KR102791823B1 - Biomarker for colon cancer diagnosis and pharmaceutical composition for preventing or treating colon cancer - Google Patents
Biomarker for colon cancer diagnosis and pharmaceutical composition for preventing or treating colon cancer Download PDFInfo
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- KR102791823B1 KR102791823B1 KR1020210052584A KR20210052584A KR102791823B1 KR 102791823 B1 KR102791823 B1 KR 102791823B1 KR 1020210052584 A KR1020210052584 A KR 1020210052584A KR 20210052584 A KR20210052584 A KR 20210052584A KR 102791823 B1 KR102791823 B1 KR 102791823B1
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Abstract
본 발명은 정상 대장세포에 비해 대장암 세포에서 CNOT2이 과발현됨을 확인하고, CNOT2의 발현 억제를 통해 p53을 활성화하여 대장암 세포의 생장을 억제하고 세포사멸을 유도함을 확인하였는바, 대장암의 진단 및 치료 타겟으로서 CNOT2를 제공할 수 있다. 또한, 본 발명은 종래 항암제와 CNOT2의 억제제의 병용 투여로 항암효과가 향상됨을 확인하였는바, 대장암의 치료약물 스크리닝 및 유전자 치료에 이용될 수 있으며, 표적 유전자 치료제로서 부작용이 감소된 항암제 등의 바이오 신약 개발에 활용될 것으로 기대된다.The present invention confirms that CNOT2 is overexpressed in colon cancer cells compared to normal colon cells, and inhibits the growth of colon cancer cells and induces apoptosis by activating p53 through inhibition of CNOT2 expression, and thus CNOT2 can be provided as a target for diagnosis and treatment of colon cancer. In addition, the present invention confirms that the anticancer effect is enhanced by combined administration of a conventional anticancer agent and a CNOT2 inhibitor, and thus can be used for screening therapeutic drugs and gene therapy for colon cancer, and is expected to be utilized in the development of new biopharmaceuticals such as anticancer agents with reduced side effects as targeted gene therapy agents.
Description
본 발명은 CNOT2 유전자를 대장암 진단을 위한 바이오마커로 제공하고, CNOT2 억제제를 포함하는 대장암 치료용 약학적 조성물 등에 관한 것으로서, 본 발명의 약학적 조성물은 CNOT2 억제를 통해 종양억제유전자인 p53을 활성화하여 대장암의 치료에 제공될 수 있다.The present invention provides the CNOT2 gene as a biomarker for diagnosing colon cancer, and relates to a pharmaceutical composition for treating colon cancer including a CNOT2 inhibitor. The pharmaceutical composition of the present invention can be provided for the treatment of colon cancer by activating p53, a tumor suppressor gene, through inhibition of CNOT2.
대장암(colon cancer)은 미국에서 암과 관련된 사망의 주요원인으로, 2010년 현재 미국에서 약 102,900명이 대장암으로 진단되고, 51,370명이 사망할 것으로 추정되는 암이다. 최근에는 서구화된 식습관으로 인해 아시아에서도 그 발병률이 증가하고 있는 추세이며, 관련된 요인으로는 과도한 동물성 지방, 당분, 알코올 섭취와 섬유소, 항산화 비타민, 야채나 과일의 섭취 부족 등이 주요 원인으로 알려져 있다. 특히 동물성 지방과 육류를 많이 섭취하면 채소나 곡물 등의 섬유질 식품 섭취와는 달리 대변 양이 적고 내용물이 대장을 통과하여 배설되는 시간이 많이 걸린다. 또한 담즙산과 스테롤의 배설이 증가하며 대장 내에 존재하는 세균종의 구성에도 변화를 일으켜 이들 물질을 화학적으로 변화시키는 세균의 종류가 증가한다. 이에 따라, 발암물질이 많이 생성됨과 동시에 대장 내에 머물고 접촉하는 시간도 길어져서 대장암이 자주 발생하게 되는 원인이 된다.대장암의 경우 혈관을 중심으로 많은 수의 세포들이 서로 덩어리진 형태로 성장하는 대표적인 고형암 세포로서 치료방법이 극히 제한적이며 완치가 어려운 암 중 하나이다. 즉, 덩어리진 대장암 세포의 중심부까지 약물Colon cancer is the leading cause of cancer-related death in the United States, with approximately 102,900 people diagnosed with colon cancer in 2010 and an estimated 51,370 deaths. Recently, the incidence has been increasing in Asia due to Westernized eating habits, and the main causes are known to be excessive intake of animal fat, sugar, and alcohol, and insufficient intake of fiber, antioxidant vitamins, vegetables, and fruits. In particular, when a lot of animal fat and meat is consumed, unlike when fibrous foods such as vegetables or grains are consumed, the amount of stool is small and it takes a long time for the contents to pass through the large intestine and be excreted. In addition, the excretion of bile acids and sterols increases, and the composition of the bacterial species present in the large intestine changes, increasing the types of bacteria that chemically change these substances. Accordingly, as a large amount of carcinogens are produced and the time they remain and come into contact with the colon is long, this becomes a cause of frequent occurrence of colon cancer. In the case of colon cancer, it is a representative solid cancer cell in which a large number of cells grow in a clump centered on blood vessels, and it is one of the cancers that is extremely limited in treatment methods and difficult to cure. In other words, the drug is injected into the center of the clumped colon cancer cells.
이 제대로 투과하지 못하기 때문에 대장암 세포를 완전히 제거하기란 쉬운 일이 아니다. 현재 약물로써 대장암을 치료할 수 있는 방법은 거의 없는 실정이고, 수술요법이나 방사선요법 등과 같은 외과적인 치료만이 대장암을 치료할 수 있는 유일한 방법이며, 이러한 치료법으로는 대장암의 완치를 기대하기 어렵다.It is not easy to completely remove colon cancer cells because they do not penetrate properly. Currently, there are almost no methods to treat colon cancer with drugs, and the only way to treat colon cancer is surgical treatment such as surgery or radiotherapy, and it is difficult to expect a complete cure for colon cancer with these treatments.
따라서 많은 연구자가 대장암 세포의 성장을 효과적으로 억제할 수 있는 방법을 개발함으로써 대장암 세포를 포함한 다양한 암세포들의 성장을 효과적으로 억제하기 위해서 많은 연구를 수행하고 있다.Therefore, many researchers are conducting a lot of research to effectively inhibit the growth of various cancer cells, including colon cancer cells, by developing a method that can effectively inhibit the growth of colon cancer cells.
세포사멸(apoptosis)은 항암제가 암세포의 증식 억제 효과를 나타내는 중요한 작용기작으로, 세포 내부에 프로그램화된 신호를 따라 여러 유전자 및 단백질들의 발현과 활성이 조절되어 일어나는 능동적인 죽음이다. 세포사멸 과정의 교란은 손상되거나 손상이 시작된 세포의 생존과 그들 세포의 성장을 유도하기 때문에 세포사멸의 억제는 암화 과정에서 중요한 역할을 한다. 아울러 암예방 효과가 있는 물질들은 이러한 비정상적인 세포의 세포사멸을 유도하며, 이들에 의한 세포사멸의 유발은 최소한 그들의 암예방 활성과 연관되어 있음이 보고되었다.Apoptosis is an important mechanism by which anticancer drugs exhibit the effect of inhibiting the proliferation of cancer cells. It is an active death that occurs when the expression and activity of various genes and proteins are regulated according to signals programmed inside the cell. Since disruption of the apoptosis process induces the survival of damaged or damaged cells and their growth, inhibition of apoptosis plays an important role in the process of carcinogenesis. In addition, it has been reported that substances with cancer prevention effects induce apoptosis of these abnormal cells, and that the induction of apoptosis by them is at least related to their cancer prevention activity.
본 발명자들은 대장암세포에서 CNOT2의 과발현을 확인하고, CNOT2가 p53의 활성을 억제하여 비정상적인 세포사멸의 억제를 통해 암세포의 증식 및 암의 성장에 기여함을 확인하고 본 발명을 완성하였다.The present inventors confirmed overexpression of CNOT2 in colon cancer cells and confirmed that CNOT2 suppresses the activity of p53, thereby contributing to the proliferation and growth of cancer cells through inhibition of abnormal apoptosis, thereby completing the present invention.
본 발명이 이루고자 하는 기술적 과제는 대장암 진단을 위한 바이오마커와 대장암 예방 및 치료용 약학적 조성물 및 대장암 치료용 약물의 스크리닝 방법을 제공하는 것이다.The technical problem to be achieved by the present invention is to provide a biomarker for diagnosing colon cancer, a pharmaceutical composition for preventing and treating colon cancer, and a screening method for a drug for treating colon cancer.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당해 기술분야의 통상의 기술자에게 명확하게 이해될 수 있을 것이다.However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.
상기 과제를 해결하기 위하여, 본 발명은 CNOT2 단백질을 포함하는 대장암 진단용 바이오마커 조성물을 제공한다.To solve the above problem, the present invention provides a biomarker composition for diagnosing colon cancer comprising CNOT2 protein.
또한, 본 발명은 CNOT2의 발현 수준을 측정할 수 있는 제제를 유효성분으로 포함하는 대장암 진단용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for diagnosing colon cancer, comprising as an active ingredient an agent capable of measuring the expression level of CNOT2.
또한, 본 발명은 CNOT2의 발현 수준을 측정할 수 있는 제제를 포함하는 대장암 진단 키트를 제공한다. In addition, the present invention provides a colon cancer diagnostic kit comprising a preparation capable of measuring the expression level of CNOT2.
본 발명의 일 구현예로서, 상기 키트에는 상기 측정 제제 이외에도 검출을 용이하게 하는 당업계에 공지된 여러 도구, 시약, 예를 들어 적합한 담체, 검출 가능한 신호를 생성할 수 있는 표지 물질, 안정화제 등이 포함될 수 있다.As one embodiment of the present invention, the kit may include, in addition to the measuring agent, various tools and reagents known in the art that facilitate detection, such as a suitable carrier, a labeling substance capable of generating a detectable signal, a stabilizer, etc.
또한, 본 발명은 대장암 진단이 필요한 개체의 대장 조직 샘플에서 CNOT2의 발현 수준을 측정하는 단계를 포함하는 대장암 진단을 위한 정보제공방법을 제공한다.In addition, the present invention provides a method for providing information for diagnosing colon cancer, which comprises a step of measuring the expression level of CNOT2 in a colon tissue sample of an individual in need of colon cancer diagnosis.
본 발명의 일 구현예로서, 상기 정보제공방법은 정상 대장 조직과 비교하여 CNOT2 단백질 발현 수준이 높은 경우 대장암이 발병하였거나 발병 가능성이 높은 것으로 판정하는 단계를 추가로 포함할 수 있다. As one embodiment of the present invention, the information providing method may additionally include a step of determining that colon cancer has developed or is likely to develop when the level of CNOT2 protein expression is high compared to normal colon tissue.
본 발명의 다른 구현예로서, 상기 CNOT2의 발현 수준을 측정할 수 있는 제제는 CNOT2 유전자의 mRNA 또는 단백질의 발현 수준을 측정할 수 있는 제제일 수 있다.As another embodiment of the present invention, the agent capable of measuring the expression level of CNOT2 may be an agent capable of measuring the expression level of mRNA or protein of the CNOT2 gene.
본 발명의 다른 구현예로서, 상기 CNOT2 mRNA의 발현 수준을 측정할 수 있는 제제는 CNOT2 mRNA와 상보적인 서열의 올리고뉴클레오티드, 프라이머, 프로브, 및/또는 항체일 수 있고, 상기 CNOT2 단백질의 발현 수준을 측정할 수 있는 제제는 CNOT2 단백질에 특이적으로 결합할 수 있는 항체, 압타머(aptamer), 및/또는 화합물일 수 있다. In another embodiment of the present invention, the agent capable of measuring the expression level of the CNOT2 mRNA may be an oligonucleotide, primer, probe, and/or antibody having a sequence complementary to CNOT2 mRNA, and the agent capable of measuring the expression level of the CNOT2 protein may be an antibody, aptamer, and/or compound capable of specifically binding to the CNOT2 protein.
본 발명의 다른 구현예로서, 상기 mRNA 발현 수준 측정은 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), 정량적 중합효소반응(quantitative RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블롯팅(Northern blotting), 또는 DNA 칩 방식으로 수행될 수 있다.As another embodiment of the present invention, the measurement of the mRNA expression level can be performed by RT-PCR, competitive RT-PCR, real-time RT-PCR, quantitative RT-PCR, RNase protection assay (RPA), Northern blotting, or DNA chip method.
또한, 본 발명은 CNOT2 억제제를 유효성분으로 포함하는 대장암 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating colon cancer, comprising a CNOT2 inhibitor as an active ingredient.
본 발명의 일 구현예로서, 상기 CNOT2 억제제는 CNOT2 유전자의 발현을 억제하는 제제 또는 CNOT2 단백질의 활성을 억제하는 제제일 수 있다. As one embodiment of the present invention, the CNOT2 inhibitor may be an agent that inhibits the expression of a CNOT2 gene or an agent that inhibits the activity of a CNOT2 protein.
본 발명의 다른 구현예로서, 상기 CNOT2 유전자 발현을 억제하는 제제는 CNOT2 mRNA의 일부와 상보적인 올리고뉴클레오티드, siRNA(small interfering RNA) 및 shRNA(small hairpin RNA)로 이루어진 군으로부터 선택된 1종 이상일 수 있다.As another embodiment of the present invention, the agent that inhibits the expression of the CNOT2 gene may be at least one selected from the group consisting of an oligonucleotide complementary to a portion of CNOT2 mRNA, a small interfering RNA (siRNA), and a small hairpin RNA (shRNA).
본 발명의 다른 구현예로서, 상기 CNOT2 유전자 발현을 억제하는 제제는 서열번호 1 및/또는 서열번호 2의 올리고뉴클레오티드일 수 있다.As another embodiment of the present invention, the agent that inhibits the expression of the CNOT2 gene may be an oligonucleotide of SEQ ID NO: 1 and/or SEQ ID NO: 2.
본 발명의 다른 구현예로서, 상기 CNOT2 발현 억제제는 대장암 세포의 세포사멸을 유도하여 생장을 억제할 수 있다.As another embodiment of the present invention, the CNOT2 expression inhibitor can inhibit the growth of colon cancer cells by inducing apoptosis.
본 발명의 다른 구현예로서, 상기 조성물은 항암제와 병용투여용 일 수 있다. As another embodiment of the present invention, the composition may be used in combination with an anticancer agent.
본 발명의 다른 구현예로서, 상기 항암제는 독소루비신, 다우노루비신, 닥티노마이신, 블레오마이신, 플리카마이신(미스라마이신), 마이토마이신 C, 스트렙토조신, 및 미톡산트론으로 이루어진 군으로부터 선택되는 1종 이상의 항암제일 수 있다.In another embodiment of the present invention, the anticancer agent may be at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, dactinomycin, bleomycin, plicamycin (misramycin), mitomycin C, streptozocin, and mitoxantrone.
또한, 본 발명은 CNOT2 억제제를 개체에 투여하는 단계를 포함하는 대장암 예방 또는 치료 방법을 제공한다. Additionally, the present invention provides a method for preventing or treating colon cancer comprising a step of administering a CNOT2 inhibitor to a subject.
본 발명의 일 구현예로서, 상기 방법은 CNOT2 억제제를 항암제와 동시에 또는 순차로 투여하는 단계를 포함하는 것일 수 있다.As one embodiment of the present invention, the method may comprise a step of administering a CNOT2 inhibitor simultaneously or sequentially with an anticancer agent.
또한, 본 발명은 대장암 예방 또는 치료 약제의 제조를 위한 CNOT2 억제제의 용도를 제공한다. The present invention also provides the use of a CNOT2 inhibitor for the manufacture of a medicament for preventing or treating colon cancer.
또한, 본 발명은 하기 단계를 포함하는 대장암 치료제 스크리닝 방법을 제공한다:In addition, the present invention provides a method for screening a colon cancer treatment agent comprising the following steps:
(1) 체외에서 세포에 후보물질을 처리하는 단계;(1) A step of treating a candidate substance to cells in vitro;
(2) 상기 세포에서 CNOT2 단백질의 발현 수준을 측정하는 단계; 및(2) a step of measuring the expression level of CNOT2 protein in the above cells; and
(3) 상기 세포의 CNOT2 단백질의 발현 수준이 후보물질을 처리하지 않은 세포보다 감소한 경우 상기 후보물질을 대장암 치료 약물로 선정하는 단계. (3) A step of selecting the candidate substance as a colorectal cancer treatment drug when the expression level of the CNOT2 protein of the above cells is reduced compared to cells not treated with the candidate substance.
본 발명의 일 구현예로서, 상기 세포는 인간 대장세포, 대장암 세포, 또는 유전공학적으로 CNOT2가 과발현 되도록 제조된 세포일 수 있다.As one embodiment of the present invention, the cell may be a human colon cell, a colon cancer cell, or a cell genetically engineered to overexpress CNOT2.
본 발명은 정상 대장세포에 비해 대장암 세포에서 CNOT2가 과발현됨을 확인하고, CNOT2의 발현 억제를 통해 p53을 활성화하여 대장암 세포의 생장을 억제하고 세포사멸을 유도함을 확인하였는바, 대장암의 진단 및 치료 타겟으로서 CNOT2를 제공할 수 있다. 또한, 본 발명은 종래 항암제와 CNOT2의 억제제의 병용 투여로 항암효과가 향상됨을 확인하였는바, 대장암의 치료약물 스크리닝 및 유전자 치료에 이용될 수 있으며, 표적 유전자 치료제로서 기존 약물의 용량을 줄임과 동시에 부작용이 감소된 항암제 등의 바이오 신약 개발에 활용될 것으로 기대된다.The present invention confirms that CNOT2 is overexpressed in colon cancer cells compared to normal colon cells, and inhibits the growth of colon cancer cells and induces apoptosis by activating p53 through inhibition of CNOT2 expression, and thus CNOT2 can be provided as a target for the diagnosis and treatment of colon cancer. In addition, the present invention confirms that the anticancer effect is enhanced by combined administration of a conventional anticancer drug and a CNOT2 inhibitor, and thus can be used for screening therapeutic drugs and gene therapy for colon cancer, and is expected to be utilized in the development of bio-new drugs such as anticancer drugs with reduced side effects while reducing the dosage of existing drugs as a targeted gene therapy agent.
도 1의 A는 대장암 세포와 정상 대장세포에서 CNOT2의 발현량을 웨스턴 블롯팅을 통해 확인한 도면이고, B는 siRNA CNOT2를 도입에 따른 세포 생존율을 나타낸 도면이다. 대장암 세포는 정상 대장세포보다 CNOT2 발현 수준이 높으며, siRNA CNOT2를 트랜스팩션(transfection)한 HCT116p53+/+ 대장암 세포주는 대조군과 비교하여 더 낮은 세포 생존율을 나타내었다.
도 2는 siRNA CNOT2를 트랜스팩션한 대장암세포주 HCT116p53+/+ 및 HCT116p53-/-의 하고 종양억제유전자인 p53 및 p21와 세포사멸 관련인자인 PARP의 발현 수준을 웨스턴 블롯팅을 통해 확인한 도면이다. siRNA CNOT2 처리 후 시간의 경과에 따라 p53 및 p21의 발현 수준이 증가하였으며, p53에 의존적으로 pro-PARP의 발현은 감소하였고, cleaved-PARP은 증가하였다.
도 3은 siRNA CNOT2 혹은 siRNA CNOT2와 siRNA p53을 동시에 트랜스팩션한 대장암세포주 HCT116p53+/+의 종양억제유전자인 p53 및 p21와 세포사멸 관련인자인 PARP의 발현 수준을 웨스턴 블롯팅을 통해 확인한 도면이다. siRNA CNOT2 및 siRNA p53을 동시에 형질전환한 대장암세포주는 siRNA CNOT2를 트랜스팩션한 대장암세포주 보다 cleaved-PARP 발현 수준이 낮았다.
도 4는 siRNA CNOT2를 트랜스팩션한 대장암세포주 HCT116p53+/+에 독소루비신(doxorubicin)을 처리한 세포와 처리하지 않은 세포에서 p53 및 p21의 발현 수준을 웨스턴 블롯팅을 통해 확인한 도면이다. CNOT2의 발현이 저해된 상태에서 독소루비신을 처리한 경우 독소루비신을 단독으로 처리한 경우보다 p53 및 p21의 발현 수준이 높았다.Figure 1A is a diagram showing the expression levels of CNOT2 in colon cancer cells and normal colon cells confirmed through Western blotting, and Figure B is a diagram showing the cell survival rate according to the introduction of siRNA CNOT2. Colon cancer cells have a higher level of CNOT2 expression than normal colon cells, and the HCT116 p53+/+ colon cancer cell line transfected with siRNA CNOT2 showed a lower cell survival rate compared to the control group.
Figure 2 is a diagram showing the expression levels of tumor suppressor genes p53 and p21 and apoptosis-related factor PARP in colon cancer cell lines HCT116 p53+/+ and HCT116 p53-/- transfected with siRNA CNOT2, as confirmed by Western blotting. The expression levels of p53 and p21 increased over time after siRNA CNOT2 treatment, the expression of pro-PARP decreased in a p53-dependent manner, and cleaved-PARP increased.
Figure 3 is a diagram showing the expression levels of tumor suppressor genes p53 and p21 and apoptosis-related factor PARP in HCT116 p53+/+ colon cancer cell line transfected with siRNA CNOT2 or siRNA CNOT2 and siRNA p53 simultaneously, as confirmed by Western blotting. The colon cancer cell line transfected with siRNA CNOT2 and siRNA p53 simultaneously showed a lower level of cleaved-PARP expression than the colon cancer cell line transfected with siRNA CNOT2 alone.
Figure 4 is a drawing showing the expression levels of p53 and p21 in cells treated with and without doxorubicin in the colon cancer cell line HCT116 p53+/+ transfected with siRNA CNOT2, as determined by Western blotting. When doxorubicin was treated while CNOT2 expression was inhibited, the expression levels of p53 and p21 were higher than when doxorubicin was treated alone.
본 발명자들은 대장암의 유전자 치료 방법을 위한 신규 타겟을 개발하기 위하여연구한 결과, 대장암세포에서 CNOT2의 과발현을 확인하고, CNOT2 siRNA를 처리한 대장암세포에서 세포사멸이 증가함을 확인하고 본 발명을 완성하였다. The present inventors conducted research to develop a novel target for a gene therapy method for colon cancer, and as a result, confirmed overexpression of CNOT2 in colon cancer cells and increased apoptosis in colon cancer cells treated with CNOT2 siRNA, thereby completing the present invention.
본 발명의 "CNOT2(CCR4-NOT transcription complex subunit 2)"는 CCR4-NOT transcription complex를 구성하는 subunit중 하나로써, CCR4-NOT transcription complex는 CNOT1, CNOT2, CNOT3, CNOT6 등의 subunit을 포함하며, 각각의 유전자마다 가지고 있는 역할과 기능이 각기 다르다. CNOT2 유전자는 유방암세포에서 전이를 조절하는 역할, 세포내에서 지방 분화의 조절자로서의 역할, 자가포식현상을 조절하는 역할 등을 하는 것으로 알려져 있으나, 대장암세포에서 대장암의 생장을 조절하는 것과 관련된 어떠한 용도도 알려진 바 없다.The "CNOT2 (CCR4-NOT transcription complex subunit 2)" of the present invention is one of the subunits constituting the CCR4-NOT transcription complex, and the CCR4-NOT transcription complex includes subunits such as CNOT1, CNOT2, CNOT3, and CNOT6, and each gene has a different role and function. The CNOT2 gene is known to play a role in regulating metastasis in breast cancer cells, a role as a regulator of fat differentiation in cells, and a role in regulating autophagy, but no use related to regulating the growth of colon cancer in colon cancer cells has been known.
본 발명은 대장암 진단을 위한 바이오마커로서 CNOT2 유전자를 제공한다. The present invention provides the CNOT2 gene as a biomarker for diagnosing colon cancer.
본 발명에서 "바이오마커"는 질병의 진행 또는 치료의 효과를 측정하기 위해 이용될 수 있는 특이적 생물 특성, 생화학적 특징 또는 양상의 분자 지표이다. 단백질 및 핵산은 예시적인 바이오마커이다.In the present invention, a "biomarker" is a molecular indicator of a specific biological characteristic, biochemical feature or pattern that can be used to measure the progression of a disease or the effectiveness of a treatment. Proteins and nucleic acids are exemplary biomarkers.
본 발명에서 "대장암의 진단용 바이오마커"는 대장암 세포를 정상 세포와 구분하여 진단할 수 있는 물질로, 정상 세포에 비해 대장암을 가진 세포에서 증가양상을 보이는 폴리펩타이드 또는 핵산(예-mRNA), 단백질을 포함하는 CNOT2 유전자의 발현산물을 의미한다.In the present invention, the “diagnostic biomarker for colon cancer” refers to an expression product of the CNOT2 gene, which includes a polypeptide or nucleic acid (e.g., mRNA) or protein that shows an increased level in cells with colon cancer compared to normal cells, and is a substance that can diagnose colon cancer cells by distinguishing them from normal cells.
본 발명은 상기 바이오마커의 발현 수준정도를 측정하여 대장암 세포와 정상 대장 세포를 구별할 수 있으며, 개체의 대장 조직 샘플에서 CNOT2의 발현 수준을 유전자 수준 또는 단백질 수준에서 측정하여 대장암 진단을 위한 정보를 제공할 수 있다. The present invention can distinguish between colon cancer cells and normal colon cells by measuring the expression level of the biomarker, and can provide information for diagnosing colon cancer by measuring the expression level of CNOT2 in a colon tissue sample of an individual at the gene level or protein level.
유전자의 발현량 변화의 측정은 당업계에 공지된 다양한 방법을 통해 실시될 수 있다. 예를 들어, RT-PCR, 노던 블롯팅, cDNA 마이크로어레이를 이용한 혼성화 반응 또는 인시투(in situ) 혼성화 반응을 이용하여 실시할 수 있다.Measurement of changes in gene expression levels can be performed using various methods known in the art. For example, it can be performed using RT-PCR, Northern blotting, hybridization using cDNA microarrays, or in situ hybridization.
RT-PCR 프로토콜에 따라 실시하는 경우에는 우선, 시료를 처리한 세포에서 총 RNA를 분리한 다음, 올리고 dT 프라이머 및 역전사효소를 이용하여 단일가닥 cDNA를 제조한다. 이어, 단일가닥 cDNA를 주형으로 이용하고, 유전자-특이적 프라이머 세트를 이용하여 PCR 반응을 실시한다. 그런 다음, PCR 증폭 산물을 전기영동하고, 형성된 밴드를 분석하여 유전자의 발현량 변화를 측정한다.When performing according to the RT-PCR protocol, first, total RNA is isolated from cells that have treated the sample, and then single-stranded cDNA is prepared using an oligo dT primer and reverse transcriptase. Next, a PCR reaction is performed using the single-stranded cDNA as a template and a gene-specific primer set. Then, the PCR amplification product is electrophoresed, and the formed band is analyzed to measure the change in gene expression level.
단백질 양의 변화는 당업계에 공지된 다양한 면역분석 방법을 통해 실시될 수 있다. 예를 들어, 방사능면역분석, 방사능면역침전, 면역침전, ELISA(enzymelinked immunosorbentassay), 캡처-ELISA, 억제 또는 경재 분석, 그리고 샌드위치 분석을 포함하지만, 이에 한정되는 것은 아니다.Changes in protein quantity can be performed by a variety of immunoassay methods known in the art, including but not limited to radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, enzymelinked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assays, and sandwich assays.
본 발명에서 “진단”이란 특정 질병 또는 질환에 대한 한 개체의 감수성(susceptibility)을 판정하는 것, 한 개체가 특정 질병 또는 질환을 현재 가지고 있는지 여부를 판정하는 것, 특정 질병 또는 질환에 걸린 한 개체의 예후(prognosis)를 판정하는 것, 또는 테라메트릭스(therametrics)(예컨대, 치료 효능에 대한 정보를 제공하기 위하여 개체의 상태를 모니터링하는 것)을 포함한다.In the present invention, “diagnosis” includes determining the susceptibility of an individual to a specific disease or condition, determining whether an individual currently has a specific disease or condition, determining the prognosis of an individual with a specific disease or condition, or therametrics (e.g., monitoring the condition of an individual to provide information about the efficacy of a treatment).
본 발명에서 예방이란 본 발명에 따른 조성물의 투여에 의해 대장 세포의 비정상적인 세포사멸 억제 또는 그에 따른 이상을 지연시키는 모든 행위를 의미하고, 치료란 본 발명에 따른 약학적 조성물의 투여에 의해 대장암의 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다. 또한, 본 발명의 대장암 치료용 약학적 조성물의 투여 대상 개체는 포유류라면 제한되지 아니하나, 바람직하게는 인간일 수 있고, 보다 구체적으로 대장암 환자일 수 있다. In the present invention, prevention means all acts of inhibiting abnormal apoptosis of colon cells or delaying abnormalities resulting therefrom by administering the composition according to the present invention, and treatment means all acts of improving or beneficially changing the symptoms of colon cancer by administering the pharmaceutical composition according to the present invention. In addition, the subject to be administered the pharmaceutical composition for colon cancer treatment of the present invention is not limited to any mammal, but may preferably be a human, and more specifically, may be a colon cancer patient.
본 발명에서 “프라이머”는 짧은 자유 수산화기를 가지는 핵산서열로서 상보적인 템플레이트와 염기쌍을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능하는 짧은 핵산서열을 말한다. 본 발명의 프라이머는 예를 들면, 포스포르아미다이트 고체 지지체 방법과 같은 당 분야에 공지된 방법을 이용하여 화학적으로 합성할 수 있다.In the present invention, the “primer” refers to a short nucleic acid sequence having a short free hydroxyl group, which can form base pairs with a complementary template and serves as a starting point for copying the template strand. The primer of the present invention can be chemically synthesized using a method known in the art, such as, for example, a phosphoramidite solid support method.
본 발명에서 “프로브”는 CNOT2 mRNA와 특이적으로 결합할 수 있는 수개 내지 수백개의 염기로 이루어진 RNA 또는 DNA 등의 핵산 단편을 의미하며 라벨링되어 있어 CNOT2 mRNA의 존재유무를 확인할 수 있다. 프로브는 올리고뉴클레오타이드 프로브, 단쇄 DNA 프로브, 이중쇄 DNA 프로브, RNA 프로브 등의 형태로 제작될 수 있고 비오틴, FITC, 로다민, DIG 등으로 표지되거나 방사선 동위 원소 등으로 표지될 수 있다.In the present invention, the “probe” refers to a nucleic acid fragment such as RNA or DNA consisting of several to several hundred bases that can specifically bind to CNOT2 mRNA and is labeled so as to be able to confirm the presence or absence of CNOT2 mRNA. The probe can be produced in the form of an oligonucleotide probe, a single-stranded DNA probe, a double-stranded DNA probe, an RNA probe, etc., and can be labeled with biotin, FITC, rhodamine, DIG, etc., or labeled with a radioisotope, etc.
본 발명에서 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트이거나, CNOT2 특이적 항체를 포함하는 LFA 키트일 수 있으나, 이에 제한되지 않는다. 일 예로서 RT-PCR 키트의 경우, 마커 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액, 데옥시뉴클레오티드(dNTPs), Taq-중합효소 및 역전사효소, DNase, RNase 억제제, DEPC-물(DEPC-water), 멸균수 등을 포함할 수 있다.In the present invention, the kit may be a kit including essential elements required for performing RT-PCR, or an LFA kit including a CNOT2 specific antibody, but is not limited thereto. As an example, in the case of an RT-PCR kit, in addition to each primer pair specific for a marker gene, the kit may include a test tube or other appropriate container, a reaction buffer, deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC-water, sterile water, and the like.
한편, 본 발명자들은 구체적인 실험을 통해 CNOT2 siRNA를 처리한 대장암세포에서 p53의 활성화와 세포사멸율 증가함을 확인하였는바, CNOT2의 억제를 통해 대장암의 예방 및 치료가 가능함을 알 수 있다. Meanwhile, the inventors of the present invention confirmed through specific experiments that p53 activation and apoptosis rate increased in colon cancer cells treated with CNOT2 siRNA, indicating that colon cancer can be prevented and treated by inhibiting CNOT2.
이에, 본 발명은 CNOT2 억제제를 유효성분으로 포함하는 대장암의 예방 또는 치료용 약학적 조성물을 제공한다.Accordingly, the present invention provides a pharmaceutical composition for preventing or treating colon cancer containing a CNOT2 inhibitor as an active ingredient.
본 발명에서 “CNOT2 억제제”란 CNOT2 유전자의 발현을 억제하거나 CNOT2 단백질의 활성을 억제하는 제제일 수 있다. CNOT2 유전자의 발현을 억제하는 제제는 CNOT2 mRNA의 생성을 억제하거나 상기 mRNA와 결합하여 그 분해를 빠르게 하거나 CNOT2 단백질로의 번역을 방해하는 물질이라면 제한되지 아니하나, CNOT2 mRNA의 일부와 상보적인 올리고뉴클레오티드, siRNA(small interfering RNA) 및 shRNA(small hairpin RNA) 등이 될 수 있다. In the present invention, the “CNOT2 inhibitor” may be an agent that inhibits the expression of the CNOT2 gene or the activity of the CNOT2 protein. The agent that inhibits the expression of the CNOT2 gene is not limited to a substance that inhibits the production of CNOT2 mRNA, binds to the mRNA to accelerate its degradation, or interferes with its translation into CNOT2 protein, but may be an oligonucleotide complementary to a portion of CNOT2 mRNA, siRNA (small interfering RNA), shRNA (small hairpin RNA), etc.
본 발명에서 "siRNA"는 이중가닥의 RNA가 다이서에 의해 절단되어 생성되는 21-25 뉴클레오티드 크기의 작은 RNA 조각으로 상보적인 서열을 갖는 CNOT2 mRNA에 특이적으로 결합하여 발현을 억제하는 것을 말한다. 본 발명의 목적상 CNOT2 mRNA에 특이적으로 결합하여 CNOT2 단백질의 발현을 억제하는 것을 의미한다. In the present invention, "siRNA" refers to a small RNA fragment of 21-25 nucleotides in size generated by cleavage of double-stranded RNA by Dicer, which specifically binds to CNOT2 mRNA having a complementary sequence and suppresses expression. For the purpose of the present invention, it means specifically binding to CNOT2 mRNA and suppressing expression of CNOT2 protein.
본 발명의 CNOT2 siRNA는 화학적으로 또는 효소학적으로 합성될 수 있다. siRNA의 제조방법으로는 특별히 한정되지 않으며, 당업계에 공지된 방법을 사용할 수 있다. 예를 들면, siRNA를 직접 화학적으로 합성하는 방법, 시험관 내(in vitro) 전사를 이용한 siRNA의 합성법, 시험관 내(in vitro) 전사에 의해 합성된 긴 이중-가닥 RNA를 효소를 이용하여 절단하는 방법, shRNA 발현 플라스미드나 바이러스성 벡터의 세포내 전달을 통한 발현법 및 PCR(polymerase chain reaction) 유도 siRNA 발현 카세트(cassette)의 세포 내 전달을 통한 발현법 등이 있으나 이에 한정되는 것은 아니다.The CNOT2 siRNA of the present invention can be synthesized chemically or enzymatically. The method for producing siRNA is not particularly limited, and methods known in the art can be used. For example, a method of directly chemically synthesizing siRNA, a method of synthesizing siRNA using in vitro transcription, a method of cleaving long double-stranded RNA synthesized by in vitro transcription using an enzyme, an expression method through intracellular delivery of an shRNA expression plasmid or viral vector, and an expression method through intracellular delivery of a PCR (polymerase chain reaction)-induced siRNA expression cassette, but are not limited thereto.
또한, 본 발명에 있어서 상기 CNOT2 억제제의 세포 내로의 도입은 미세 주입법, 칼슘 포스페이트 공동-침전법, 전기천공법 및 리포좀 이용법 등을 이용하여 실시할 수 있으나, 이에 제한되는 것은 아니다.In addition, in the present invention, introduction of the CNOT2 inhibitor into cells can be performed using, but is not limited to, a microinjection method, a calcium phosphate co-precipitation method, an electroporation method, and a liposome method.
한편, 본 발명자들은 구체적인 실험을 통해 CNOT2의 발현 억제된 대장암 세포에 항암제를 처리하는 경우 암세포의 세포사멸이 활발해 짐을 확인하였는바, 본 발명의 약학적 조성물은 공지의 항암제와 병용투여 되어 그 효능을 증가시시키 위한 용도로 제공될 수 있다.Meanwhile, the inventors of the present invention have confirmed through specific experiments that when anticancer drugs are treated on colon cancer cells in which the expression of CNOT2 is suppressed, apoptosis of cancer cells is activated. Therefore, the pharmaceutical composition of the present invention can be provided for the purpose of increasing the efficacy of a known anticancer drug by co-administration with the drug.
본 발명에서 “약학적 조성물(pharmaceutical composition)”은 약학적 조성물의 제조에 통상적으로 사용되는 적절한 담체, 부형제, 및 희석제를 더 포함할 수 있다. The “pharmaceutical composition” in the present invention may further include suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions.
본 발명에서 "담체(carrier)"란 비이클(vehicle)이라고도 불리며, 세포 또는 조직 내로의 단백질 또는 펩타이드의 부가를 용이하게 하는 화합물을 의미하는 것으로서, 예를 들어 디메틸술폭사이드(DMSO)는 생물체의 세포 또는 조직 내로의 많은 유기물의 투입을 용이하게 하는 통상 사용되는 담체이다.In the present invention, the term "carrier", also called a vehicle, means a compound that facilitates the addition of a protein or peptide into a cell or tissue. For example, dimethyl sulfoxide (DMSO) is a commonly used carrier that facilitates the introduction of many organic substances into the cells or tissues of a living organism.
본 발명에서 "희석제(diluent)"란 대상 단백질 또는 펩타이드의 생물학적 활성 형태를 안정화시킬 뿐만 아니라, 단백질 또는 펩타이드를 용해시키게 되는 물에서 희석되는 화합물로 정의된다. 버퍼 용액에 용해되어 있는 염은 당해 분야에서 희석제로 사용된다. 통상 사용되는 버퍼 용액은 포스페이트 버퍼 식염수이며, 이는 인간 용액의 염 상태를 모방하고 있기 때문이다. 버퍼 염은 낮은 농도에서 용액의 pH를 제어할 수 있기 때문에, 버퍼 희석제가 화합물의 생물학적 활성을 변형하는 일은 드물다. 여기에 사용된 아젤라산을 함유하는 화합물들은 인간 환자에게 그 자체로서, 또는 결합 요법에서와 같이 다른 성분들과 함께 또는 적당한 담체나 부형제와 함께 혼합된 약학적 조성물로서, 투여될 수 있다.In the present invention, a "diluent" is defined as a compound that is diluted in water, which not only stabilizes the biologically active form of the target protein or peptide, but also dissolves the protein or peptide. A salt dissolved in a buffer solution is used as a diluent in the art. A commonly used buffer solution is phosphate buffered saline, because it mimics the salt state of human solutions. Since the buffer salt can control the pH of the solution at low concentrations, it is rare for a buffer diluent to modify the biological activity of the compound. The compounds containing azelaic acid used herein can be administered to a human patient as such, or as a pharmaceutical composition mixed with other ingredients or with suitable carriers or excipients, as in combination therapy.
또한, 본 발명에 따른 비알코올 지방간염 예방 또는 치료용 약학적 조성물은 각각 통상의 방법에 따라 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 시럽, 에어로졸 등의 외용제 및 멸균 주사용액의 형태로 제형화하여 사용될 수 있으며, 상기 조성물에 포함될 수 있는 담체, 부형제 및 희석제로는 락토즈, 덱스트로즈, 수크로스, 올리고당, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유를 들 수 있다. 제제화할 경우에는 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 분해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 조제된다. 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제된다. 또한 단순한 부형제 이외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다. 비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁제, 유제, 동결건조제제, 좌제가 포함된다. 비수성용제, 현탁제로는 프로필렌글리콜 (propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈 (tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.In addition, the pharmaceutical composition for preventing or treating non-alcoholic steatohepatitis according to the present invention can be formulated and used in the form of external preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc. and sterile injection solutions according to conventional methods, and carriers, excipients and diluents that can be included in the composition include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.
본 발명의 약학적 조성물은 경구 또는 비경구, 바람직하게는 비경구로 투여할 수 있고, 비경구 투여인 경우에는 근육 주입, 정맥내 주입, 피하 주입, 복강 주입, 국소 투여, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, can be administered by intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, topical administration, transdermal administration, etc.
본 발명의 약학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. The appropriate dosage of the pharmaceutical composition of the present invention can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 대용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention can be manufactured in the form of a unit dose or can be manufactured by placing it in a large-capacity container by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and by being formulated. At this time, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granules, tablets or capsules, and may additionally contain a dispersant or stabilizer.
본 발명은 다양한 변환을 가할 수 있고 여러 가지 실시예를 가질 수 있는 바, 이하 특정 실시예들을 도면에 예시하고 상세한 설명에 상세하게 설명하고자 한다. 그러나, 이는 본 발명을 특정한 실시 형태에 대해 한정하려는 것이 아니며, 본 발명의 사상 및 기술 범위에 포함되는 모든 변환, 균등물 내지 대체물을 포함하는 것으로 이해되어야 한다. 본 발명을 설명함에 있어서 관련된 공지 기술에 대한 구체적인 설명이 본 발명의 요지를 흐릴 수 있다고 판단되는 경우 그 상세한 설명을 생략한다.The present invention can be modified in various ways and has various embodiments, and thus specific embodiments are illustrated in the drawings and described in detail in the detailed description below. However, this is not intended to limit the present invention to specific embodiments, and it should be understood that all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention are included. In describing the present invention, if it is determined that a specific description of a related known technology may obscure the gist of the present invention, the detailed description thereof will be omitted.
[실시예][Example]
실시예 1. 대장암세포주와 대장세포에서의 CNOT2 발현양 비교Example 1. Comparison of CNOT2 expression levels in colon cancer cell lines and colon cells
본 발명에서 쓰인 CNOT2 유전자의 기본 발현을 대장암세포와 정상 대장세포에서 비교하였다. 각 세포주를 수거하여 NP-40 lysis buffer를 이용 단백질을 추출해 Western blotting을 통하여 CNOT2, β-actin 발현을 확인하였다. 도 1의 A에서 확인할 수 있듯이, 정상 대장세포와 비교시 대장암세포에서 CNOT2의 발현이 높은 것을 알 수 있었다.The basic expression of the CNOT2 gene used in the present invention was compared between colon cancer cells and normal colon cells. Each cell line was collected, proteins were extracted using NP-40 lysis buffer, and CNOT2 and β-actin expression was confirmed through Western blotting. As can be confirmed in Fig. 1A, the expression of CNOT2 was higher in colon cancer cells compared to normal colon cells.
실시예 2. 대장암 세포주에 siRNA CNOT2의 도입에 따른 대장암세포의 생장 억제 효과 확인Example 2. Confirmation of the growth inhibition effect of colon cancer cells by introduction of siRNA CNOT2 into colon cancer cell lines
본 발명의 CNOT2 유전자 억제제는 CNOT2 단백질을 코딩하는 유전자의 안티센스 올리고뉴클레오타이드, siRNA (small interfering RNA)를 이용, CNOT2 유전자의 발현을 조절할 수 있는 물질을 사용하였다. 본 발명에서 사용한 안티센스 올리고뉴클레오타이드는 특정 mRNA의 서열에 상보적인 핵산 서열을 함유하고 있는 DNA 또는 RNA 이들의 유도체를 의미하고, mRNA내의 상보적인 셔열 부분에 결합, mRNA에서 단백질로의 번역을 저해하는 작용을 한다. 본 발명에서 사용된 siRNA는 바이오니아 ((아이디번호: 4848-1), 대전, 한국)에서 주문 및 구입해서 사용했다. 해당 CNOT2 siRNA와 대조군으로 사용하는 control siRNA (바이오니아 (아이디번호: SN-1002), 대전, 한국)를 대장암 세포주 HCT116p53+/+과 HCT116p53-/-에 도입한 후 해당 유전자의 발현 저하에 따른 암세포주의 성장 속도 및 세포사멸 (apoptosis)를 관찰하였다.The CNOT2 gene inhibitor of the present invention uses a substance capable of controlling the expression of the CNOT2 gene using an antisense oligonucleotide, siRNA (small interfering RNA) of a gene encoding CNOT2 protein. The antisense oligonucleotide used in the present invention refers to a derivative of DNA or RNA containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and binds to a complementary sequence portion in the mRNA, thereby inhibiting the translation of mRNA into protein. The siRNA used in the present invention was ordered and purchased from Bioneer ((ID number: 4848-1), Daejeon, Korea). After introducing the CNOT2 siRNA and control siRNA (Bioneer (ID number: SN-1002), Daejeon, Korea) into the colon cancer cell lines HCT116 p53+/+ and HCT116 p53-/-, the growth rate and apoptosis of the cancer cell lines according to the decrease in the expression of the gene were observed.
[siRNA CNOT2 올리고뉴클레오타이드][siRNA CNOT2 oligonucleotide]
sense(서열번호 1): 5'-CAGCUAUAAAGAUCCAACA-3'sense(SEQ ID NO: 1): 5'-CAGCUAUAAAGAUCCAACA-3'
antisense(서열번호 2): 5'-UGUUGGAUCUUUAUAGCUG-3'antisense (SEQ ID NO: 2): 5'-UGUUGGAUCUUUAUAGCUG-3'
실시예 3. siRNA CNOT2가 도입된 HCT116Example 3. HCT116 with siRNA CNOT2 introduced p53+/+p53+/+ 과 HCT116and HCT116 p53-/-p53-/- 대장암세포주에서의 세포증식 억제효과 확인Confirmation of cell proliferation inhibition effect in colon cancer cell lines
각 세포주를 96 well plate에 1X104/ml로 세포로 배양 후, siRNA는 80 nM 농도로 TurboFect trasfection reagent (Thermo Fisher Scientific, Waltham, MA, USA)를 이용하여 도입하였다. 도입 후 0시간, 48시간 후에 CCK8 assay를 사용하여 흡광도에서 450 nM에서 측정하였다. 도 1에 B에서 보는 것과 같이 control siRNA를 처리한 군과 비교했을 때, CNOT2의 발현을 저해한 군에서 대장암 세포의 생장을 억제하는 것으로 확인되었다. After culturing each cell line in a 96-well plate at 1X104 /ml, siRNA was introduced at a concentration of 80 nM using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). After introduction, the absorbance was measured at 450 nM using a CCK8 assay 0 and 48 hours later. As shown in Fig. 1B, compared to the group treated with control siRNA, the group in which the expression of CNOT2 was inhibited inhibited the growth of colon cancer cells.
실시예 4. siRNA CNOT2가 도입된 HCT116Example 4. HCT116 with siRNA CNOT2 introduced p53+/+p53+/+ 과 HCT116and HCT116 p53-/-p53-/- 대장암세포주에서의 p53, p53의 타겟유전자 p21, 그리고 세포사멸 관련 단백질 PARP의 활성변화 확인 Confirmation of changes in the activity of p53, p53 target gene p21, and apoptosis-related protein PARP in colon cancer cell lines
각 세포주를 6 well plate에 7X104/ml로 세포를 배양 후, siRNA는 80 nM 농도로 TurboFect trasfection reagent (Thermo Fisher Scientific, Waltham, MA, USA) 를 이용 도입하였다. 도입 후 72시간 후에 수거하여 단백질을 추출하여 Western blotting을 사용하여 p53, p21, pro-PARP, cleaved-PARP의 단백질 발현을 확인하였다. 그 결과를 도 2에 나타내었다. Each cell line was cultured in a 6-well plate at 7X104 /ml, and siRNA was introduced at a concentration of 80 nM using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). After 72 hours of introduction, the cells were harvested, proteins were extracted, and protein expression of p53, p21, pro-PARP, and cleaved-PARP was confirmed using Western blotting. The results are shown in Fig. 2.
도 2에서 확인되는 것과 같이, CNOT2의 유전자를 저해시키고 시간 의존적으로 관찰했을 때, 종양억제유전자인 p53의 활성이 시간 의존적으로 증가하는 경향을 보였다. p53의 타겟 유전자인 p21 또한 시간 의존적으로 증가하는 경향을 보였다. 흥미로운 사실은, 세포사멸의 대표적인 인자인 PARP의 발현 변화인데, HCT116p53+/+세포에서는 cleaved-PARP가 증가하고 pro-PARP가 감소하는 경향으로 나타나는 반면 HCT116p53-/-세포에서는 상대적으로 발현이 약한 것으로 관찰되어 p53 의존적인 세포세포사멸이 일어나는 것을 알 수 있다..As confirmed in Fig. 2, when the gene of CNOT2 was inhibited and observed in a time-dependent manner, the activity of the tumor suppressor gene p53 tended to increase in a time-dependent manner. The target gene of p53, p21, also tended to increase in a time-dependent manner. An interesting fact is that the expression of PARP, a representative factor of apoptosis, changes in which cleaved-PARP increases and pro-PARP decreases in HCT116 p53+/+ cells, whereas relatively weak expression was observed in HCT116 p53-/- cells, indicating that p53-dependent apoptosis occurs.
실시예 5. siRNA CNOT2와 siRNA p53이 도입된 HCT116Example 5. HCT116 cells introduced with siRNA CNOT2 and siRNA p53 p53+/+p53+/+ 대장암세포주에서의 세포사멸 비교Comparison of apoptosis in colon cancer cell lines
세포주를 6 well plate에 7X104/ml로 세포로 배양 후, siRNA는 80 nM 농도로 TurboFect trasfection reagent (Thermo Fisher Scientific, Waltham, MA, USA)를 이용하여 도입하였다. 도입 후 72시간 후에 수거하여 단백질을 추출하여 Western blotting을 사용하여 p53, p21, Pro-PARP, cleaved-PARP, CNOT2의 단백질 발현을 확인하였다. 도 3의 결과에서 확인할 수 있듯이, siRNA를 이용해 CNOT2의 발현을 저해한 군과 siRNA를 이용해 p53과 CNOT2의 발현을 동시에 저해한 군을 비교했을 때, 동시에 처리한 군이 세포사멸로 가는 것이 줄어드는 것을 확인해 볼 수 있다. 즉, CNOT2의 발현 저해를 통한 세포사멸은 종양억제유전자인 p53의 활성화를 통해 유도됨을 알 수 있다.After culturing the cell line in a 6-well plate at 7X104 /ml, siRNA was introduced at a concentration of 80 nM using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cells were harvested 72 hours after introduction, and proteins were extracted and analyzed for the protein expression of p53, p21, Pro-PARP, cleaved-PARP, and CNOT2 using Western blotting. As can be seen in the results in Fig. 3, when comparing the group in which CNOT2 expression was inhibited using siRNA and the group in which the expression of p53 and CNOT2 was simultaneously inhibited using siRNA, it can be confirmed that the group treated simultaneously was less likely to proceed to apoptosis. That is, it can be seen that apoptosis through inhibition of CNOT2 expression is induced through the activation of p53, a tumor suppressor gene.
실시예 6. siRNA CNOT2를 HCT116Example 6. siRNA CNOT2 into HCT116 p53+/+p53+/+ 대장암세포주에 도입하고 doxorubicin 처리후 p53 및 p21의 발현 수준 비교Comparison of expression levels of p53 and p21 after introduction into colon cancer cell lines and doxorubicin treatment
세포주를 6 well plate에 7X104/ml로 세포로 배양 후, siRNA는 80 nM 농도로 TurboFect trasfection reagent (Thermo Fisher Scientific, Waltham, MA, USA)를 이용하여 도입하였다. 도입 후 48시간 후에 새로운 media에 doxorubicin 0.1 μM을 24시간 처리 후 세포를 수거, 단백질을 추출하여 Western blotting을 사용하여 p53, p21, CNOT2의 단백질 발현을 확인하였다. After culturing the cell line in a 6-well plate at 7X104 /ml, siRNA was introduced at a concentration of 80 nM using TurboFect transfection reagent (Thermo Fisher Scientific, Waltham, MA, USA). 48 hours after introduction, the cells were treated with 0.1 μM doxorubicin in fresh media for 24 hours, harvested, and proteins were extracted to confirm the protein expression of p53, p21, and CNOT2 using Western blotting.
그 결과, 도 4의 결과에서 볼 수 있듯이 siRNA를 처리해 CNOT2의 발현을 낮추고 대장암 치료제로도 사용되고 있는 doxorubicin을 처리했을 때, CNOT2가 있는 상태로 doxorubicin을 처리한 군에 비해 p53과 그 타겟 유전자인 p21의 발현이 더 높아지는 것을 관찰할 수 있었다. 다시 말해, 상승효과를 볼 수 있었다.As a result, as can be seen in the results in Fig. 4, when siRNA was treated to lower the expression of CNOT2 and doxorubicin, which is also used as a treatment for colon cancer, was treated, it was observed that the expression of p53 and its target gene p21 was higher than in the group treated with doxorubicin in the presence of CNOT2. In other words, a synergistic effect was observed.
상기 결과들을 종합해 보면, 대장암세포에서 CNOT2의 발현 저해는 대장암세포의 생장을 억제하고 세포사멸을 유도하는데, 바로 종양 억제 유전자인 p53의 활성을 통해 이루어진다는 사실과 CNOT2의 발현 저해가 종래의 대장암 치료제와 같이 쓰일 때 상승 효과작용을 이뤄낸다는 사실을 알 수 있었다.In summary, the above results show that inhibition of CNOT2 expression in colon cancer cells inhibits the growth of colon cancer cells and induces apoptosis, which is achieved through the activation of the tumor suppressor gene p53, and that inhibition of CNOT2 expression has a synergistic effect when used together with conventional colon cancer treatments.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시 양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
<110> University-Industry Cooperation Group of Kyung Hee University <120> Biomarker for colon cancer diagnosis and pharmaceutical composition for preventing or treating colon cancer <130> DHP21-K179 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CNOT2 siRNA_sense <400> 1 cagcuauaaa gauccaaca 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CNOT2 siRNA_antisense <400> 2 uguuggaucu uuauagcug 19 <110> University-Industry Cooperation Group of Kyung Hee University <120> Biomarker for colon cancer diagnosis and pharmaceutical composition for preventing or treating colon cancer <130> DHP21-K179 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CNOT2 siRNA_sense <400> 1 cagcuauaaaa gauccaaca 19 <210> 2 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> CNOT2 siRNA_antisense <400> 2 uguuggaucu uuauagcug 19
Claims (15)
상기 CNOT2 억제제는 CNOT2 mRNA의 일부와 상보적인 올리고뉴클레오티드, siRNA(small interfering RNA) 및 shRNA(small hairpin RNA)로 이루어진 군으로부터 선택된 1종 이상이며,
상기 CNOT2 억제제는 서열번호 1 및/또는 서열번호 2의 올리고뉴클레오티드인 것을 특징으로 하는, 약학적 조성물. A pharmaceutical composition for preventing or treating colon cancer containing a CNOT2 inhibitor as an active ingredient,
The above CNOT2 inhibitor is at least one selected from the group consisting of oligonucleotides, siRNA (small interfering RNA), and shRNA (small hairpin RNA) complementary to a portion of CNOT2 mRNA.
A pharmaceutical composition, characterized in that the CNOT2 inhibitor is an oligonucleotide having sequence number 1 and/or sequence number 2.
상기 조성물은 대장암 세포에서 p53를 활성화하여 세포사멸을 유도하는 것을 특징으로 하는, 약학적 조성물. In Article 6,
A pharmaceutical composition characterized in that the composition induces apoptosis by activating p53 in colon cancer cells.
상기 조성물은 항암제와 병용투여용인 것을 특징으로 하는, 약학적 조성물. In Article 6,
A pharmaceutical composition characterized in that the composition is for combined administration with an anticancer agent.
상기 조성물은 항암제와 병용투여되어 대장암 세포의 세포사멸을 촉진하는 것을 특징으로 하는, 약학적 조성물. In Article 11,
A pharmaceutical composition characterized in that the composition promotes apoptosis of colon cancer cells when administered in combination with an anticancer agent.
상기 항암제는 독소루비신, 다우노루비신, 닥티노마이신, 블레오마이신, 플리카마이신(미스라마이신), 마이토마이신 C, 스트렙토조신, 및 미톡산트론으로 이루어진 군으로부터 선택되는 1종 이상의 항암제인 것을 특징으로 하는, 약학적 조성물. In Article 11,
A pharmaceutical composition, characterized in that the anticancer agent is at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, dactinomycin, bleomycin, plicamycin (misramycin), mitomycin C, streptozocin, and mitoxantrone.
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