into an expression vector for mammalian cells. Examples of such expression vectors include viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpesviruses, and Sendai viruses, and animal cell expression plasmids.
Furthermore, when the expression inhibitor is an RNA molecule, it may be chemically modified to improve stability. Examples of chemically modified RNA include RNA containing nucleic acid analogs such as phosphorothioate, morpholinophosphorodiamidate, boranophosphate, and LNA (Locked Nucleic Acid), 2'-O-methylated RNA, and 2'-O- Examples include methoxyethylated RNA.
本発明の「がんの増殖抑制剤」とは、がんの増殖を抑制できる剤のことであり、ｓｎｏＲＮＡの発現を抑制する剤を有効成分として使用することによって達成することができるものを言う。
本発明の「がん治療剤」とは、がんの増殖を抑制することにより、がん細胞のアポトーシスを完全に又は不完全に惹起するか、あるいは増殖が止まったがん細胞を生体の免疫反応によって完全に又は不完全に死滅させる治療剤のことを言う。
本発明の増殖抑制剤及び治療剤は、担癌患者の治療に使用されるだけでなく、担癌患者から癌を摘出した後に、再発や転移を予防する目的で投与してもよく、癌の切除後の患者における予後の改善剤又は癌の転移予防剤としても使用することもできる。The "cancer growth inhibitor" of the present invention refers to an agent that can inhibit cancer growth, and refers to something that can be achieved by using an agent that inhibits snoRNA expression as an active ingredient. .
The "cancer therapeutic agent" of the present invention is a drug that completely or incompletely induces apoptosis in cancer cells by suppressing cancer proliferation, or suppresses cancer cells whose proliferation has stopped by the immune system of the living body. A therapeutic agent that is completely or incompletely killed by a reaction.
The growth inhibitor and therapeutic agent of the present invention can be used not only for the treatment of cancer-bearing patients, but also for the purpose of preventing recurrence and metastasis after the cancer has been removed from the cancer-bearing patient. It can also be used as an agent for improving the prognosis of patients after resection or as an agent for preventing cancer metastasis.
また、本発明の増殖抑制剤及び治療剤は、以下のように使用される。
（用量、用法）
投与方法としては、本発明の増殖抑制剤及び治療剤を生体内で癌にデリバリーできることを限度として特に制限されないが、例えば、血管内（動脈内又は静脈内）注射、持続点滴、皮下投与、局所投与、筋肉内投与等が挙げられる。これらの中でも、好ましくは動静脈内投与が挙げられる。
投与量は、使用する有効成分の種類、患者の症状の程度、患者の性別、年齢等に応じて、標的分子の発現抑制（機能阻害を含む）が可能な範囲で適宜設定すればよい。
本発明の増殖抑制剤及び治療剤は、単独で使用してもよいが、抗腫瘍作用を有する１種又は２種以上の他の薬剤及び／又は放射線療法と併用してもよい。
（製剤形態）
本発明の増殖抑制剤及び治療剤は、その製剤形態に応じて、薬学的に許容される担体や添加剤を加えて製剤化される。例えば、固形製剤の場合であれば、賦形剤、結合剤、崩壊剤、滑沢剤等を用いて製剤化することができる。また、液状製剤の場合であれば、生理食塩水、緩衝液等を用いて製剤化することができる。
また、本発明の増殖抑制剤及び治療剤において、有効成分として核酸分子を使用する場合であれば、当該核酸分子が癌細胞内に移行され易いように、核酸導入補助剤と共に製剤化されていることが望ましい。核酸導入補助剤としては、具体的には、リポフェクタミン、オリゴフェクタミン、ＲＮＡｉフェクト、リポソーム、ポリアミン、ＤＥＡＥデキストラン、リン酸カルシウム、デンドリマー等が挙げられる。Moreover, the growth inhibitor and therapeutic agent of the present invention are used as follows.
(Dose, usage)
The administration method is not particularly limited as long as the growth inhibitor and therapeutic agent of the present invention can be delivered to cancer in vivo, but examples include intravascular (intraarterial or intravenous) injection, continuous drip, subcutaneous administration, and local administration. administration, intramuscular administration, etc. Among these, intraarteriovenous administration is preferred.
The dosage may be appropriately determined depending on the type of active ingredient used, the severity of the patient's symptoms, the patient's sex, age, etc., within a range that allows suppression of expression (including functional inhibition) of the target molecule.
The growth inhibitor and therapeutic agent of the present invention may be used alone, or may be used in combination with one or more other drugs having antitumor effects and/or radiotherapy.
(Preparation form)
The growth inhibitor and therapeutic agent of the present invention are formulated by adding pharmaceutically acceptable carriers and additives, depending on the formulation form. For example, in the case of a solid preparation, it can be formulated using excipients, binders, disintegrants, lubricants, etc. Moreover, in the case of a liquid preparation, it can be formulated using physiological saline, a buffer solution, or the like.
In addition, in the case where a nucleic acid molecule is used as an active ingredient in the proliferation inhibitor and therapeutic agent of the present invention, the nucleic acid molecule is formulated together with a nucleic acid introduction aid so that the nucleic acid molecule is easily transferred into cancer cells. This is desirable. Specific examples of the nucleic acid introduction aid include lipofectamine, oligofectamine, RNAifect, liposome, polyamine, DEAE dextran, calcium phosphate, and dendrimer.
以下、実施例および試験例に基づいて本発明をより具体的に説明するが、本発明はこれによって限定されるものではない。 Hereinafter, the present invention will be explained in more detail based on Examples and Test Examples, but the present invention is not limited thereto.
（実施例１）がん細胞に特有の高発現のｓｎｏＲＮＡの同定
（１）方法：
ａ）データベース：
Ｎａｔｉｏｎａｌ Ｃａｎｓｅｒ Ｉｎｓｔｉｔｕｔｅ（ＮＣＩ）（米国国立がん研究所) とＮａｔｉｏｎａｌ Ｈｕｍａｎ Ｇｅｎｏｍｅ Ｒｅｓｅａｒｃｈ Ｉｎｓｔｉｔｕｔｅ（ＮＨＧＲＩ）（米国国立ヒトゲノム研究所)によるプロジェクトで収集したさまざまな組織におけるがんのゲノムやエピゲノム、トランスクリプトーム、変異情報などのデータがＴｈｅ Ｃａｎｓｅｒ Ｇｅｎｏｍｅ Ａｔｌａｓ（ＴＣＧＡ）に集約されている。
ｂ）同定方法：
ＴＣＧＡの収載データから、正常検体と癌検体の比較が可能であり、かつｓｎｏＲＮＡ発現情報を含むデータの抽出を行った。そして、正常検体に比べて癌検体で発現が有意に増加しているｓｎｏＲＮＡを抽出し、さらにＣｏｘ比例ハザードモデルにてｓｎｏＲＮＡの高発現癌患者群の生命予後が低発現癌患者群の生命予後と比べて不良をしめすｓｎｏＲＮＡを、発癌ｓｎｏＲＮＡとして同定した。(Example 1) Identification of highly expressed snoRNA specific to cancer cells (1) Method:
a) Database:
Cancer genomes, epigenomes, and transcriptomes from various tissues collected through a project by the National Cancer Institute (NCI) and the National Human Genome Research Institute (NHGRI), Data such as mutation information is aggregated in The Cancer Genome Atlas (TCGA).
b) Identification method:
From the data listed in TCGA, we extracted data that allows comparison between normal and cancer samples and that includes snoRNA expression information. Then, we extracted snoRNAs whose expression was significantly increased in cancer samples compared to normal samples, and then used a Cox proportional hazards model to predict that the life prognosis of cancer patients with high snoRNA expression was different from that of cancer patients with low snoRNA expression. SnoRNAs that performed poorly in comparison were identified as oncogenic snoRNAs.
（２）結果：
上記検討の結果、がん細胞に特有の高発現のｓｎｏＲＮＡとして、表１に示される発癌ｓｎｏＲＮＡを同定することが出来た。例えば、表１の腎癌の場合、ＳＮＯＲＤ９９が健常人の１３倍も高発現していた。そして、図２に示されるように、ＳＮＯＲＤ９９が高発現している癌患者の予後が悪かった。(2) Results:
As a result of the above studies, the oncogenic snoRNAs shown in Table 1 were identified as highly expressed snoRNAs specific to cancer cells. For example, in the case of renal cancer shown in Table 1, SNORD99 was expressed 13 times more highly than in healthy individuals. As shown in FIG. 2, cancer patients with high expression of SNORD99 had a poor prognosis.
浸潤性乳癌（ｂｒｅａｓｔ ｉｎｖａｓｉｖｅ ｃａｒｃｉｎｏｍａ）
結腸癌（ｃｏｌｏｎ ａｄｅｎｏｃａｒｃｉｎｏｍａ）
腎淡明細胞癌（ｋｉｄｎｅｙ ｒｅｎａｌ ｃｌｅａｒ ｃｅｌｌ ｃａｒｃｉｎｏｍａ）
肝細胞癌（ｌｉｖｅｒ ｈｅｐａｔｏｃｅｌｌｕｌａｒ ｃａｒｃｉｎｏｍａ）
肺腺癌（ｌｕｎｇ ａｄｅｎｏｃａｒｃｉｎｏｍａ）
肺扁平上皮癌（ｌｕｎｇ ｓｑｕａｍｏｕｓ ｃｅｌｌ ｃａｒｃｉｎｏｍａ）
胃癌（ｓｔｏｍａｃｈ ａｄｅｎｏｃａｒｃｉｎｏｍａ）
invasive breast cancer
colon cancer
kidney clear cell carcinoma
liver hepatocellular carcinoma
lung adenocarcinoma
Lung squamous cell carcinoma
Stomach adenocarcinoma
上記表１に示されるように、浸潤性乳癌では、ＳＮＯＲＡ５０Ｃが健常人と比べて発現量が４．６倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０５以下と有意差があった。ＳＮＯＲＡ５６が健常人と比べて発現量が５．７倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。ＳＮＯＲＤ４２Ｂが健常人と比べて発現量が７．０倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。
結腸癌では、ＳＮＯＲＡ５６が健常人と比べて発現量が５．９倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。ＳＮＯＲＡ６９が健常人と比べて発現量が３．７倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。
腎淡明細胞癌では、ＳＮＯＲＤ１１１が健常人と比べて発現量が４．０倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。ＳＮＯＲＤ９９が健常人と比べて発現量が１３．０倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。As shown in Table 1 above, in invasive breast cancer, the expression level of SNORA50C is 4.6 times that of healthy subjects, and the Cox proportional hazard analysis between the expression level and life prognosis showed a significant p value of 0.05 or less. There was a difference. The expression level of SNORA56 was 5.7 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less. The expression level of SNORD42B was 7.0 times that of healthy individuals, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
In colon cancer, the expression level of SNORA56 was 5.9 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less. The expression level of SNORA69 was 3.7 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
In renal clear cell carcinoma, the expression level of SNORD111 was 4.0 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less. The expression level of SNORD99 was 13.0 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
肝細胞癌では、ＳＮＯＲＡ７４Ａが健常人と比べて発現量が５．７倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。ＳＮＯＲＤ９９が健常人と比べて発現量が４．６倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０５以下と有意差があった。
肺腺癌では、ＳＮＯＲＤ１０５Ｂが健常人と比べて発現量が５．３倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０５以下と有意差があった。ＳＮＯＲＤ３７が健常人と比べて発現量が８．６倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。
肺扁平上皮癌では、ＳＮＯＲＡ７４Ａが健常人と比べて発現量が４．３倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０５以下と有意差があった。ＳＮＯＲＤ５４が健常人と比べて発現量が５．７倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。
胃癌では、ＳＮＯＲＡ６９が健常人と比べて発現量が１３．０倍で発現量と生命予後とのＣｏｘ比例ハザード解析でｐ値が０．０１以下と有意差があった。In hepatocellular carcinoma, the expression level of SNORA74A was 5.7 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less. The expression level of SNORD99 was 4.6 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.05 or less.
In lung adenocarcinoma, the expression level of SNORD105B was 5.3 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.05 or less. The expression level of SNORD37 was 8.6 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
In squamous cell lung cancer, the expression level of SNORA74A was 4.3 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.05 or less. The expression level of SNORD54 was 5.7 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
In gastric cancer, the expression level of SNORA69 was 13.0 times that of healthy subjects, and Cox proportional hazard analysis between the expression level and life prognosis showed a significant difference with a p value of 0.01 or less.
同定された発癌ｓｎｏＲＮＡの塩基配列と、その発現を抑制するｓｉＲＮＡの塩基配列の一例を、表２、表３に示す。 Examples of the base sequences of the identified oncogenic snoRNAs and the base sequences of siRNAs that suppress their expression are shown in Tables 2 and 3.
ヒトｓｎｏＲＮＡの配列は、米国のＮＣＢＩで構築しているデータベースより抽出した。なお、ｓｎｏＲＮＡに対するｓｉＲＮＡは、ｓｎｏＲＮＡ配列に基づき、そのｓｉＲＮＡを設計し、５０％以上の発現抑制効果がある配列を、上記表２、表３に示した。なお、上記ｓｉＲＮＡはアンチセンスＲＮＡとしても機能し得る。また、デコイ様に機能してもよい。 The human snoRNA sequence was extracted from a database constructed by NCBI in the United States. In addition, siRNA for snoRNA was designed based on the snoRNA sequence, and sequences having an expression suppression effect of 50% or more are shown in Tables 2 and 3 above. Note that the above siRNA can also function as antisense RNA. It may also function like a decoy.
（実施例２）腎淡明細胞癌で高発現（正常細胞の１３．０倍）のＳＮＯＲＤ９９が欠損したヒト腎癌細胞株と欠損の無いヒト腎癌細胞株の増殖性の比較評価
腎癌（腎淡明細胞癌：ｋｉｄｎｅｙ ｒｅｎａｌ ｃｌｅａｒ ｃｅｌｌ ｃａｒｃｉｎｏｍａ）患者では、図２に示すようにｓｎｏＲＮＡとして、ＳＮＯＲＤ９９が特に顕著に発現しており、その腎癌患者の予後が悪いことを見出している。そこで、腎癌で高発現しているＳＮＯＲＤ９９のがんにおける機能を明らかにするため、ＳＮＯＲＤ９９が欠損したヒト腎がん細胞株と、ポジティブ・コントロールのヒト腎がん細胞株を用いて、ヌードマウスの皮下に他家移植を行った。
（１）材料・試薬
・ポジテイブ・コントロールのヒト腎癌細胞株７８６Ｏ ｍｏｃｋ
・ＳＮＯＲＤ９９が欠損したヒト腎癌細胞株７８６Ｏ９９ＫＯ
上記ポジテイブ・コントロール細胞株およびＳＮＯＲＤ９９欠損細胞株の作成方法は、次の通りである。
７８６Ｏ ｍｏｃｋおよび７８６Ｏ９９ＫＯは、Ｃｒｉｓｐｒ－Ｃａｓ９法により作成した。ヒト腎癌細胞株７８６Ｏ細胞にＣａｓ９を安定過剰発現させた後、ｇｕｉｄｅ ＲＮＡ（ｇｃｃｇｇｇｃｃｔｔｃｃａａｃｃｃｇｇｔ）をレンチウイルスにより導入しピューロマイシン耐性選択を行い７８６Ｏ９９ＫＯを作成した。ＳＮＯＲＤ９９遺伝子欠損は定量ＰＣＲおよびゲノムＤＮＡ配列決定により確認した。７８６Ｏ ｍｏｃｋは７８６ＯにＣａｓ９を安定過剰発現させた後、ｇｕｉｄｅ ＲＮＡなしレンチウイルスを導入し薬剤耐性選択を行い作成した。
（２）方法
ヌードマウス（雄性、４週令）の皮下に、ＳＮＯＲＤ９９が欠損したヒト腎癌細胞株（７８６Ｏ９９ＫＯ）とポジテイブ・コントロールのヒト腎癌細胞株（７８６Ｏ ｍｏｃｋ）を、５ｘ１０６個注入した。６週後の腎癌形成の様子を目視で観察し、移植腎癌細胞の直径を計測した。
（３）結果
図３に示されるように、６週後に、ＳＮＯＲＤ９９が欠損したヒト腎癌細胞株では、ポジテイブ・コントロールのヒト腎癌細胞株に比べて、腎癌形成が抑制されていた。移植腎癌の直径を、以下の表４に示す。(Example 2) Comparative evaluation of proliferation of a human renal cancer cell line deficient in SNORD99, which is highly expressed (13.0 times that of normal cells) in renal clear cell carcinoma, and a human renal cancer cell line without the deficiency Renal cancer ( As shown in FIG. 2, SNORD99 is particularly prominently expressed as an snoRNA in kidney clear cell carcinoma (kidney renal clear cell carcinoma) patients, and it has been found that these kidney cancer patients have a poor prognosis. Therefore, in order to clarify the function of SNORD99, which is highly expressed in renal cancer, we used a human renal cancer cell line deficient in SNORD99 and a positive control human renal cancer cell line to test nude mice. Allogeneic transplantation was performed subcutaneously.
(1) Materials/reagents/positive control human kidney cancer cell line 786O mock
・Human renal cancer cell line 786O99KO lacking SNORD99
The method for creating the positive control cell line and SNORD99-deficient cell line is as follows.
786O mock and 786O99KO were created by Crispr-Cas9 method. After stably overexpressing Cas9 in human renal cancer cell line 786O cells, guide RNA (gccgggccttccaacccggt) was introduced by lentivirus, and puromycin resistance selection was performed to create 786O99KO. SNORD99 gene deletion was confirmed by quantitative PCR and genomic DNA sequencing. 786O mock was created by stably overexpressing Cas9 in 786O, introducing lentivirus without guide RNA, and performing drug resistance selection.
(2) Method 5x10 6 cells of a human renal cancer cell line lacking SNORD99 (786O99KO) and a positive control human renal cancer cell line (786O mock) were subcutaneously injected into nude mice (male, 4 weeks old). . Six weeks later, the state of renal cancer formation was visually observed, and the diameter of the transplanted renal cancer cells was measured.
(3) Results As shown in FIG. 3, after 6 weeks, renal cancer formation was suppressed in the SNORD99-deficient human renal cancer cell line compared to the positive control human renal cancer cell line. The diameters of transplanted renal cancers are shown in Table 4 below.
上記表４に示すように、６週間後の移植腎癌の体積は、ＳＮＯＲＤ９９が欠損したヒト腎がん細胞株（７８６Ｏ９９ＫＯ）では２１ｍｍ３であり、ポジテイブ・コントロール（７８６Ｏｍｏｃｋ）では、４３６ｍｍ３（ｐ＜０．０１）であった。
以上のように、ＳＮＯＲＤ９９が欠損したヒト腎がん細胞株では、ヌードマウスに対するがん細胞の生着・増殖が顕著に抑制されることを見出した。このことは、腎癌で高発現のＳＮＯＲＤ９９を抑制すると、腎癌の増殖が抑制できることを示しており、ＳＮＯＲＤ９９の発現を抑制すれば、がん治療が可能であることを示している。As shown in Table 4 above, the volume of the transplanted renal cancer after 6 weeks was 21 mm 3 in the SNORD99-deficient human renal cancer cell line (786O99KO), and 436 mm 3 (p <0.01).
As described above, it was found that in a human renal cancer cell line lacking SNORD99, the engraftment and proliferation of cancer cells in nude mice was significantly suppressed. This shows that suppressing SNORD99, which is highly expressed in renal cancer, can suppress the growth of renal cancer, and that suppressing the expression of SNORD99 makes it possible to treat cancer.
本発明のがん治療剤は、がん細胞で特異的に高発現するｓｎｏＲＮＡを発現抑制することによって、効果的にがん細胞の増殖を抑制し、新たな作用機序でがん治療を行うことが出来る。本発明のがん増殖抑制剤は、単剤で使用することができ、更には免疫賦活化のがん治療剤と併用して、その効果を高めることが出来る。それ故、本発明のがん増殖抑制剤としては、ｓｎｏＲＮＡを発現抑制ができるものとして、ｓｉＲＮＡやデコイ等の核酸医薬を使用することができ、新たながんの増殖抑制剤、更にはがんの治療剤として利用できる。 The cancer therapeutic agent of the present invention effectively suppresses the proliferation of cancer cells by suppressing the expression of snoRNA that is highly expressed specifically in cancer cells, and performs cancer treatment with a new mechanism of action. I can do it. The cancer growth inhibitor of the present invention can be used as a single agent, or can be used in combination with an immunostimulatory cancer treatment agent to enhance its effect. Therefore, as the cancer growth inhibitor of the present invention, nucleic acid medicines such as siRNA and decoys can be used as agents capable of suppressing the expression of snoRNA, and can be used as new cancer growth inhibitors or even cancer growth inhibitors. It can be used as a therapeutic agent.
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