KR20160021058A - Use of LYPD1 for cancer diagnostic or therapeutic marker - Google Patents
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- KR20160021058A KR20160021058A KR1020150114771A KR20150114771A KR20160021058A KR 20160021058 A KR20160021058 A KR 20160021058A KR 1020150114771 A KR1020150114771 A KR 1020150114771A KR 20150114771 A KR20150114771 A KR 20150114771A KR 20160021058 A KR20160021058 A KR 20160021058A
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Abstract
Description
본 발명은 LYPD1(LY6/PLAUR domain containing 1) 유전자의 발현 억제제, LYPD1 단백질의 활성 억제제 또는 이의 혼합물을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물, LYPD1 유전자 mRNA 또는 이의 단백질의 수준을 측정하는 제제를 포함하는, 암 진단용 조성물, 상기 진단용 조성물을 포함하는 암 진단용 키트, 및 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 측정하여 암 진단을 위한 정보를 제공하는 방법 또는 암 치료제의 스크리닝 방법에 관한 것이다.
The present invention relates to a pharmaceutical composition for preventing or treating cancer, comprising an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of LYPD1 protein activity or a mixture thereof as an active ingredient, a level of LYPD1 gene mRNA or its protein A cancer diagnosis kit comprising the diagnostic composition, and a method of providing information for cancer diagnosis by measuring the level of mRNA or a protein of the LYPD1 gene or a method of screening a cancer therapeutic agent .
암은, 세포주기가 조절되지 않아 세포분열을 계속하는 질병으로, 폐암, 식도암, 자궁암, 유방암, 대장암, 전립선암 등이 있다. 그 중 대장암은, 병리학적으로는 대부분이 선암(adenocarcinoma)이며, 부위별로는 크게 결장암과 직장암으로 구분된다. 부위별 발생 빈도는 하부 대장, 즉 직장에서 발생하는 경우가 약 50%로 가장 높다. 대장암은 미국 및 유럽 등지에서 암 관련 사망의 두 번째 요인으로 작용하는 것으로 알려져 있다(American Cancer Society statics for 2009). 또한, 대장암의 치료는 외과적 절제술을 근간으로 항암화학요법 및 방사선요법이 병행되고 있다. 이러한 수술적 요법, 항암화학요법 및 방사선요법 등의 진보에도 불구하고, 특정한 증상이 없이 진행되는 대장암의 특성으로 인하여 다른 장기로 전이된 후 진단되는 경우가 빈번하여, 수술 시점을 놓침으로써 높은 사망율을 나타내고 있다. 따라서 이러한 이유로 대장암의 효과적인 진단 및 치료 표적의 개발이 요구되고 있는 상황이다. Cancer is a disease in which cell cycle is not controlled and cell division continues, such as lung cancer, esophageal cancer, uterine cancer, breast cancer, colon cancer and prostate cancer. Among them, colorectal cancer is most commonly adenocarcinoma, and it is divided into colon cancer and rectal cancer. The incidence of each site is highest in the lower colon, or rectum, about 50%. Colon cancer is the second leading cause of cancer-related deaths in the United States and Europe (American Cancer Society statics for 2009). The treatment of colorectal cancer is based on surgical resection, and chemotherapy and radiotherapy are combined. Despite advances such as surgical treatment, chemotherapy and radiotherapy, it is frequently diagnosed after metastasis to other organs due to the characteristics of colorectal cancer progressing without specific symptoms, . Therefore, effective diagnosis and treatment targets for colorectal cancer are required for this reason.
또한, 전립선암은 전립선에서 발생하는 악성 종양(malignant tumor)으로, 서양의 경우 남성암 중 가장 흔한 암으로서 높은 발생 빈도를 보인다. 그러나 전립선암은 특이한 증상이 없어 조기 발견이 어려우므로, 암 조직이 커지면서 배뇨에 지장을 받거나, 전립선암이 뼈 등의 다른 장기로 전이되어 골 동통 등의 증상이 나타난 후에야 병원을 찾는 경우가 많다. 따라서 이러한 이유로 전립선암의 효과적인 진단 및 치료 표적의 개발이 요구되고 있는 상황이다.
In addition, prostate cancer is a malignant tumor originating from the prostate gland. In the western world, it is the most common cancer among men. However, since prostate cancer is uncommon, it is difficult to detect it early. Therefore, it is often difficult to find a hospital after the cancer tissue is enlarged or the prostate cancer is transferred to other organs such as bone or the like. Therefore, it is required to develop an effective diagnosis and treatment target of prostate cancer for this reason.
한편, LYPD1(LY6/PLAUR domain containing 1) 유전자는 침팬치, 개, 소, 쥐 및 닭 등에서 발견되며, 인간의 LYPD1 유전자는 81 종의 생물체와 오솔로그(orthologs)를 가진다. 현재 LYPD1에 대한 논문은 거의 발표된 것이 없으며, Hela cell에서 종양 억제 역할을 함이 보고된 바 있으나(Exp Cell Res, 2006, 312(6): 865-76), 그 외 암과 관련하여 LYPD1의 기능에 대하여 알려진 바가 없다.
On the other hand, LYPD1 (LY6 / PLAUR domain containing 1) gene is found in chimpanzee, dog, cattle, mouse and chicken, and human LYPD1 gene has 81 organisms and orthologs. Although there is no published report on LYPD1, it has been reported that Hela cells have a tumor suppressor role (Exp Cell Res, 2006, 312 (6): 865-76) There is no known function.
이러한 배경 하에, 본 발명자들은 암의 진단 및 치료를 위한 마커를 개발하기 위해 예의 노력한 결과, 암 진단 마커로서 LYPD1이 효과적으로 사용될 수 있을 뿐만 아니라, LYPD1 유전자의 발현을 억제하면 암세포의 침윤, 세포이동성 및 세포성장이 억제되는 반면, LYPD1 유전자의 발현을 강화하면 암세포의 침윤 및 발암과 관련된 주요 세포신호전달이 증가하므로, 이의 발현 또는 활성을 억제하는 경우, 암의 예방 또는 치료에 효과적임을 확인하고 본 발명을 완성하였다.
Under these circumstances, the present inventors have made intensive efforts to develop a marker for diagnosis and treatment of cancer. As a result, LYPD1 can be effectively used as a cancer diagnostic marker. In addition, suppression of the expression of LYPD1 gene leads to invasion of cancer cells, Cell growth is inhibited, while the expression of LYPD1 gene is enhanced, leading to an increase in major cell signal transduction associated with cancer cell infiltration and carcinogenesis. Therefore, when the expression or activity of the LYPD1 gene is inhibited, it is effective in prevention or treatment of cancer, .
본 발명의 하나의 목적은 LYPD1 유전자의 발현 억제제, LYPD1 단백질의 활성 억제제 또는 이의 혼합물을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.It is an object of the present invention to provide a pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.
본 발명의 다른 목적은 LYPD1 유전자의 발현 억제제, LYPD1 단백질의 활성 억제제 또는 이의 혼합물을 유효성분으로 포함하는, 암의 예방 또는 개선용 건강기능식품을 제공하는 것이다. Another object of the present invention is to provide a health functional food for preventing or ameliorating cancer comprising an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.
본 발명의 또 다른 목적은 LYPD1 유전자 mRNA 또는 이의 단백질의 수준을 측정하는 제제를 포함하는, 암 진단용 조성물을 제공하는 것이다.It is still another object of the present invention to provide a cancer diagnostic composition comprising an agent for measuring the level of LYPD1 gene mRNA or a protein thereof.
본 발명의 또 다른 목적은 상기 조성물을 포함하는 암 진단용 키트를 제공하는 것이다.Yet another object of the present invention is to provide a cancer diagnostic kit comprising the composition.
본 발명의 또 다른 목적은 (a) 암이 의심되는 개체의 분리된 시료로부터 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 정상 대조군 시료의 유전자의 mRNA 또는 이의 단백질의 수준보다 높은지 여부를 비교하는 단계를 포함하는, 암 진단을 위한 정보를 제공하는 방법 또는 암 진단 방법을 제공하는 것이다.Yet another object of the present invention is to provide a method for detecting cancer, comprising: (a) measuring the level of the mRNA of LYPD1 gene or a protein thereof from a separate sample of a subject suspected of having cancer; And (b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a level of the mRNA of the gene of the normal control sample, or a method of diagnosing cancer .
본 발명의 또 다른 목적은 (a) 암 치료용 후보물질을 LYPD1 유전자를 발현하는 분리된 시료에 처리한 다음, 상기 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 상기 시료를 처리하지 않은 대조군 시료의 수준보다 낮은 경우 암 치료제로 선택하는 단계를 포함하는, 암 치료제의 스크리닝 방법을 제공하는 것이다.
It is still another object of the present invention to provide a method for treating cancer, which comprises the steps of (a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA of the LYPD1 gene or a protein thereof; And (b) when the level of the mRNA of the gene or the protein thereof is lower than that of the control sample not treated with the sample, a method for screening a cancer therapeutic agent.
상기 과제를 해결하기 위한 본 발명의 하나의 양태는 LYPD1(LY6/PLAUR domain containing 1) 유전자의 발현 억제제, LYPD1 단백질의 활성 억제제 또는 이의 혼합물을 유효성분으로 포함하는, 암의 예방 또는 치료용 약학적 조성물을 제공한다.
One aspect of the present invention for solving the above problems is a pharmaceutical composition for preventing or treating cancer comprising an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of LYPD1 protein activity or a mixture thereof as an active ingredient Lt; / RTI >
본 발명에 따르면, 암세포에서 LYPD1 유전자의 발현 또는 활성이 억제되면 암세포의 침윤, 세포 이동성 및 세포성장이 감소하므로, LYPD1 유전자의 발현 또는 상기 단백질의 활성을 억제하면 암의 예방 또는 치료가 가능하다.
According to the present invention, when the expression or activity of LYPD1 gene is inhibited in cancer cells, the infiltration of cancer cells, cell mobility and cell growth are reduced. Thus, prevention or treatment of cancer can be achieved by inhibiting the expression of LYPD1 gene or the activity of the protein.
본 발명에서 사용되는 용어 "LYPD1"이란 "LY6/PLAUR domain containing 1"의 약어로서, 상기 LYPD1은 Hela cell에서 종양 억제 역할을 함이 보고된 바 있으나(Yu DH, et al., Exp Cell Res, 2006), 그 외 암과 관련하여 LYPD1의 기능에 대하여 알려진 바가 없다. As used herein, the term "LYPD1" is an abbreviation of "LY6 / PLAUR domain containing 1 ", and LYPD1 has been reported to inhibit the tumor in Hela cells (Yu DH, et al., Exp Cell Res, 2006), and the function of LYPD1 in relation to other cancers is unknown.
오히려, 본 발명에서는 LYPD1이 암세포의 침윤, 세포 이동성 및 세포성장을 촉진시키는 활성이 있어, 이의 발현을 억제시키는 경우 암세포의 침윤, 세포 이동성 및 세포성장이 억제되므로, 암 진단 마커 또는 암 치료제로서 사용 가능함을 확인하였다. Rather, in the present invention, LYPD1 has an activity of promoting invasion of cancer cells, cell mobility and cell growth, and inhibiting the expression thereof inhibits invasion of cancer cells, cell mobility and cell growth, so that it is used as a cancer diagnostic marker or cancer therapeutic agent .
상기 LYPD1의 유전자 및 단백질 정보는 NCBI 유전자 은행과 같은 공지의 데이터베이스를 통하여 용이하게 확인할 수 있으며, 그 예로 NCBI 유전자 은행의 Gene ID: 116372인 LYPD1일 수 있으나, 이에 제한되지 않는다. 또한, 상기 LYPD1은 서열번호 7로 표시되는 아미노산 서열을 가질 수 있으며, 또한 특별히 이에 제한되지 않으나, 서열번호 1로 표시되는 염기서열을 가질 수 있다.The gene and protein information of LYPD1 can be easily confirmed through a known database such as NCBI gene bank, for example, but not limited to, LYPD1 which is Gene ID: 116372 of NCBI gene bank. The LYPD1 may have the amino acid sequence shown in SEQ ID NO: 7, and may have the nucleotide sequence shown in SEQ ID NO: 1, though it is not particularly limited thereto.
이러한 LYPD1의 서열을 바탕으로 LYPD1의 발현 억제제 또는 활성 억제제를 설계할 수 있으며, 상기 서열은 이러한 설계에 있어 일정 정도 변형이 가능하다. 본 기술 분야의 당업자라면 이러한 인위적인 변형에 의해 80% 이상, 구체적으로는 90% 이상, 보다 구체적으로는 95% 이상, 보다 더 구체적으로는 98%의 상동성이 유지되는 서열 역시 사용할 수 있음은 자명하다.
Based on the sequence of LYPD1, an inhibitor of LYPD1 expression or an activity inhibitor can be designed, and the sequence can be modified to some extent in this design. Those skilled in the art will appreciate that sequences that retain more than 80%, in particular more than 90%, more specifically more than 95%, and more particularly 98% Do.
구체적으로, 본 발명의 일 실시예에서는 대장암 세포인 SW480sub 세포주에서 siRNA에 의해 LYPD1의 발현이 억제되는 경우 세포의 침윤, 이동성 및 세포 성장이 감소함을 확인하였고(실시예 8), LYPD1이 대장암 세포에서 발현이 강화되는 경우 세포의 침윤 능력이 증가함을 확인하였다(도 5).Specifically, in one embodiment of the present invention, it was confirmed that when the expression of LYPD1 is inhibited by siRNA in SW480sub cell line, which is a colorectal cancer cell, cell infiltration, mobility and cell growth are reduced (Example 8) It was confirmed that when the expression is enhanced in the cancer cells, the infiltration ability of the cells is increased (FIG. 5).
또한, 본 발명의 다른 일 실시예에서는 SW480 및 HEK293E 세포를 형질전환한 후, 웨스턴 블랏을 통해, 공통적으로 JNK의 인산화반응 및 전사인자 AP-1(activator protein 1)의 구성성분인 ATF-2의 인산화반응이 증가됨을 확인하였다(도 6). 그 결과 SW480에서는 간엽세포 마커인 비멘틴(vimentin)의 발현이 증가되었고, 또한 cyclin D1 및 cyclin A의 발현이 증가되었으며, HEK293E에서는 ATF-2 외에 AP-1의 구성성분인 c-Jun의 인산화반응 및 cyclin E, cyclin A의 발현이 증가되었음을 알 수 있었다. In another embodiment of the present invention, SW480 and HEK293E cells were transfected and then Western blotted, commonly used for the phosphorylation of JNK and ATF-2, a component of transcription factor AP-1 (activator protein 1) Phosphorylation was increased (FIG. 6). As a result, in SW480, the expression of vimentin, vimentin, which is a mesenchymal cell marker, and the expression of cyclin D1 and cyclin A were increased. In addition to ATF-2 in HEK293E, phosphorylation of c-Jun, And cyclin A, cyclin A were increased in the control group.
또한, 본 발명의 또 다른 일 실시예에서는 HEK293E 세포주에서 루시퍼라제 분석법을 통해 전사인자 AP-1의 활성 변화를 확인한 결과, LYPD1 발현에 의해 AP-1의 활성이 증가함을 확인하였다(도 7). 이를 통해 AP-1 활성은 암세포 활성(성장 및 침윤)과 밀접한 관련이 있으므로, LYPD1 발현에 의한 AP-1 활성 조절이 침윤/세포 이동성 및 세포성장 조절에 기여함을 나타내는 것이다. In addition, in another embodiment of the present invention, the activity of AP-1 was confirmed by the luciferase assay in the HEK293E cell line, and the activity of AP-1 was increased by the expression of LYPD1 (FIG. 7) . Thus, AP-1 activity is closely related to cancer cell activity (growth and invasion), indicating that AP-1 activity regulation by LYPD1 expression contributes to invasion / cell migration and cell growth regulation.
결론적으로, 대장암세포를 포함한 다양한 암세포에서 본 발명의 LYPD1의 발현이 감소되면, 그에 따라 AP-1 활성도 감소되어 암세포의 침윤/세포 이동성 및 세포성장이 감소됨을 알 수 있었다. 즉, 본 발명의 LYPD1은 다양한 암세포의 침윤/세포 이동성 및 세포성장과 관련된 암 진단 또는 치료 표적으로서, LYPD1의 발현을 측정함으로써 암을 진단할 수 있고, 또한 이의 발현 또는 활성을 억제함으로써 암을 치료할 수 있다.In conclusion, when the expression of LYPD1 of the present invention is decreased in various cancer cells including colon cancer cells, AP-1 activity is decreased, and the infiltration / cell mobility and cell growth of cancer cells are decreased. That is, the LYPD1 of the present invention can diagnose cancer by measuring the expression of LYPD1 as a diagnostic or therapeutic target of cancer associated with invasion / cell mobility and cell growth of various cancer cells, and can also treat cancer by suppressing its expression or activity .
또한, 본 발명의 또 다른 일 실시예에서는 전립선암 세포주인 PC3에서 siRNA에 의해 LYPD1의 발현이 억제되는 경우 세포 성장 및 LYPD1 발현이 감소되고(도 9 및 10), 세포 이동성이 감소함을 확인하였다(도 11). 이는 본 발명의 LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 시사하는 것이다.
In another embodiment of the present invention, cell growth and LYPD1 expression are decreased when the expression of LYPD1 is inhibited by siRNA in the prostate cancer cell line PC3 (FIGS. 9 and 10), and cell mobility is decreased (Fig. 11). This suggests that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on cancer treatment.
본 발명에서 사용되는 용어 "LYPD1 유전자의 발현 억제제"은 LYPD1의 발현 또는 활성을 감소시키는 물질을 통칭하는 의미로 사용되며, 보다 구체적으로는 LYPD1 의 발현을 전사 수준 또는 단백질 수준에서 LYPD1 의 발현을 감소시키는 모든 물질을 포함할 수 있다. 상기 LYPD1 발현을 저해하는 물질은 LYPD1을 표적으로 하여 LYPD1 의 발현 또는 활성을 억제할 수 있는 화합물, 핵산, 펩타이드, 바이러스 또는 상기 핵산을 포함하는 벡터 등 그 형태에 제한없이 사용 가능하다. As used herein, the term "inhibitor of LYPD1 gene expression" is used collectively to refer to a substance that decreases the expression or activity of LYPD1. More specifically, the expression of LYPD1 is decreased at the transcription level or protein level Which may include all materials which make it possible. The substance that inhibits the expression of LYPD1 may be used in any form such as a compound, a nucleic acid, a peptide, a virus, or a vector containing the nucleic acid, which can target LYPD1 and inhibit the expression or activity of LYPD1.
구체적으로, 상기 LYPD1 유전자의 발현 억제제는 LYPD1 유전자의 안티센스 올리고뉴클레오타이드, siRNA, shRNA 및 microRNA로 구성된 군으로부터 선택되는 하나 이상일 수 있다.Specifically, the LYPD1 gene expression inhibitor may be at least one selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the LYPD1 gene.
또한, 구체적으로 상기 LYPD1 유전자의 발현 억제제는 서열번호 2 내지 5로 이루어지는 군에서 선택되는 하나 이상의 서열과 결합할 수 있다. 상기 서열번호 2 내지 5의 염기서열은 LYPD1 유전자에서 상기 억제제와 결합할 수 있는 염기서열 부분이다. Specifically, the LYPD1 gene expression inhibitor may bind to one or more sequences selected from the group consisting of SEQ ID NOS: 2 to 5. The nucleotide sequence of SEQ ID NO: 2 to 5 is a nucleotide sequence part capable of binding to the inhibitor in the LYPD1 gene.
이러한 발현 억제제는 당업계에 통상적으로 알려진 기술에 따라 당업자가 LYPD1의 발현 억제를 가지고 올 수 있도록 용이하게 설계될 수 있다.
Such an expression inhibitor can be easily designed so that a person skilled in the art can come to restrain the expression of LYPD1 according to techniques commonly known in the art.
또한, 상기 LYPD1 단백질의 활성 억제제는 LYPD1 유전자로부터 발현되는 단백질에 특이적으로 결합하는 항체 또는 앱타머 등일 수 있으나, 이에 제한되지 않는다.In addition, the activity inhibitor of the LYPD1 protein may be an antibody or an aptamer that specifically binds to a protein expressed from the LYPD1 gene, but is not limited thereto.
이러한 항체는 다클론 항체, 단일클론 항체 또는 항원 결합성을 갖는 것이면 상기 항체의 단편들도 본 발명의 항체에 포함된다. 나아가, 본 발명의 항체에는 인간화 항체 등의 특수 항체, 및 인간 항체 등도 포함하며, 신규한 항체 외에 이미 당해 기술분야에서 공지된 항체들도 포함될 수 있다. 상기 항체는 LYPD1 유전자로부터 발현되는 단백질을 특이적으로 인식하는 결합의 특성을 갖는 한, 2개의 중쇄와 2개의 경쇄의 전체 길이를 가지는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체의 분자의 기능적인 단편이란, 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab')2및 Fv 등이 있으나, 이에 제한되는 것은 아니다.
Such an antibody may be a polyclonal antibody, a monoclonal antibody or fragments of the antibody as long as the antibody has antigen binding ability. Furthermore, the antibody of the present invention includes a special antibody such as a humanized antibody, and a human antibody. In addition to the novel antibody, antibodies that are already known in the art may also be included. The antibody comprises a functional fragment of an antibody molecule as well as a complete form having the full length of two heavy chains and two light chains, so long as the antibody has the property of binding specifically recognizing the protein expressed from the LYPD1 gene. The functional fragment of the molecule of the antibody refers to a fragment having at least an antigen binding function, and includes, but is not limited to, Fab, F (ab ') 2, F (ab') 2 and Fv.
또한 본 발명의 조성물은 단독으로 사용할 수 있지만, 치료 효율을 증가시키기 위해 방사선 요법 또는 화학요법(세포 성장 정지 또는 세포 독성 물질, 항생 물질형 물질, 알킬화제, 항대사성 물질, 호르몬제, 면역제, 인터페론형 물질, 사이클로옥시게나제 억제제, 메탈로매트릭스프로테아제 억제제, 텔로머라제 억제제, 티로신 키나제 억제제, 항성장인자 수용체 물질, 항-HER 물질, 항-EGFR 물질, 항-혈관생성 물질, 파르네실 트랜스퍼라제 억제제, ras-raf 시그날 전도 경로 억제제, 세포 주기 억제제, 기타 cdk 억제제, 튜불린 결합체, 토포이소머라제Ⅰ 억제제, 토포이소머라제 Ⅱ 억제제 등)과 같은 다른 항암 치료법과 병용하여 사용할 수 있으나, 이에 제한되지 않는다. In addition, the composition of the present invention may be used alone, but it may be used alone or in combination with radiation therapy or chemotherapy (including cell growth arrest or cytotoxic substance, antibiotic substance, alkylating agent, antimetabolite, hormone, An anti-EGFR agent, an anti-angiogenic agent, a paroxetine transfer agent, an anti-angiogenic agent, an anti-angiogenesis inhibitor, a metallo-matrix protease inhibitor, a telomerase inhibitor, a tyrosine kinase inhibitor, Such as, but not limited to, inhibitors, ras-raf signaling pathway pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binders, topoisomerase I inhibitors, topoisomerase II inhibitors, It is not limited.
본 발명의 목적상 상기 암은 LYPD1 유전자의 발현 또는 상기 단백질의 활성을 억제하여 예방 또는 치료가 가능한 암이라면 제한이 없으나, 구체적으로, 대장암, 전립선암, 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종 등일 수 있으며, 더욱 구체적으로 대장암 또는 전립선암일 수 있으나, 이에 제한되지 않는다. For the purpose of the present invention, the cancer is not limited as long as it is a cancer that can be prevented or treated by inhibiting the expression of the LYPD1 gene or the activity of the protein. Specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, , Pancreatic cancer, skin cancer, head or neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, stomach cancer, anal cancer, colon cancer, breast cancer, endometrial carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, Hodgkin's disease, Esophageal cancer, Small intestine cancer, Endocrine cancer, Thyroid cancer, Parathyroid cancer, Adenocarcinoma, Soft tissue sarcoma, Urethral cancer, Penile cancer, Chronic or acute leukemia, Lymphocytic lymphoma, Bladder cancer, Cell carcinoma, renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically Colon cancer, or prostate cancer.
상기 대장암은 대장의 가장 안쪽 표면인 점막에서 발생한 암으로, 직장암, 결장암 및 항문암을 통칭한 것을 의미한다.The colon cancer is a cancer arising from the mucosa, which is the innermost surface of the large intestine, and refers to rectal cancer, colon cancer and anal cancer.
상기 전립선암은 전립선에 발생한 암을 의미한다.The prostate cancer refers to a cancer that occurs in the prostate gland.
본 발명에서 사용되는 용어 "예방"이란, 상기 조성물을 개체에 투여하여 암의 발병을 억제시키거나 지연시키는 모든 행위를 의미한다. 본 발명에서 사용되는 용어 "치료"란, 상기 조성물을 암 질환 개체에 투여하여 암 증세가 호전되도록 하거나 이롭게 되도록 하는 모든 행위를 의미한다.
As used herein, the term "prevention" refers to any act that inhibits or delays the onset of cancer by administering the composition to an individual. The term "treatment" as used in the present invention means all the actions that cause the cancer symptom to be improved or benefited by administering the composition to a cancerous subject.
본 발명의 조성물은 약제학적으로 허용되는 담체와 함께 투여될 수 있고, 경구 투여시에는 결합체, 활택제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등을 사용할 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등을 혼합하여 사용할 수 있으며, 국소 투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등을 사용할 수 있다. 본 발명의 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭시르, 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수 회 투약 형태로 제조될 수 있다.The composition of the present invention may be administered together with a pharmaceutically acceptable carrier. In oral administration, a conjugate, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, In the case of injections, a buffer, a preservative, an anhydrous agent, a solubilizer, an isotonic agent, a stabilizer and the like may be mixed. In the case of topical administration, a base, excipient, lubricant and preservative may be used. Formulations of the compositions of the present invention may be prepared in a variety of ways by mixing with pharmaceutically acceptable carriers as described above. For example, oral administration may be in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc. In the case of injections, unit dosage ampoules or multiple dose forms may be prepared.
본 발명에서 사용되는 용어, 투여는 어떠한 적절한 방법으로 환자에게 본 발명의 조성물을 도입하는 것을 의미하며, 본 발명의 조성물의 투여경로는 목적 조직에 도달할 수 있는 한 어떠한 일반적인 경로를 통하여 투여될 수 있다. 경구 투여, 복강 내 투여, 정맥 내 투여, 근육 내 투여, 피하 투여, 피 내 투여, 비 내 투여, 폐 내 투여, 직장 내 투여, 강 내 투여, 복강 내 투여, 경막 내 투여가 이루어질 수 있으나, 이에 제한되지는 않는다. 본 발명의 조성물의 유효 투여량의 범위는 성별, 체표면적, 질환의 종류 및 중증도, 연령, 약물에 대한 민감도, 투여경로 및 배출비율, 투여시간, 치료기간, 표적세포, 발현 수준 등 기타 의학 분야에 잘 알려진 다양한 요인에 따라 달라질 수 있으며, 당 분야의 전문가들에 의해 용이하게 결정될 수 있다.
As used herein, the term administration refers to the introduction of a composition of the present invention to a patient in any suitable manner, and the route of administration of the composition of the present invention may be administered via any conventional route so long as it can reach the target tissue have. Intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intraperitoneal, intramuscular, subcutaneous, intradermal, But is not limited thereto. The effective dose range of the composition of the present invention can be varied depending on the sex, body surface area, kind and severity of disease, age, sensitivity to the drug, administration route and discharge rate, administration time, treatment period, target cell, And may be readily determined by one of ordinary skill in the art.
본 발명의 또 하나의 양태는 LYPD1 유전자의 발현 억제제, LYPD1 단백질의 활성 억제제 또는 이의 혼합물을 유효성분으로 포함하는, 암의 예방 또는 개선용 건강기능식품을 제공한다. Another aspect of the present invention provides a health functional food for preventing or ameliorating cancer comprising an inhibitor of LYPD1 gene expression, an inhibitor of LYPD1 protein activity, or a mixture thereof as an active ingredient.
상기 식품의 종류는 특별히 제한되지 아니하며, 통상적인 의미에서의 식품을 모두 포함한다. 상기 물질을 첨가할 수 있는 식품의 비제한적인 예로는 육류, 소세지, 빵, 초콜릿, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등을 들 수 있다.The kind of the food is not particularly limited, and includes food in a conventional sense. Non-limiting examples of foods to which the material can be added include dairy products including meats, sausages, breads, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, ice creams, , A drink, an alcoholic beverage, and a vitamin complex.
본 발명의 상기 건강기능식품이 음료 조성물인 경우, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상기 천연 탄수화물의 비제한적인 예로 포도당, 과당과 같은 모노사카라이드; 말토스, 수크로오스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 들 수 있다. 상기 첨가되는 추가 성분의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.When the health functional food of the present invention is a beverage composition, it may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Non-limiting examples of such natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; Natural sweetening agents such as dextrin, cyclodextrin; Synthetic sweetening agents such as saccharin and aspartame, and the like. The proportion of the additional component added may be appropriately determined by a person skilled in the art.
상기 외에 본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강 기능 식품 조성물은 천연 과일 주스, 과일 음료 또는 야채 음료 등의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 사용되거나 2 이상을 조합하여 사용할 수 있다. 이러한 첨가물의 비율 또한 당업자에 의해 적절히 선택될 수 있다.
In addition to the above, the health functional food of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the health functional food composition of the present invention may contain pulp for the production of natural fruit juice, fruit drink or vegetable drink. These components may be used independently or in combination of two or more. The ratios of these additives can also be suitably selected by those skilled in the art.
본 발명의 다른 양태는 LYPD1(LY6/PLAUR domain containing 1) 유전자 mRNA 또는 이의 단백질의 수준을 측정하는 제제를 포함하는, 암 진단용 조성물을 제공한다. Another aspect of the present invention provides a cancer diagnostic composition comprising an agent for measuring the level of LYPD1 (LY6 / PLAUR domain containing 1) gene mRNA or a protein thereof.
상기 LYPD1에 대해서는 앞서 설명한 바와 같다.The LYPD1 is as described above.
또한, 상기 기술한 바와 같이 LYPD1의 유전자 및 단백질 정보는 NCBI 유전자 은행과 같은 공지의 데이터베이스를 통하여 용이하게 확인할 수 있으며, 그 예로 NCBI 유전자 은행의 Gene ID: 116372인 LYPD1일 수 있으나, 이에 제한되지 않는다. 또한, 상기 LYPD1은 서열번호 7로 표시되는 아미노산 서열을 가질 수 있으며, 또한 특별히 이에 제한되지 않으나, 서열번호 1로 표시되는 염기서열을 가질 수 있다. As described above, the gene and protein information of LYPD1 can be easily confirmed through a known database such as NCBI gene bank, for example, LYPD1 which is Gene ID: 116372 of NCBI gene bank, but is not limited thereto . The LYPD1 may have the amino acid sequence shown in SEQ ID NO: 7, and may have the nucleotide sequence shown in SEQ ID NO: 1, though it is not particularly limited thereto.
이러한 LYPD1의 서열을 바탕으로 LYPD1의 발현 수준을 측정하여, 암의 진행 또는 발병 여부를 진단할 수 있다. 상기 서열은 암의 진행 또는 발병 여부를 진단하는데 있어서 일정 정도 변형이 가능하다. 본 기술 분야의 당업자라면 이러한 인위적인 변형에 의해 80% 이상, 구체적으로는 90% 이상, 보다 구체적으로는 95% 이상, 보다 더 구체적으로는 98%의 상동성이 유지되는 서열이 본 발명에서 목적하는 암 진단 마커로서 사용될 수 있으며, 또한 정상 개체와 암이 의심되는 개체 간에 발현량 차이를 유의있게 비교 가능하게 하는 한, 본 발명의 상기 서열과 균등한 것임을 쉽게 이해할 것이다.
Based on the sequence of LYPD1, the expression level of LYPD1 can be measured to diagnose cancer progression or onset. The sequence may be modified to some extent in diagnosing cancer progression or onset. Those skilled in the art will appreciate that sequences which retain more than 80%, in particular more than 90%, more specifically more than 95%, more specifically more than 98% homology by such an artificial modification, It will be easily understood that the present invention can be used as a cancer diagnostic marker and is equivalent to the above sequence of the present invention as long as the difference between the normal individuals and the suspected cancer can be significantly compared.
본 발명에서 사용되는 용어 "진단"이란, 병리 상태의 존재 또는 특징을 확인하는 것을 의미한다. 본 발명에 있어서, 상기 진단은 암의 진행 또는 발병 여부를 확인하는 것으로 해석될 수 있다.
As used herein, the term "diagnosis" means identifying the presence or characteristic of a pathological condition. In the present invention, the diagnosis can be interpreted as confirming whether or not the cancer has progressed or developed.
본 발명에서 사용되는 용어 "마커 또는 진단용 마커"란 암세포 또는 암 질환을 가진 개체를 정상세포 또는 정상 개체와 구분하여 진단할 수 있는 물질로, 정상 세포에 비하여 암이 진행 또는 발병된 세포 또는 개체에서 증가 또는 감소를 보이는 폴리펩티드, 단백질 또는 핵산(예: mRNA 등), 지질, 당지질, 당단백질 또는 당(단당류, 이당류, 올리고당류 등) 등과 같은 유기 생체 분자들을 포함한다. 본 발명의 목적상, 본 발명의 암 진단 마커는 LYPD1으로, 암 세포에서 발현이 증가하는 유전자이다.
As used herein, the term "marker or diagnostic marker" refers to a substance capable of diagnosing a cancer cell or an individual having cancer by distinguishing it from a normal cell or a normal cell. The cell or object Such as polypeptides, proteins or nucleic acids (such as mRNA), lipids, glycolipids, glycoproteins or sugars (monosaccharides, disaccharides, oligosaccharides, etc.) that exhibit increased or decreased activity. For the purpose of the present invention, the cancer diagnostic marker of the present invention is LYPD1, which is a gene whose expression is increased in cancer cells.
본 발명에서 사용되는 용어 "LYPD1 유전자 mRNA의 수준을 측정하는 제제"란, 시료에 포함된 LYPD1의 발현 여부를 확인하는 방법에 사용되는 제제를 의미하는데, 바람직하게는 RT-PCR, 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR(Real-time RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블럿팅(Northern blotting), 유전자 칩 분석법 등의 방법에 사용되는 표적 유전자에 특이적으로 결합할 수 있는 프라이머 또는 프로브가 될 수 있으나, 특별히 이에 제한되지는 않는다.The term "agent for measuring the level of LYPD1 gene mRNA" used in the present invention means a preparation used for confirming the expression of LYPD1 contained in a sample, preferably RT-PCR, competitive RT-PCR A target gene used in methods such as competitive RT-PCR, real-time RT-PCR, RNase protection assay (RPA), Northern blotting, But it is not particularly limited thereto.
본 발명에서 사용되는 용어 "프라이머"란, 짧은 자유 3말단 수산화기(free 3' hydroxyl group)를 가지는 핵산 서열로 상보적인 템플레이트(template)와 염기쌍(base pair)을 형성할 수 있고 템플레이트 가닥 복사를 위한 시작 지점으로 기능을 하는 짧은 핵산 서열을 의미한다. 프라이머는 적절한 완충용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재하에서 DNA 합성이 개시할 수 있다. The term "primer" as used herein refers to a nucleic acid sequence having a short free 3 'hydroxyl group and capable of forming a base pair with a complementary template, Refers to a short nucleic acid sequence that functions as a starting point. The primers can initiate DNA synthesis in the presence of reagents and four different nucleoside triphosphates for polymerization reactions (i. E., DNA polymerase or reverse transcriptase) at appropriate buffer solutions and temperatures.
본 발명에서 사용되는 용어 "프로브"란, 유전자 또는 mRNA와 특이적 결합을 이룰 수 있는 짧게는 수 염기 내지 길게는 수백 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하는데, 올리고뉴클레오티드(oligonucleotide) 프로브, 단쇄 DNA(single stranded DNA) 프로브, 이중쇄 DNA(double stranded DNA) 프로브, RNA 프로브 등의 형태로 제작될 수 있고, 보다 용이하게 검출하기 위하여 라벨링될 수 있으나 이에 제한되는 것은 아니다.As used herein, the term "probe" refers to a nucleic acid fragment such as RNA or DNA corresponding to a few nucleotides or hundreds of nucleotides, which can specifically bind to a gene or mRNA. An oligonucleotide, A probe, a single stranded DNA probe, a double stranded DNA probe, an RNA probe, or the like, and may be labeled for easier detection, but the present invention is not limited thereto.
본 발명의 프라이머 또는 프로브는 포스포르아미다이트 고체 지지체 방법, 또는 기타 널리 공지된 방법을 사용하여 화학적으로 합성할 수 있다. 이러한 핵산서열은 또한 당해 분야에 공지된 많은 수단을 이용하여 변형시킬 수 있다. 이러한 변형의 비-제한적인 예로는 메틸화, 캡화, 천연 뉴클레오타이드 하나 이상의 동족체로의 치환, 및 뉴클레오타이드 간의 변형, 예를 들면, 하전되지 않은 연결체 (예: 메틸 포스포네이트, 포스소트리에스테르, 포스포로아미데이트, 카바메이트 등) 또는 하전된 연결체 (예: 포스포로티오에이트, 포스포로디티오에이트 등)로의 변형이 있다. 핵산은 하나 이상의 부가적인 공유 결합된 잔기, 예를 들면, 단백질(예: 뉴클레아제, 독소, 항체, 시그날 펩타이드, 폴리-L-리신 등), 삽입제(예: 아크리딘, 프소랄렌 등), 킬레이트화제(예: 금속, 방사성 금속, 철, 산화성 금속 등), 및 알킬화제를 함유할 수 있다. 본 발명의 핵산 서열은 또한 검출 가능한 시그날을 직접적으로 또는 간접적으로 제공할 수 있는 표지를 이용하여 변형시킬 수 있다. 표지의 예로는 방사성 동위원소, 형광성 분자, 바이오틴 등이 있다.
The primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences may also be modified using many means known in the art. Non-limiting examples of such modifications include, but are not limited to, methylation, capping, substitution of one or more natural nucleotides with one or more homologues, and modifications between nucleotides, such as uncharged linkers (e.g., methylphosphonate, phosphotriester, (E.g., phosphoramidate, carbamate, etc.) or charged linkages (e.g., phosphorothioate, phosphorodithioate, etc.). The nucleic acid can be in the form of one or more additional covalently linked residues such as a protein such as a nuclease, a toxin, an antibody, a signal peptide, a poly-L-lysine, an intercalator such as acridine, ), Chelating agents (e.g., metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. The nucleic acid sequences of the present invention can also be modified using labels that can directly or indirectly provide a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, biotin, and the like.
본 발명에서 사용되는 용어 "단백질의 수준 측정"이란 암을 진단하기 위하여 생물학적 시료에서 암 마커 유전자로부터 발현된 단백질의 존재 여부와 발현 정도를 확인하는 과정으로, 구체적으로는, 상기 유전자의 단백질에 대하여 특이적으로 결합하는 항체를 이용하여 단백질의 양을 확인할 수 있다. As used herein, the term "measurement of protein level" refers to a process for determining the presence and expression level of a protein expressed from a cancer marker gene in a biological sample in order to diagnose cancer. Specifically, The amount of the protein can be confirmed by using a specifically binding antibody.
본 발명에서, "항체"란 항원성 부위에 대해서 지시되는 특이적인 단백질 분자를 의미한다. 본 발명의 목적상, 항체는 마커 단백질에 대해 특이적으로 결합하는 항체를 의미하며, 다클론 항체, 단클론 항체 및 재조합 항체를 모두 포함한다. 상기한 바와 같이 암 마커 단백질이 규명되었으므로, 이를 이용하여 항체를 생성하는 것은 당업계에 널리 공지된 기술을 이용하여 용이하게 제조할 수 있다. In the present invention, "antibody" means a specific protein molecule directed against an antigenic site. For purposes of the present invention, an antibody refers to an antibody that specifically binds to a marker protein and includes both polyclonal antibodies, monoclonal antibodies, and recombinant antibodies. Since the cancer marker protein has been identified as described above, the production of the antibody using the cancer marker protein can be easily performed using techniques well known in the art.
다클론 항체는 상기한 대장암 또는 전립선암 마커 단백질 항원을 동물에 주사하고 동물로부터 채혈하여 항체를 포함하는 혈청을 수득하는 당업계에 널리 공지된 방법에 의해 생산할 수 있다. 이러한 다클론 항체는 염소, 토끼, 양, 원숭이, 말, 돼지, 소 개 등의 임의의 동물 종 숙주로부터 제조 가능하다. Polyclonal antibodies can be produced by methods well known in the art for obtaining serum containing antibodies by injection of the colon cancer or prostate cancer marker protein antigen into an animal and blood sampling from the animal. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, small dogs, and the like.
단일클론 항체는 당업계에 널리 공지된 하이브리도마 방법(hybridoma method)(Kohler 및 Milstein (1976) European Jounral of Immunology 6:511-519 참조), 또는 파지 항체 라이브러리(Clackson et al, Nature, 352:624-628, 1991; Marks et al, J. Mol. Biol., 222:58, 1-597, 1991) 기술을 이용하여 제조될 수 있다. 상기 방법으로 제조된 항체는 겔 전지영동, 투석, 염 침전, 이온교환 크로마토그래피, 친화성 크로마토그래피 등의 방법을 이용하여 분리, 정제할 수 있다. Monoclonal antibodies can be obtained from the hybridoma method (see Kohler and Milstein (1976) European Jounal of Immunology 6: 511-519), or the phage antibody library (Clackson et al, Nature, 352: 1992, Marks et al., J. Mol. Biol., 222: 58, 1-597, 1991). The antibody prepared by the above method can be isolated and purified by gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, affinity chromatography, and the like.
또한 본 발명의 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 완전한 형태뿐만 아니라, 항체 분자의 기능적인 단편을 포함한다. 항체 분자의 기능적인 단편이란 적어도 항원 결합 기능을 보유하고 있는 단편을 뜻하며, Fab, F(ab'), F(ab') 2 및 Fv 등이 있다.The antibodies of the present invention also include functional fragments of antibody molecules as well as complete forms with two full-length light chains and two full-length heavy chains. A functional fragment of an antibody molecule refers to a fragment having at least an antigen binding function, and includes Fab, F (ab ') 2, F (ab') 2 and Fv.
상기 항체를 이용하여 이와 결합한 표적 단백질의 양을 확인하기 위한 분석 방법으로는 웨스턴 블랏, 엘라이자(enzyme linked immunosorbent assay,ELISA), 방사선면역분석(RIA: Radioimmunoassay), 방사 면역 확산법(radioimmunodiffusion), 오우크테로니(Ouchterlony) 면역 확산법, 로케트(rocket) 면역전기영동, 조직면역 염색, 면역침전 분석법(Immunoprecipitation Assay), 보체 고정 분석법(Complement Fixation Assay), 유세포분석(Fluorescence Activated Cell Sorter, FACS), 단백질 칩(protein chip) 등이 있으나, 이로 제한되는 것은 아니다.
Methods for determining the amount of the target protein bound to the antibody using the antibody include Western blotting, enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), radioimmunodiffusion Immunoprecipitation Assay, Complement Fixation Assay, Fluorescence Activated Cell Sorter (FACS), Protein Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Immunoprecipitation Assay, Fluorescence Activated Cell Sorter Chip, and the like, but are not limited thereto.
본 발명의 다른 하나의 양태는 상기 암 진단용 조성물을 포함하는 암 진단용 키트를 제공한다. Another aspect of the present invention provides a cancer diagnostic kit comprising the cancer diagnostic composition.
본 발명의 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준은 암세포가 정상세포보다 높으므로, 상기 키트로 측정한 시료와 정상세포의 상기 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 비교하여, 시료가 정상세포보다 상기 수준이 높으면 암 세포로 진단할 수 있다.
Since the level of the mRNA or the protein of the LYPD1 gene of the present invention is higher than that of the normal cells, the level of the mRNA or the protein of the LYPD1 gene of the normal cell is compared with that of the normal cell. If the level is high, cancer cells can be diagnosed.
상기 키트는 암의 발병이 의심되는 개체의 시료로부터 LYPD1의 발현 수준을 측정함으로써 암을 진단하는데 사용될 수 있는데, 특별히 이에 제한되지 않으나, 상기 LYPD1의 발현 수준을 측정하기 위한 프라이머 또는 프로브 뿐만 아니라 분석 방법에 적합한 한 종류 또는 그 이상의 다른 구성 성분 조성물, 용액 또는 장치가 포함될 수도 있다. The kit can be used to diagnose cancer by measuring the expression level of LYPD1 from a sample of a subject suspected of having cancer, including, but not limited to, a primer or a probe for measuring the expression level of LYPD1, One or more other component compositions, solutions or devices suitable for use in the present invention may be included.
구체적으로, 본 발명의 LYPD1의 발현 수준을 측정하기 위한 진단 키트는 RT-PCR을 수행하기 위해 필요한 필수 요소를 포함하는 키트일 수 있다. RT-PCR 키트는, 상기 유전자에 대한 특이적인 각각의 프라이머 쌍 외에도 테스트 튜브 또는 다른 적절한 컨테이너, 반응 완충액(pH 및 마그네슘 농도는 다양), 데옥시뉴클레오타이드(dNTPs), Taq-폴리머라아제 및 역전사효소와 같은 효소, DNase, RNAse 억제제, DEPC-수(DEPC-water), 멸균수 등을 포함할 수 있다. 또한, 정량 대조군으로 사용되는 유전자에 특이적인 프라이머 쌍을 포함할 수 있다. Specifically, the diagnostic kit for measuring the expression level of LYPD1 of the present invention may be a kit containing essential elements necessary for conducting RT-PCR. The RT-PCR kit can be used in combination with a test tube or other appropriate container, a reaction buffer (pH and magnesium concentration varies), deoxynucleotides (dNTPs), Taq polymerase and reverse transcriptase , DNase, RNAse inhibitor, DEPC-water, sterile water, and the like. It may also contain a primer pair specific for the gene used as a quantitative control.
또한, 구체적으로 본 발명의 키트는 유전자 칩 분석법을 수행하기 위해 필요한 필수 요소를 포함할 수 있다. 유전자 칩 분석용 키트는, 유전자 또는 그의 단편에 해당하는 cDNA가 프로브로 부착되어 있는 기판, 및 형광표식 프로브를 제작하기 위한 시약, 제제, 효소 등을 포함할 수 있다. 또한, 기판은 정량 대조군 유전자 또는 그의 단편에 해당하는 cDNA를 포함할 수 있다.In particular, the kit of the present invention may contain essential elements necessary for performing gene chip analysis. The kit for gene chip analysis may include a substrate on which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. In addition, the substrate may contain a cDNA corresponding to a quantitative control gene or a fragment thereof.
또한, 구체적으로본 발명의 키트는 ELISA를 수행하기 위해 필요한 필수 요소를 포함할 수 있다. ELISA 키트는 마커 단백질에 대한 특이적인 항체를 포함한다. 항체는 각 마커 단백질에 대한 특이성 및 친화성이 높고 다른 단백질에 대한 교차 반응성이 거의 없는 항체로, 단클론 항체, 다클론 항체 또는 재조합 항체이다. 또한 ELISA 키트는 대조군 단백질에 특이적인 항체를 포함할 수 있다. 그 외 ELISA 키트는 결합된 항체를 검출할 수 있는 시약, 예를 들면, 표지된 2차 항체, 발색단(chromophores), 효소(예: 항체와 컨주게이트됨) 및 그의 기질 또는 항체와 결합할 수 있는 다른 물질 등을 포함할 수 있다. In addition, specifically, the kit of the present invention can include essential elements necessary for performing ELISA. ELISA kits contain antibodies specific for the marker protein. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each marker protein and little cross reactivity to other proteins. The ELISA kit may also include antibodies specific for the control protein. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.
또한, 본 발명의 목적상 상기 암은 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준이 정상세포보다 높은한 제한이 없으나, 구체적으로, 대장암, 전립선암, 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종 등일 수 있으며, 더욱 구체적으로 대장암 또는 전립선암일 수 있으나, 이에 제한되지 않는다.
For the purpose of the present invention, the cancer is not limited as long as the level of the mRNA or the protein of the LYPD1 gene is higher than that of a normal cell, but specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, non- Ovarian cancer, rectal cancer, stomach cancer, anorectal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, ovarian cancer, ovarian cancer, ovarian cancer, Cancer of the kidney, cancer of the urethra, cancer of the urethra, cancer of the urethra, cancer of the urethra, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cancer, endometrioid cancer, , Renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically, Amil it is, but is not limited thereto.
본 발명의 또 다른 하나의 양태는 상기 진단용 조성물 또는 키트를 이용하여 암 진단을 위한 정보를 제공하는 방법을 제공하는데, 구체적으로, (a) 암이 의심되는 개체의 분리된 시료로부터 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 정상 대조군 시료의 유전자의 mRNA 또는 이의 단백질의 수준보다 높은지 여부를 비교하는 단계를 포함할 수 있다. 또한, 암 진단을 위한 정보를 제공하는 방법은 암 진단 방법일 수 있다. Another aspect of the present invention provides a method for providing information for cancer diagnosis using the diagnostic composition or kit. Specifically, there is provided a method for diagnosing cancer, comprising the steps of: (a) isolating a mRNA of LYPD1 gene Or measuring the level of the protein thereof; And (b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a protein thereof. In addition, the method of providing information for cancer diagnosis may be a cancer diagnosis method.
상기 방법은 또한 추가로 (c) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 정상 대조군 시료의 유전자의 mRNA 또는 이의 단백질의 수준보다 높으면 암이 발병하는 것으로 판단하는 단계를 포함할 수 있다.
The method may further comprise the step of (c) determining that the cancer is onset if the level of the mRNA of the gene or the protein thereof is higher than the mRNA of the gene of the normal control sample or the protein thereof.
암이 의심되는 개체에서 mRNA를 분리하는 과정은 공지의 공정을 이용하여 수행할 수 있으며, 본 발명에서 사용되는 용어 "개체의 시료"란 암 마커인 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준이 차이나는 조직, 세포, 혈액, 혈청, 혈장, 타액, 객담, 뇌척수액 또는 뇨와 같은 시료 등을 포함하나, 이에 제한되지 않는다. The process of isolating mRNA from an individual suspected of having cancer can be performed using a known process. The term "sample of an individual" used in the present invention refers to a gene having a different level of the mRNA of LYPD1 gene or its protein But are not limited to, tissue, cells, blood, serum, plasma, saliva, sputum, cerebrospinal fluid or urine.
상기 mRNA 수준은 다양한 방법으로 측정할 수 있으며, 구체적으로 상기 LYPD1 유전자에 특이적으로 결합하는 프라이머 또는 프로브를 이용할 수 있다. The mRNA level can be measured by various methods. Specifically, a primer or a probe that specifically binds to the LYPD1 gene can be used.
또한, 구체적으로 상기 mRNA 수준은 역전사효소 중합효소반응(RT-PCR), 경쟁적 역전사효소 중합효소반응(competitive RT-PCR), 실시간 역전사효소 중합효소반응(real time quantitative RT-PCR), RNase 보호 분석법(RNase protection method), 노던 블랏팅(Northern blotting) 또는 유전자 칩에 의하여 측정되는 것일 수 있으나, 이로 제한되는 것은 아니다. Specifically, the mRNA level may be measured by RT-PCR, competitive RT-PCR, real-time quantitative RT-PCR, RNase protection assay But are not limited to, those determined by RNase protection method, Northern blotting or gene chips.
상기 LYPD1 단백질 수준의 측정 방법은 웨스턴 블랏, ELISA, 방사선면역분석, 방사 면역 확산법, 오우크테로니 면역 확산법, 로케트 면역전기영동, 조직면역 염색, 면역침전 분석법, 보체 고정 분석법, FACS, 단백질 칩 등이 있으나 이로 제한되는 것은 아니다.Methods for measuring the LYPD1 protein level include Western blotting, ELISA, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc. But is not limited thereto.
상기 분석 방법들을 통하여, 정상 대조군에서의 항원-항체 복합체의 형성량과 암 의심 환자에서의 항원-항체 복합체의 형성량을 비교할 수 있고, 암 마커 유전자에서 단백질로의 유의한 발현량의 증가여부를 판단하여, 암 의심 환자의 실제 암 발병 여부를 진단할 수 있다. Through the above analysis methods, it is possible to compare the amount of the antigen-antibody complex formed in the normal control group with the amount of the antigen-antibody complex formed in the suspected cancer patient, and whether the expression level of the cancer marker gene is significantly increased Thus, it is possible to diagnose the actual cancer incidence in a patient suspected of cancer.
본 발명에서 용어 "항원-항체 복합체"란 암 마커 단백질과 이에 특이적인 항체의 결합물을 의미하고, 항원-항체 복합체의 형성량은 검출 라벨(detection label)의 시그널의 크기를 통해서 정량적으로 측정 가능하다. The term "antigen-antibody complex" in the present invention means a combination of a cancer marker protein and an antibody specific thereto, and the amount of the antigen-antibody complex formed can be quantitatively measured through a signal label of a detection label Do.
이러한 검출 라벨은 효소, 형광물, 리간드, 발광물, 미소입자(microparticle), 레독스 분자 및 방사선 동위원소로 이루어진 그룹중에서 선택할 수 있으며, 반드시 이로 제한되는 것은 아니다. 검출 라벨로서 효소가 사용되는 경우 이용 가능한 효소에는 β-글루쿠로니다제, β-D-글루코시다제, β-D-갈락토시다제, 우레아제, 퍼옥시다아제 또는 알칼라인 포스파타아제, 아세틸콜린에스테라제, 글루코즈 옥시다제, 헥소키나제와 GDPase, RNase, 글루코즈 옥시다제와 루시페라제, 포스포프럭토키나제, 포스포에놀피루베이트 카복실라제, 아스파르테이트 아미노트랜스페라제, 포스페놀피루베이트 데카복실라제, β-라타마제 등이 있으며 이로 제한되지 않는다. 형광물에는 플루오레신, 이소티오시아네이트, 로다민, 피코에리테린, 피코시아닌, 알로피코시아닌, o-프탈데히드, 플루오레스카민 등이 있으며 이로 제한되지 않는다. 리간드에는 바이오틴 유도체 등이 있으며 이로 제한되지 않는다. 발광물에는 아크리디늄 에스테르, 루시페린, 루시퍼라아제 등이 있으며 이로 제한되지 않는다. 미소입자에는 콜로이드 금, 착색된 라텍스 등이 있으며 이로 제한되지 않는다. 레독스 분자에는 페로센, 루테늄 착화합물, 바이올로젠, 퀴논, Ti 이온, Cs 이온, 디이미드, 1,4-벤조퀴논, 하이드로퀴논, K4W(CN)8,[Os(bpy)3]2+,[RU(bpy)3]2+,[MO(CN)8]4-등이 포함되며 이로 제한되지 않는다. 방사선동위원소에는 3H,14C,32P,35S,36Cl,51Cr,57Co,58Co,59Fe,90Y,125I,131I,186Re등이 포함되며 이로 제한되지 않는다.Such detection labels may be selected from the group consisting of enzymes, chromophores, ligands, emitters, microparticles, redox molecules, and radioisotopes, but are not necessarily limited thereto. When an enzyme is used as the detection label, available enzymes include? -Glucuronidase,? -D-glucosidase,? -D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholine Glucoamylase, terazo, glucose oxidase, hexokinase and GDPase, RNase, glucose oxidase and luciferase, phosphofructokutase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphoenolpyruvate decar ≪ / RTI > beta-lactamase, and the like. The minerals include, but are not limited to, fluorescein, isothiocyanate, rhodamine, picoeriterine, picocyanin, allophycocyanin, o-phthaldehyde, fluororescamine and the like. Ligands include, but are not limited to, biotin derivatives. Emitters include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Fine particles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include ferrocene, ruthenium complex compounds, Biology hydrogen, quinone, Ti ions, Cs ions, diimide, 1,4-benzoquinone, hydroquinone, K 4 W (CN) 8 , [Os (bpy) 3] 2+ , [RU (bpy) 3 ] 2+ , [MO (CN) 8 ] 4-, and the like. Radioisotope includes include 3 H, 14 C, 32 P , 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re are not limited to .
단백질 발현수준 측정은 구체적으로는, ELISA법을 이용하는 것이다. ELISA는 고체 지지체에 부착된 항원을 인지하는 표지된 항체를 이용하는 직접적 ELISA, 고체 지지체에 부착된 항원을 인지하는 항체의 복합체에서 포획 항체를 인지하는 표지된 항체를 이용하는 간접적 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 표지된 또 다른 항체를 이용하는 직접적 샌드위치 ELISA, 고체 지지체에 부착된 항체와 항원의 복합체에서 항원을 인지하는 또 다른 항체와 반응시킨 후 이 항체를 인지하는 표지된 2차 항체를 이용하는 간접적 샌드위치 ELISA 등 다양한 ELISA 방법을 포함한다. 보다 바람직하게는, 고체 지지체에 항체를 부착시키고 시료를 반응시킨 후 항원-항체 복합체의 항원을 인지하는 표지된 항체를 부착시켜 효소적으로 발색시키거나 항원-항체 복합체의 항원을 인지하는 항체에 대해 표지된 2차 항체를 부착시켜 효소적으로 발색시키는 샌드위치 ELISA 방법에 의해서 검출한다. 암 마커 단백질과 항체의 복합체 형성 정도를 확인하여, 암 발병 여부를 확인할 수 있다.Specifically, the expression level of the protein is measured by an ELISA method. ELISAs include direct ELISA using labeled antibodies that recognize the antigen attached to the solid support, indirect ELISA using labeled antibodies that recognize the capture antibody in a complex of antibodies recognizing the antigen attached to the solid support, A direct sandwich ELISA using another labeled antibody that recognizes an antigen in the complex of antibody and antigen, a method of reacting with another antibody recognizing an antigen in a complex of an antibody and an antigen attached to a solid support, Indirect sandwich ELISA using a secondary antibody, and various ELISA methods. More preferably, the antibody is attached to a solid support, the sample is reacted, and the labeled antibody recognizing the antigen of the antigen-antibody complex is adhered to produce an enzyme, or an antibody that recognizes the antigen of the antigen-antibody complex Is detected by a sandwich ELISA method in which a labeled secondary antibody is attached and the enzyme is developed. The degree of complex formation between the cancer marker protein and the antibody can be confirmed to confirm the onset of cancer.
또한, 구체적으로는, 상기 암 마커에 대한 하나 이상의 항체를 이용한 웨스턴 블랏을 이용하는 것이다. 시료에서 전체 단백질을 분리하고, 이를 전기영동하여, 단백질을 크기에 따라 분리한 다음, 니트로셀루로즈 막으로 이동시켜 항체와 반응시킨다. 생성된 항원-항체 복합체의 양을 표지된 항체를 이용하여 확인하는 방법으로 유전자의 발현에 의해 생성된 단백질의 양을 확인하여, 암 발병 여부를 확인할 수 있다. 상기 검출 방법은 대조군에서의 마커 유전자의 발현량과 암이 발병한 세포에서의 마커 유전자의 발현량을 조사하는 방법으로 이루어진다. mRNA 또는 단백질 수준은 상기한 마커 단백질의 절대적(예: ㎍/㎖) 또는 상대적(예: 시그널의 상대 강도) 차이로 나타낼 수 있다. Specifically, western blotting using one or more antibodies against the cancer marker is used. The whole protein is separated from the sample, electrophoresed, the protein is separated according to the size, and then transferred to the nitrocellulose membrane to react with the antibody. The amount of the protein produced by expression of the gene can be confirmed by confirming the amount of the produced antigen-antibody complex using the labeled antibody, and it is possible to confirm whether or not the cancer has occurred. The detection method comprises a method of examining the expression level of a marker gene in a control group and the expression level of a marker gene in a cancer-causing cell. The level of mRNA or protein may be expressed as the absolute (e.g., [mu] g / ml) or relative (e.g., the relative intensity of the signal) difference of the marker protein.
또한, 구체적으로는, 상기 암 마커에 대한 하나 이상의 항체를 이용한 면역조직 염색을 실시하는 것이다. 정상 대장 상피 조직 및 암으로 의심되는 조직을 채취 및 고정한 후, 당업계에서 널리 공지된 방법으로 파라핀 포매 블록을 제조한다. 이들을 수 um 두께의 절편으로 만들어 유리 슬라이드에 붙인 후, 이와 상기의 항체 중 선택된 1개와 공지의 방법에 의하여 반응시킨다. 이후, 반응하지 못한 항체는 세척하고, 상기에 언급한 검출라벨 중의 하나로 표지하여 현미경 상에서 항체의 표지 여부를 판독한다.More specifically, immunostaining is performed using one or more antibodies against the cancer marker. Normal colon epithelial tissues and suspected cancerous tissues are collected and fixed, and paraffin-embedded blocks are prepared by methods well known in the art. They are made into sections with a thickness of several um and attached to a glass slide, and then reacted with a selected one of the above antibodies by a known method. Thereafter, the unreacted antibody is washed and labeled with one of the above-mentioned detection labels, and the labeling of the antibody is read on a microscope.
또한, 구체적으로는, 상기 암 마커에 대한 하나 이상의 항체가 기판 위의 정해진 위치에 배열되어 고밀도로 고정화되어 있는 단백질 칩을 이용하는 것이다. 단백질 칩을 이용하여 시료를 분석하는 방법은, 시료에서 단백질을 분리하고, 분리한 단백질을 단백질 칩과 혼성화시켜서 항원-항체 복합체를 형성시키고, 이를 판독하여, 단백질의 존재 또는 발현 정도를 확인하여, 암 발병 여부를 확인할 수 있다.More specifically, a protein chip in which at least one antibody against the cancer marker is arranged at a predetermined position on a substrate and immobilized at a high density is used. A method of analyzing a sample using a protein chip is a method of separating a protein from a sample, hybridizing the separated protein with a protein chip to form an antigen-antibody complex, reading the protein, It can be confirmed whether or not the cancer has occurred.
또한, 본 발명의 목적상 상기 암은 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준이 정상세포보다 높은 한 제한이 없으나, 구체적으로, 대장암, 전립선암, 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종 등일 수 있으며, 더욱 구체적으로 대장암 또는 전립선암일 수 있으나, 이에 제한되지 않는다.
For the purpose of the present invention, the cancer is not limited as long as the level of the mRNA or the protein of the LYPD1 gene is higher than that of a normal cell, but specifically, the cancer is not limited to colon cancer, prostate cancer, lung cancer, non- Ovarian cancer, rectal cancer, stomach cancer, anorectal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical carcinoma, vaginal carcinoma, vulvar carcinoma, ovarian cancer, ovarian cancer, ovarian cancer, Cancer of the kidney, cancer of the urethra, cancer of the urethra, cancer of the urethra, cancer of the urethra, chronic or acute leukemia, lymphocytic lymphoma, bladder cancer, kidney or ureteral cancer, kidney cancer, endometrioid cancer, , Renal pelvic carcinoma, central nervous system (CNS) tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma or pituitary adenoma, and more specifically, Amil it is, but is not limited thereto.
본 발명의 또 다른 하나의 양태는 (a) 암 치료용 후보물질을 LYPD1 유전자를 발현하는 분리된 시료에 처리한 다음, 상기 LYPD1 유전자의 mRNA 또는 이의 단백질의 수준을 측정하는 단계; 및 (b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 상기 시료를 처리하지 않은 대조군 시료의 수준보다 낮은 경우 암 치료제로 선택하는 단계를 포함하는, 암 치료제의 스크리닝 방법을 제공한다. Another aspect of the present invention is a method for treating cancer, comprising the steps of: (a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA of the LYPD1 gene or a protein thereof; And (b) when the level of the mRNA or the protein of the gene is lower than that of the control sample not treated with the sample, a method for screening a cancer therapeutic agent.
암을 예방 또는 치료할 수 있는 후보 물질의 부재 하에 LYPD1 유전자를 발현하는 세포에서 본 발명의 LYPD1의 수준을 측정하고, 또한, 상기 후보 물질의 존재 하에서 본 발명의 상기 LYPD1의 수준을 측정하여 양자를 비교한 후, 상기 후보 물질이 존재할 때의 본 발명의 상기 LYPD1의 발현 수준을 상기 후보 물질의 부재 하에서의 수준보다 감소시키는 물질을 암 예방 또는 치료용 제제로 예측할 수 있다.The level of LYPD1 of the present invention is measured in a cell expressing the LYPD1 gene in the absence of a candidate substance capable of preventing or treating cancer and the level of the LYPD1 of the present invention is measured in the presence of the candidate substance, , A substance which reduces the level of expression of the LYPD1 of the present invention in the absence of the candidate substance in the absence of the candidate substance can be predicted by an agent for the prophylaxis or treatment of cancer.
또한, 구체적으로 상기 LYPD1 유전자를 발현하는 세포는 대장암, 전립선암, 폐암, 비소세포성 폐암, 결장암, 골암, 췌장암, 피부암, 두부 또는 경부 암, 피부 또는 안구내 흑색종, 자궁암, 난소암, 직장암, 위암, 항문부근암, 결장암, 유방암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병(Hodgkin's disease), 식도암, 소장암, 내분비선암, 갑상선암, 부갑상선암, 부신암, 연조직 육종, 요도암, 음경암, 만성 또는 급성 백혈병, 림프구 림프종, 방광암, 신장 또는 수뇨관암, 신장세포 암종, 신장골반 암종, 중추신경계(CNS; central nervous system) 종양, 1차 중추신경계 림프종, 척수 종양, 뇌간 신경교종 또는 뇌하수체 선종 세포일 수 있으며, 더욱 구체적으로, 대장암 또는 전립선암 세포일 수 있으고, 이에 따라 본 발명은 대장암 또는 전립선암 치료제의 스크리닝 방법일 수 있으나, 이에 제한되지 않는다.
Specifically, the cell expressing the LYPD1 gene may be a colon, prostate, lung, non-small cell lung, colon, bone, pancreatic, skin, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, pituitary adenocarcinoma, adenocarcinoma, adenocarcinoma of the uterine cervix, adenocarcinoma, vulvar carcinoma, vulvar carcinoma, (CNS) tumors, primary central nervous system (CNS) neoplasms, renal cell carcinomas, renal pelvic carcinomas, central nervous system (CNS) tumors, Lymphoma, spinal cord tumor, brainstem glioma, or pituitary adenoma cell. More specifically, it may be a colon cancer or a prostate cancer cell. Accordingly, the present invention provides a method for treating colon cancer or prostate cancer But the present invention is not limited thereto.
본 발명은 현재까지 그 기능이 잘 알려지지 않은 LYPD1(LY6/PLAUR domain containing 1) 유전자가 암세포의 침윤, 세포 이동성 및 세포성장을 조절하며, 또한 세포신호전달을 조절함을 규명한바, 본 발명의 LYPD1을 이용하여 암 진단용 조성물, 상기 조성물을 포함하는 암 진단용 키트, 암 진단을 위한 정보를 제공하는 방법, 암 치료제의 스크리닝 방법; 및 암의 예방 또는 치료용 약학적 조성물 등을 제공할 수 있다.
The present inventors have confirmed that a LYPD1 (LY6 / PLAUR domain containing 1) gene, whose function is not well known until now, regulates the infiltration of cancer cells, cell mobility and cell growth and regulates cell signal transduction. A cancer diagnosis kit comprising the composition, a method of providing information for cancer diagnosis, a screening method of cancer treatment agent, And a pharmaceutical composition for preventing or treating cancer and the like.
도 1은 대장암 세포주에서 LYPD1의 발현을 siRNA로 억제한 결과 암세포의 침윤 및 세포 이동성 감소함을 보여주는 도이다. a는 상층 챔버에서 하층 챔버로 이동한 대장암세포를 크리스탈 바이올렛 용액으로 염색한 결과를 보여주는 사진이다. b는 상층 챔버에서 하층 챔버로 이동한 대장암세포의 상대적인 비율을 나타내는 그래프이다.
도 2는 대장암 세포주에서 LYPD1의 발현을 siRNA로 억제한 결과 암세포의 성장이 감소됨을 보여주는 도이다.
도 3은 대장암 세포주에서 LYPD1의 발현을 siRNA로 48 시간(a) 및 72 시간(b) 동안 억제한 결과, LYPD1 발현이 감소됨을 보여주는 도이다.
도 4는 정상세포에 LYPD1 발현 벡터(p3XFlagCMV14-LYPD1 및 pFlagCMV1-LYPD1)의 형질전환을 통해 LYPD1 발현 정도를 확인한 도이다.
도 5는 대장암 세포주에서 LYPD1의 발현을 증가시킨 결과(a), 침윤이 능력이 증가함(b)을 보여주는 도이다.
도 6은 정상세포(a) 및 대장암 세포주(b)에서 LYPD1의 발현을 각각 증가시킨 경우, 세포신호전달의 활성화 여부를 보여주는 도이다.
도 7은 정상세포에서 LYPD1의 발현에 따른 AP-1 리포터 활성 증가를 보여주는 도이다.
도 8은 6종의 대장암 세포주와 2종의 전립선암 세포주에서 LYPD1의 발현을 RT-qPCR 기법으로 확인한 결과를 나타낸 그래프이다.
도 9는 전립선암 세포주에서 LYPD1의 발현을 siRNA로 48 시간(a) 및 72 시간(b) 동안 억제한 결과, 암세포의 성장이 감소됨을 보여주는 도이다.
도 10은 전립선암 세포주에서 LYPD1의 발현을 siRNA로 억제한 결과 LYPD1 발현이 감소됨을 보여주는 도이다.
도 11은 전립선암 세포주에서 LYPD1의 발현을 siRNA로 억제한 결과 암세포의 이동성이 감소함을 보여주는 도이다. a는 상층 챔버에서 하층 챔버로 이동한 전립선암 세포를 크리스탈 바이올렛 용액으로 염색한 결과를 보여주는 사진이다. b는 상층 챔버에서 하층 챔버로 이동한 전립선암 세포의 상대적인 비율을 나타내는 그래프이다.FIG. 1 shows that the inhibition of LYPD1 expression by siRNA in colorectal cancer cell lines leads to a decrease in cancer cell infiltration and cell mobility. a is a photograph showing the result of staining a colon cancer cell which migrated from an upper chamber to a lower chamber with a crystal violet solution. and b is a graph showing the relative proportion of colon cancer cells transferred from the upper chamber to the lower chamber.
FIG. 2 is a graph showing that the growth of cancer cells is reduced as a result of suppression of expression of LYPD1 by siRNA in colon cancer cell lines.
FIG. 3 is a graph showing that LYPD1 expression is reduced as a result of suppression of expression of LYPD1 by siRNA for 48 hours (a) and 72 hours (b) in a colon cancer cell line.
FIG. 4 is a graph showing the degree of LYPD1 expression by transforming LYPD1 expression vectors (p3XFlagCMV14-LYPD1 and pFlagCMV1-LYPD1) into normal cells.
FIG. 5 shows (a) the increase in the expression of LYPD1 in the colon cancer cell line and (b) the increase in the infiltration ability. FIG.
FIG. 6 is a graph showing the activation of cell signaling when the expression of LYPD1 is increased in the normal cell (a) and the colon cancer cell line (b), respectively.
FIG. 7 shows the increase in AP-1 reporter activity according to the expression of LYPD1 in normal cells.
Fig. 8 is a graph showing the results of RT-qPCR expression of LYPD1 expression in 6 colon cancer cell lines and 2 prostate cancer cell lines.
FIG. 9 shows that the growth of cancer cells is reduced as a result of inhibiting the expression of LYPD1 in prostate cancer cell lines with siRNA for 48 hours (a) and 72 hours (b).
FIG. 10 is a graph showing that LYPD1 expression is reduced as a result of inhibition of LYPD1 expression by siRNA in a prostate cancer cell line. FIG.
11 is a graph showing that the mobility of cancer cells is reduced as a result of suppression of expression of LYPD1 by siRNA in the prostate cancer cell line. a is a photograph showing the results of staining of prostate cancer cells that have moved from the upper chamber to the lower chamber with a crystal violet solution. and b is a graph showing the relative proportion of prostate cancer cells transferred from the upper chamber to the lower chamber.
이하, 본 발명을 하기 실시예에서 보다 구체적으로 설명한다. 그러나 이들 예는 본 발명의 이해를 돕기 위한 것일 뿐, 이들에 의해 본 발명이 한정되는 것은 아니다.
Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.
실시예Example
1. 세포배양 1. Cell culture
모든 인간 세포주는 American Type Culture Collection(USA)에서 제공받았다. 대장암 세포주인 SW480, HCT116, HT29, HCT15, Lovo, Colo205 세포주 및 전립선암 세포주인 DU145, PC3 세포주는 10% FBS, 페니실린-스트렙토마이신, L-글루타민, 소디움 피루베이트가 포함된 RPMI1640(GIBCO, USA) 배지에서, HEK293E 세포는 DMEM 배지에서 37℃, 5% CO2조건에서 배양하였다. SW480sub는 SW480으로부터 얻은 subpopulation이었다.
All human cell lines were obtained from the American Type Culture Collection (USA). The colon cancer cell lines SW480, HCT116, HT29, HCT15, Lovo, Colo205 and prostate cancer cell lines DU145 and PC3 cell lines were cultured in RPMI1640 (GIBCO, USA) containing 10% FBS, penicillin-streptomycin, L-glutamine and sodium pyruvate ) Medium, HEK293E cells were cultured in DMEM medium at 37 ° C and 5% CO 2 . SW480sub was a subpopulation obtained from SW480.
실시예Example
2. 2.
siRNAsiRNA
또는 플라스미드의 형질감염 Or transfection of the plasmid
SW480 및 PC3 세포를 Microporation 기법(Neon Transfection system, Invitrogen)을 설명서대로 이용하여 형질감염시켰다. 세포를 수확한 뒤, PBS로 세척하고, R 버퍼에 3 x105cell/12㎕의 농도로 재부유시킨 뒤, 40 μM siRNA 2㎕ 또는 1 μg 플라스미드를 첨가하고 Microporator 기기를 이용하여 형질감염시켰다. 48시간 후, 세포를 수확하여 침윤실험 및 세포를 용균시켜 웨스턴 블랏 실험을 수행하였다. 사용한 LYPD1 특이적 siRNA는 Dharmacon에서 구입하였다(4 종의 mixture). 표적 염기서열 4종은 표 1과 같다(5' → 3' 방향).
SW480 and PC3 cells were transfected using the Microporation technique (Neon Transfection system, Invitrogen) as described. After harvesting the cells, washed with PBS, after which 3 x10 5 material in a concentration of cell / 12㎕ suspended in buffer R, the addition of 40
인터그린 알파5(integrin alpha5) 특이적 siRNA 서열은 5'-GGACCAAGGCAGAAGGCAG-3'(서열번호 6)이고, 안정성을 위해 3'말단에 TT를 추가하여 사용하였다.The integrin alpha5-specific siRNA sequence was 5'-GGACCAAGGCAGAAGGCAG-3 '(SEQ ID NO: 6) and TT was added at the 3' end for stability.
또한, HEK293E 세포에 PEI(polyethyleneimine)을 이용하여 플라스미드를 도입하였다(6 웰 기준 플라스미드 2 μg + PEI 4 μg 사용).
In addition, plasmid was introduced into HEK293E cells using PEI (polyethyleneimine) (using 2 μg of 6-well plasmid + 4 μg of PEI).
실시예Example
3. 3.
LYPD1LYPD1
발현 Expression
컨스트럭트Construct
제작 making
LYPD1을 코딩하는 영역(LYPD1 아미노산 서열(서열번호 7, NP_653187.3)의 1번부터 141번까지를 코딩하는 유전자)를 PCR로 얻어 p3XFlag-CMV14(Sigma)의 HindIII-BamHI에 서브클로닝하였다. 프라이머 세트는 포워드 프라이머로 5'-aacccaagcttgccgccatgtgggtcctaggcatcg-3'(서열번호 8) 및 리버스 프라이머로 5'-aacccggatccgca gtg tgc cga gaa gag g-3'(서열번호 9)을 사용하였다.The region coding for LYPD1 (the gene coding for
LYPD1를 코딩하는 영역 중 일부(신호 펩타이드를 제외한 서열번호 7의 아미노산 서열 23번부터 141번까지를 코딩하는 유전자)를 PCR로 얻어 pFlag-CMV1(Sigma)의 HindIII-BamHI에 서브클로닝하였다. 프마이머 세트는 포워드 프라이머로 5'-aagcccaagcttatccagtgctaccagtgtgaag-3'(서열번호 10) 및 리버스 프라이머로 5'-aacccggatccctatca gca gtg tgc cga gaa g-3'(서열번호 11)를 사용하였다.A part of the region encoding LYPD1 (the gene coding for amino acid sequence 23 to 141 of SEQ ID NO: 7 except for the signal peptide) was subcloned into HindIII-BamHI of pFlag-CMV1 (Sigma) by PCR. The primer set used 5'-aagcccaagcttatccagtgctaccagtgtgaag-3 '(SEQ ID NO: 10) as the forward primer and 5'-aacccggatccctatca gca gtg tgc cga gaa g-3' (SEQ ID NO: 11) as the reverse primer.
이때, PCR에서 사용한 기질(LYPD1를 코딩하는 영역이 서브클로닝된 플라스미드)는 한국인간유전자은행(genbank.kribb.re.kr)에서 구매하였고, 최종 얻어진 플라스미드는 시퀀싱을 통해 확인하였다.
At this time, the substrate used in the PCR (plasmid in which the region encoding LYPD1 was subcloned) was purchased from the Korean Human Gene Bank (genbank.kribb.re.kr), and the final plasmid was confirmed by sequencing.
실시예 4. 침윤 분석(Invasion assay) 및 세포 이동 분석(cell migration assay)
Example 4. Invasion assay and cell migration assay.
암세포 침윤능력의 변화를 분석하기 위하여, control siRNA는 센스 스트랜드 5'-CUU ACG CUG AGU ACU UCG A-3'(서열번호 12), 안티센스 스트랜드 5'-UCG AAG UAC UCA GCG UAA G-3'(서열번호 13)의 서열에 3' 말단에 TT를 각각 추가하여 제작하였고(siGL3; 에스티팜), 상기 control siRNA 또는 LYPD1 특이적 siRNA를 SW480sub 세포주에 형질전환하였다. 또한, LYPD1 발현 벡터 vs empty 벡터로 SW480 세포주를 형질전환하였다. 48시간 뒤 침윤 분석을 수행하였다. 구체적으로, 24-웰 트렌스웰 플레이트(8 포어 사이즈; Costar, 미국)의 다공성 막을 무혈청 배지로 희석된 250 ㎍/㎖ 농도의 100 ㎕ 마트리젤(BD Biosciences, 미국)로 코팅하였고, 실온에서 1시간 동안 방치하여 고형화시켰다. 트렌스웰 플레이트의 하층은 화학유인물(chemoattractant)로서 5 ㎍/㎖ 농도의 콜라겐 타입 I(Sigma) 100 ㎕를 이용하여 코팅하였다. In order to analyze the change of the cancer cell infiltration ability, the control siRNA was synthesized from the sense strand 5'-CUU ACG CUG AGU ACU UCG A-3 '(SEQ ID NO: 12), the antisense strand 5'-UCG AAG UAC UCA GCG UAA G-3' SEQ ID NO: 13), and the control siRNA or LYPD1-specific siRNA was transformed into the SW480sub cell line. In addition, SW480 cell line was transformed with LYPD1 expression vector vs empty vector. Infiltration analysis was performed 48 hours later. Specifically, the porous membrane of a 24-well transfer plate (8 pore size; Costar, USA) was coated with 100 μl of a matrigel (BD Biosciences, USA) at a concentration of 250 μg / ml diluted in serum-free medium, ≪ / RTI > for a period of time. The lower layer of the Transwell plate was coated with 100 [mu] l of collagen type I (Sigma) at a concentration of 5 [mu] g / ml as a chemoattractant.
무혈청 배지에 재부유시킨 3×104개의 세포를 상층 챔버에 분주하였고, 37 ℃, 5% CO2조건에서 48시간 동안 배양하면서 상층 챔버에서 하층 챔버로 이동하도록 하였다. 이동하지 않은 세포는 상층 챔버의 표면에서 제거하였다. 하층 챔버로 전이한 세포는 PBS에 녹인 3.7 % 파라포름알데하이드로 고정하였고, 2 % 크리스탈 바이올렛 용액으로 염색하였다. 여분의 크리스탈 바이올렛 용액을 증류수로 세척한 뒤, 선택 면적(×100)의 사진을 찍었고, 이동한 세포수는 5개의 선택 면적에서 계수하였다. 평균값을 구하고, 상대적 %를 계산하였다.
3 × 10 4 cells resuspended in serum-free medium were dispensed into the upper chambers and allowed to migrate from the upper chamber to the lower chamber while incubating for 48 h at 37 ° C and 5% CO 2 . Unmoved cells were removed from the surface of the upper chamber. Cells transformed into the lower chamber were fixed with 3.7% paraformaldehyde dissolved in PBS and stained with 2% crystal violet solution. The extra crystal violet solution was washed with distilled water, and a selected area (× 100) was photographed. The number of migrated cells was counted in five selected areas. The average value was calculated, and the relative percentage was calculated.
세포 이동능력의 변화를 분석하기 위하여, 마트리젤 코팅없이 1.5×104개의 세포를 상층 챔버에 분주하였다.
To analyze the changes in cell migration ability, 1.5 x 10 4 cells were dispensed into the upper chamber without matrigel coating.
실시예 5. 역전사 폴리머라제 연쇄 반응(Reverse transcription-polymerase chain reaction) Example 5. Reverse transcription Polymerase chain reaction (reverse transcription-polymerase chain reaction)
RNA 전체를 TRIzol(Invitrogen)을 사용하여 추출하였고, 상보적 DNA를 역전사효소(Bioneer, 대전, 한국)를 사용하여 합성하였다. LYPD1 특이적 프라이머(5'-CCCGAGTTCATTGTGAATTG-3'(서열번호 14) 및 5'-ACAGGACTTGCGGTACATGA-3'(서열번호 15)); 및 글리세르알데하이드 3-포스페이트 디하이드로제나제 특이적 프라이머(5'-CATGACCACAGTCCATGCCAT-3'(서열번호 16) 및 5'-AAGGCCATGCCAGTGAGCTTC-3'(서열번호 17))로 SYBR Green(PKT, 서울, 한국)를 사용하여 Real-time quantitative PCR을 수행하였다(어닐링 온도 58℃).
The entire RNA was extracted using TRIzol (Invitrogen), and complementary DNA was synthesized using reverse transcriptase (Bioneer, Daejeon, Korea). LYPDl specific primers (5'-CCCGAGTTCATTGTGAATTG-3 '(SEQ ID NO: 14) and 5'-ACAGGACTTGCGGTACATGA-3' (SEQ ID NO: 15)); And SYBR Green (PKT, Seoul, Korea) with the glyceraldehyde 3-phosphate dehydrogenase specific primer (5'-CATGACCACAGTCCATGCCAT-3 '(SEQ ID NO: 16) and 5'- AAGGCCATGCCAGTGAGCTTC- ) Was used to perform real-time quantitative PCR (annealing temperature 58 ° C).
실시예Example
6. 6.
웨스턴Western
블랏Blat
분석(Western blot analysis) Western blot analysis
세포를 RIPA buffer(10 mM Tris, pH 7.2, 150 mM NaCl, 1 % deoxycholate, 1 % Triton X-100, 0.1 % SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, 컴플릿 프로테아제 저해제)에서 용해시켰다. 세포 용해물을 변형된 브래드포드 분석법(Bradford assay; Bio-Rad Laboratories, Hercules, CA)으로 정량하였다. 세포 용해물 30 ㎍을 SDS 시료 버퍼와 혼합하여 가열하였고, 8-15 % SDS-PAGE 겔에 영동하였다. 분리된 단백질을 니트로셀룰로오스 막(nitrocellulose membrane)에 이동시키고, 5 % 스킴 밀크(skim milk)로 차단하였다. 이 후 anti-beta-actin, anti-cyclin E, anti-cyclin A(1:1000 희석; Santa Cruz), anti-phospho-ERK1/2, anti-ERK1/2, anti-phospho-JNK, anti-JNK, anti-phospho-ATF2, anti-ATF2, anti-c-Jun, anti-c-Jun, anti-cyclin D1(1:1000; Cell signaling technology), anti-flag, anti-vimentin(1:1000; Sigma)와 함께 반응시켰고, 홀스레디쉬 퍼올시데이즈(horseradish peroxidase)가 결합된 2차 항체와 반응시켰다. 이 후 ECL 키트(ECL Plus, Amersham, USA)를 제조사의 방법대로 처리하였다.
Cells were lysed in RIPA buffer (10 mM Tris, pH 7.2, 150 mM NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 1 mM sodium orthovanadate, 50 mM NaF, 1 mM PMSF, complete protease inhibitor) . Cell lysates were quantitated by the modified Bradford assay (Bio-Rad Laboratories, Hercules, CA). 30 μg of cell lysate was mixed with SDS sample buffer, heated, and subjected to 8-15% SDS-PAGE gels. The separated proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk. After that, anti-cyclin A, anti-cyclin A (1: 1000 dilution; Santa Cruz), anti-phospho-ERK1 / 2, anti-ERK1 / 2, anti-phospho-JNK, anti-JNK , anti-phospho-ATF2, anti-ATF2, anti-c-Jun, anti-c-Jun, anti-cyclin D1 (1: ) And reacted with a secondary antibody conjugated with horseradish peroxidase. The ECL kit (ECL Plus, Amersham, USA) was then processed according to the manufacturer's instructions.
실시예Example
7. 프로모터 리포터 분석(Promoter reporter assay) 7. Promoter reporter assay
리포펙타민 200(Invitrogen)으로 세포를 형질전환시켰다. 형질전환을 위해, 2×105세포를 6 웰 플레이트에 시딩하여 24 시간 배양 후, 2 ㎍ 리포터 플라스미드(AP-1 리포터 DNA; AP-1 cis-Reporting system, pAP-1-Luc; Stratagene) 및 1.8 ㎍ LYPD1 발현 벡터를 함께 형질전환시켰다. 형질 주입 후 48 시간 또는 72 시간 째에 반딧불 루시퍼라제 활성을 Dual-luciferase reporter assay system(Promega)를 통해 측정하였다. 함께 형질전환된 2 ㎍ 레닐라 루시퍼라제 벡터 pRL-TK(Promega)에 의해 코딩되는 레닐라 루시퍼라제 활성을 측정하여 형질전환 효율을 분석하였다.Cells were transformed with Lipofectamine 200 (Invitrogen). For transfection, 2 × 10 5 cells after the seeded in 6 well plates cultivation for 24 hours, 2 ㎍ reporter plasmid (AP-1 reporter DNA; AP-1 cis-Reporting system, pAP-1-Luc; Stratagene) , and 1.8 < RTI ID = 0.0 > pg < / RTI > LYPD1 expression vector. The activity of firefly luciferase was measured 48 hours or 72 hours after the transfection by Dual-luciferase reporter assay system (Promega). The transformation efficiency was analyzed by measuring the Renilla luciferase activity, which was coded by the 2 [mu] g Renilla luciferase vector pRL-TK (Promega) transformed together.
실시예Example
8. 대장암 세포주에서 8. In colorectal cancer cell lines
LYPD1LYPD1
의 발현을 Expression
siRNAsiRNA
로 억제한 결과 Suppressed by
실시예Example
8-1. 암세포의 침윤 및 세포 이동성 감소 8-1. Infiltration of cancer cells and decrease of cell mobility
SW480sub 세포주에 LYPD1 특이적 siRNA을 Microporation 기법으로 도입한지 48 시간 후 세포를 수확하여 Transwell을 통해 침윤 및 세포 이동성을 분석하였다.Forty - eight hours after the introduction of LYPD1 - specific siRNA into the SW480sub cell line by microporation technique, the cells were harvested and analyzed for invasion and cell migration through Transwell.
그 결과, 침윤은 약 34 %, 세포 이동성은 약 45 % 감소하였고, 양성 대조군으로 인터그린 알파 5 특이적 siRNA를 사용한 경우, 침윤은 약 29 %, 세포 이동성은 32 % 감소하였다(도 1).
As a result, the infiltration was reduced by about 34% and the cell mobility by about 45%. In the case of using intergreen alpha 5 -specific siRNA as a positive control, infiltration was reduced by about 29% and cell migration by 32% (FIG.
이를 통해 LYPD1의 발현을 억제하는 경우 인터그린 알파 5의 발현을 억제하는 것보다 대장암 세포의 침윤 및 세포 이동성이 더 높게 억제됨을 알 수 있으므로, 이는 본 발명의 LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 시사하는 것이다.
This suggests that suppression of LYPD1 expression inhibits the invasion and cell mobility of colon cancer cells more than suppression of intergreen alpha 5 expression, Suggesting that there is a significant effect on
실시예Example
8-2. 암세포의 세포 성장 감소 8-2. Reduced cell growth of cancer cells
SW480sub세포주에 LYPD1 특이적 siRNA을 microporation기법으로 도입한지 48 시간 후 세포를 수확하여, 96 웰 플레이트에 3000 cells/100 ㎕/웰로 깔고(10% FBS 포함 배지), 배양 후 48 시간 및 72 시간에 세포 성장을 비색 기법으로 측정하였다. CCK-8 시약을 10 ㎕를 넣고 2시간 후, OD450nm - OD650nm 값을 측정한 결과, LYPD1-siRNA(siLYPD1)에 의해 세포성장이 각각 34 % 및 46 % 감소함을 확인하였다(도 2).Cells were harvested 48 hours after introducing LYPD1-specific siRNA into SW480sub cell line by microporation technique. Cells were plated on 96-well plates at 3000 cells / 100 μl / well (medium containing 10% FBS) Growth was measured by colorimetric techniques. As a result of measuring the OD450nm-OD650nm value after 2 hours from adding 10 CC of CCK-8 reagent, it was confirmed that the cell growth was reduced by 34% and 46% by LYPD1-siRNA (siLYPD1) (Fig. 2).
또한, siRNA에 의한 LYPD1의 발현 억제를 real time quantitative PCR로 확인한 결과, LYPD1 발현이 51 % 감소됨을 확인하였다(도 3).
In addition, real-time quantitative PCR confirmed the inhibition of LYPD1 expression by siRNA, indicating that LYPD1 expression was reduced by 51% (FIG. 3).
그 결과, LYPD1의 발현이 억제되면 암세포의 성장이 감소함을 알 수 있었고, 이를 통해 본 발명의 LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 확인하였다.
As a result, it was found that the inhibition of the expression of LYPD1 reduced the growth of cancer cells. Thus, it was confirmed that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on the cancer treatment.
실시예Example
9. 9.
LYPD1LYPD1
의 발현을 증가시킨 결과And the expression of
실시예Example
9-1. 정상세포에서 9-1. In normal cells
LYPD1LYPD1
의 발현 정도 확인The expression level of
실시예 3에서 제작한 LYPD1 발현 벡터(p3XFlagCMV14-LYPD1 및 pFlagCMV1-LYPD1)의 LYPD1 발현 정도를 확인하기 위해, 상기 벡터를 정상 세포인 HEK293E 세포에 형질전환하고, 48 시간 후 용해하여 용해물 전체를 얻거나, 무혈청 배지로 바꾼 후 48 시간 동안 배양하여 조정 배지(conditioned medium)를 얻었다. 용해물 및 조정 배지를 anti-flag로 웨스턴 블랏 분석하였다(도 4).
In order to confirm the level of LYPD1 expression of the LYPD1 expression vectors (p3XFlagCMV14-LYPD1 and pFlagCMV1-LYPD1) prepared in Example 3, the vector was transformed into HEK293E cells as normal cells and after 48 hours, the whole lysate was obtained Or changed into serum-free medium, and cultured for 48 hours to obtain a conditioned medium. The lysates and conditioned media were analyzed by Western blot with anti-flag (Fig. 4).
그 결과, p3XFlagCMV14-LYPD1에 의해 LYPD1이 뚜렷하게 발현됨을 확인하였다. 또한, 조정 배지에서 signal이 검출됨을 확인하였고, 이를 통해 LYPD1의 전체 또는 일부분 영역이 세포 외로 분비 또는 쉐딩(shedding)됨을 알 수 있었다.
As a result, it was confirmed that LYPD1 was clearly expressed by p3XFlagCMV14-LYPD1. Also, it was confirmed that the signal was detected in the conditioned medium, indicating that all or part of LYPD1 was secreted or shedded outside the cell.
실시예Example
9-2. 암세포의 침윤능력의 증가 9-2. Increased invasive capacity of cancer cells
LYPD1 발현 증가에 따른 암세포의 침윤 능력의 변화를 분석하기 위해, SW480세포에 LYPD1 발현벡터(p3XFLAG-CMV14-LYPD1)를 일시적으로 형질전환한 후 48 시간 후에 세포를 수확하여 Transwell을 이용한 침윤실험을 수행한 결과, 침윤이 26 % 증가함을 확인하였다(도 5).
In order to analyze the change of invasion capacity of cancer cells according to the increase of LYPD1 expression, SW480 cells were transiently transfected with LYPD1 expression vector (p3XFLAG-CMV14-LYPD1), and after 48 hours, cells were harvested and infiltrated with Transwell As a result, infiltration increased by 26% (FIG. 5).
실시예Example
9-3. 주요 세포신호전달의 활성화 9-3. Activation of key cell signaling
또한, SW480 및 HEK293E 세포를 형질전환한 후, 48 시간 및 72 시간에 세포를 용해하여 웨스턴 블랏을 수행한 결과, 공통적으로 JNK의 인산화반응 및 전사인자 AP-1(activator protein 1)의 구성성분인 ATF-2의 인산화반응이 증가됨을 확인하였다(도 6). In addition, SW480 and HEK293E cells were transformed and the cells were lysed at 48 hours and 72 hours. Western blotting revealed that the phosphorylation of JNK and the constitutive component of transcription factor AP-1 (activator protein 1) It was confirmed that the phosphorylation reaction of ATF-2 was increased (FIG. 6).
그 결과, SW480에서는 간엽세포 마커인 비멘틴(vimentin)의 발현이 증가되었고, 또한 cyclin D1 및 cyclin A의 발현이 증가되었으며, HEK293E에서는 ATF-2 외에 AP-1의 구성성분인 c-Jun의 인산화반응 및 cyclin E, cyclin A의 발현이 증가되었음을 알 수 있었다. As a result, expression of cyclin D1 and cyclin A was increased in SW480, and expression of cyclin D1 and cyclin A was increased. In addition to ATF-2 in HEK293E, phosphorylation of c-Jun, a component of AP-1 And the expression of cyclin E and cyclin A was increased.
이는 LYPD1의 세포성장 및 세포 이동성을 조절하는 경로와 관련 있는 세포신호전달 경로를 시사하는 것이다.
This suggests a cell signaling pathway associated with pathways regulating LYPD1 cell growth and cell migration.
실시예Example
10. 10.
LYPD1LYPD1
의 발현에 따른 AP-1 reporter 활성 조절Regulation of AP-1 reporter activity
HEK293E 세포주에 LYPD1 발현 벡터 또는 empty vector(p3XFLAG-CMV14)를 LF2000을 이용하여 도입한 지 48 시간 후, 루시퍼라제 분석법을 통해 전사인자 AP-1의 활성 변화를 확인하였다(도 7).After 48 hours of introduction of the LYPD1 expression vector or empty vector (p3XFLAG-CMV14) into the HEK293E cell line using LF2000, the activity of the transcription factor AP-1 was confirmed through the luciferase assay (Fig. 7).
그 결과, LYPD1 발현에 의해 AP-1의 활성이 증가함을 확인하였다(48 시간째 2배 증가, 72 시간째 45% 증가). 이러한 결과는 웨스턴 블랏 분석에서, LYPD1 발현에 의한 phospho-c-Jun 등의 증가와 일치하는 결과이다.
As a result, it was confirmed that the activity of AP-1 was increased by LYPD1 expression (2-fold increase at 48 hours and 45% increase at 72 hours). These results are consistent with the increase of phospho-c-Jun by LYPD1 expression in Western blot analysis.
이를 통해, AP-1 활성은 암세포 활성(성장 및 침윤)과 밀접한 관련이 있으므로, LYPD1 발현에 의한 AP-1 활성 조절이 침윤/세포 이동성 및 세포성장 조절에 기여함을 알 수 있었다.
As a result, AP-1 activity is closely related to cancer cell activity (growth and invasion), so that the regulation of AP-1 activity by LYPD1 expression contributes to invasion / cell migration and cell growth regulation.
결론적으로, 대장암세포를 포함한 다양한 암세포에서 본 발명의 LYPD1의 발현이 감소되면, 그에 따라 AP-1 활성도 감소되어 암세포의 침윤/세포 이동성 및 세포성장이 감소됨을 알 수 있었다. 즉, 본 발명의 LYPD1은 다양한 암세포의 침윤/세포 이동성 및 세포성장과 관련된 암 진단 마커로서, LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 알 수 있었다.
In conclusion, when the expression of LYPD1 of the present invention is decreased in various cancer cells including colon cancer cells, AP-1 activity is decreased, and the infiltration / cell mobility and cell growth of cancer cells are decreased. That is, LYPD1 of the present invention is a cancer diagnostic marker related to invasion / cell mobility and cell growth of various cancer cells, and it was found that inhibiting the expression of LYPD1 has a remarkable effect on cancer treatment.
실시예Example
11. 전립선암 세포주에서 11. Prostate cancer cell lines
LYPD1LYPD1
의 발현을 Expression
siRNAsiRNA
로 억제한 결과 Suppressed by
실시예Example
11-1. 대장암 세포주 및 전립선암 세포주에서 11-1. In colorectal cancer cell lines and prostate cancer cell lines
LYPD1LYPD1
의 발현 분석Expression analysis
다양한 암세포주에서 LYPD1 유전자의 발현을 확인하기 위하여 상기 실시예 1의 조건으로 배양한 다양한 세포주로부터 Trizol(Invitrogen)을 이용하여 전체 RNA를 분리하고, 이를 이용하여 cDNA를 합성하였다. 또한, 상기 실시예 5의 real time quantitative PCR 을 수행하였다. In order to confirm the expression of LYPD1 gene in various cancer cell lines, total RNA was isolated from various cell lines cultured under the conditions of Example 1 using Trizol (Invitrogen), and cDNA was synthesized using this. Also, the real time quantitative PCR of Example 5 was performed.
그 결과, 대장암 세포주 및 전립선암 세포주에서 LYPD1의 발현수준이 다양함을 확인하였다(도 8).
As a result, it was confirmed that the expression level of LYPD1 was varied in the colon cancer cell line and the prostate cancer cell line (FIG. 8).
실시예Example
11-2. 암세포의 세포 성장 감소 11-2. Reduced cell growth of cancer cells
PC3 세포주에 LYPD1 특이적 siRNA을 microporation기법으로 도입한지 48 시간 후 세포를 수확하여, 96 웰 플레이트에 3000 cells/100 ㎕/웰로 깔고(10% FBS 포함 배지), 배양 후 48 시간 및 72 시간에 세포 성장을 비색 기법으로 측정하였다. CCK-8 시약을 10 ㎕를 넣고 2시간 후, OD450nm - OD650nm 값을 측정한 결과, LYPD1-siRNA(siLYPD1)에 의해 세포성장이 각각 21 % 및 27 % 감소함을 확인하였다(도 9). Cells were harvested 48 hours after introducing LYPD1-specific siRNA into the PC3 cell line by microporation technique. Cells were plated in 96-well plates at 3000 cells / 100 μl / well (medium containing 10% FBS) Growth was measured by colorimetric techniques. As a result of measuring the OD450 nm-OD650 nm value after 2 hours from adding 10 μl of CCK-8 reagent, it was confirmed that cell growth was reduced by 21% and 27% by LYPD1-siRNA (siLYPD1), respectively (FIG.
또한, siRNA에 의한 LYPD1의 발현 억제를 real time quantitative PCR로 확인한 결과, LYPD1 발현이 70 % 감소됨을 확인하였다(도 10).
In addition, the inhibition of LYPD1 expression by siRNA was confirmed by real time quantitative PCR, and LYPD1 expression was reduced by 70% (FIG. 10).
이를 통해, LYPD1의 발현이 억제되면 전립선암 세포의 성장이 감소함을 알 수 있었고, 이를 통해 본 발명의 LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 확인하였다.
As a result, it was found that the inhibition of LYPD1 expression decreased the growth of prostate cancer cells. Thus, it was confirmed that the inhibition of LYPD1 expression of the present invention had a remarkable effect on cancer treatment.
실시예Example
11-3. 암세포의 이동성 감소 11-3. Reduced mobility of cancer cells
PC3 세포주에 LYPD1 특이적 siRNA을 Microporation 기법으로 도입한지 48 시간 후 세포를 수확하여 Transwell을 통해 세포의 이동성을 분석하였다.Cells were harvested 48 hours after introduction of LYPD1 specific siRNA into PC3 cell line by Microporation technique and cell mobility was analyzed through Transwell.
그 결과, 세포 이동성은 약 21 % 감소하였다(도 11).
As a result, cell mobility decreased by about 21% (Fig. 11).
이를 통해, LYPD1의 발현을 억제하는 경우 전립선암 세포의 이동성이 매우 높게 억제됨을 알 수 있으므로, 이는 본 발명의 LYPD1의 발현을 억제하는 것이 암 치료에 현저한 효과가 있음을 시사하는 것이다.
Thus, it can be seen that when the expression of LYPD1 is inhibited, the mobility of prostate cancer cells is highly suppressed, suggesting that inhibiting the expression of LYPD1 of the present invention has a remarkable effect on cancer treatment.
이상의 설명으로부터, 본 발명이 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며, 한정적인 것이 아닌 것으로서 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허청구범위의 의미 및 범위, 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, as well as all changes or modifications derived from the meaning and scope of the appended claims and their equivalents.
<110> Korea Research Institute of Bioscience and Biotechnology <120> Use of LYPD1 for cancer diagnostic or therapeutic marker <130> KPA140674-KR-P1 <150> KR 10-2014-0105989 <151> 2014-08-14 <160> 17 <170> KopatentIn 2.0 <210> 1 <211> 2683 <212> DNA <213> Homo sapiens LYPD1 <400> 1 agggcggtgt caatgcaccc tccagcggtg cgcgcaggcg ggagaaggga gggcggcccg 60 ggcaagtgag acagttaagg cagtgtcccc accacacccc cacccagatt ggccacgccg 120 agctggttct tgacagaagg ccttcgcgga ggaagagggg gcacagctgc acaggacacc 180 ctacggagcc tgcgggcgtg gaactttgcc aggcgcacgg gaacgcgcgc ccttcctgtc 240 agcctcccgg ggcgccaggc tcccgcggcc cgcagcggga cagcctcagt tgtgtgggct 300 ggacccagtc gctggggtac cgaccagtcc tggaaggcgc agaggacgtg gagtggggag 360 gctgccttcc tatgtgcgaa gggccagccg ggcacgcagt cctcagaccc tagtccgcac 420 ccggcaggtc cccacggcac ctgctgcgcc ctcctcgccg ctcccccaac ctccccatct 480 cagaaaacta ccagttctct cccgcccccc ggcgcccctt tcccaggaac gtgcggaggc 540 gggagaagag gaagacagga agggggtggg gatgtgaagc gaccgtccca gccttccccg 600 cccgccaccc ccaccccaac tcggcagccg tcacgtgatg cctggagtgg gaggtgggga 660 gaaaaggcga gacttttgtg ggtgctcccg atcgccagta gttccttcag tctcagccgc 720 caactccgga ggcgcggtgc tcggcccggg agcgcgagcg ggaggagcag agacccgcag 780 ccgggagccc gagcgcgggc gatgcaggct ccgcgagcgg cacctgcggc tcctctaagc 840 tacgaccgtc gtctccgcgg cagcagcgcg ggccccagca gcctcggcag ccacagccgc 900 tgcagccggg gcagcctccg ctgctgtcgc ctcctctgat gcgcttgccc tctcccggcc 960 ccgggactcc gggagaatgt gggtcctagg catcgcggca actttttgcg gattgttctt 1020 gcttccaggc tttgcgctgc aaatccagtg ctaccagtgt gaagaattcc agctgaacaa 1080 cgactgctcc tcccccgagt tcattgtgaa ttgcacggtg aacgttcaag acatgtgtca 1140 gaaagaagtg atggagcaaa gtgccgggat catgtaccgc aagtcctgtg catcatcagc 1200 ggcctgtctc atcgcctctg ccgggtacca gtccttctgc tccccaggga aactgaactc 1260 agtttgcatc agctgctgca acacccctct ttgtaacggg ccaaggccca agaaaagggg 1320 aagttctgcc tcggccctca ggccagggct ccgcaccacc atcctgttcc tcaaattagc 1380 cctcttctcg gcacactgct gaagctgaag gagatgccac cccctcctgc attgttcttc 1440 cagccctcgc ccccaacccc ccacctccct gagtgagttt cttctgggtg tccttttatt 1500 ctgggtaggg agcgggagtc cgtgttctct tttgttcctg tgcaaataat gaaagagctc 1560 ggtaaagcat tctgaataaa ttcagcctga ctgaattttc agtatgtact tgaaggaagg 1620 aggtggagtg aaagttcacc cccatgtctg tgtaaccgga gtcaaggcca ggctggcaga 1680 gtcagtcctt agaagtcact gaggtgggca tctgcctttt gtaaagcctc cagtgtccat 1740 tccatccctg atgggggcat agtttgagac tgcagagtga gagtgacgtt ttcttagggc 1800 tggagggcca gttcccactc aaggctccct cgcttgacat tcaaacttca tgctcctgaa 1860 aaccattctc tgcagcagaa ttggctggtt tcgcgcctga gttgggctct agtgactcga 1920 gactcaatga ctgggactta gactggggct cggcctcgct ctgaaaagtg cttaagaaaa 1980 tcttctcagt tctccttgca gaggactggc gccgggacgc gaagagcaac gggcgctgca 2040 caaagcgggc gctgtcggtg gtggagtgcg catgtacgcg caggcgcttc tcgtggttgg 2100 cgtgctgcag cgacaggcgg cagcacagca cctgcacgaa cacccgccga aactgctgcg 2160 aggacaccgt gtacaggagc gggttgatga ccgagctgag gtagaaaaac gtctccgaga 2220 aggggaggag gatcatgtac gcccggaagt aggacctcgt ccagtcgtgc ttgggtttgg 2280 ccgcagccat gatcctccga atctggttgg gcatccagca tacggccaat gtcacaacaa 2340 tcagccctgg gcagacacga gcaggaggga gagacagaga aaagaaaaac acagcatgag 2400 aacacagtaa atgaataaaa ccataaaata tttagcccct ctgttctgtg cttactggcc 2460 aggaaatggt accaattttt cagtgttgga cttgacagct tcttttgcca caagcaagag 2520 agaatttaac actgtttcaa acccggggga gttggctgtg ttaaagaaag accattaaat 2580 gctttagaca gtgtatttat accagttgat gtctgttaat tttaaaaaaa tgttttcatt 2640 ggtgtttgtt tgcgtatcca gaaagcagtt catgttatcc ata 2683 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 gcacacugcu gaacugaag 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <400> 3 gaacguucaa gacaugugu 19 <210> 4 <211> 19 <212> RNA <213> Homo sapiens <400> 4 ggagcaaagu gccgggauc 19 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 caaguccugu gcaucauca 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> integrin alpha5-specific siRNA <400> 6 ggaccaaggc agaaggcag 19 <210> 7 <211> 141 <212> PRT <213> Homo sapiens LYPD1 <400> 7 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu Leu 1 5 10 15 Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu Phe Gln 20 25 30 Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn Cys Thr Val 35 40 45 Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu Gln Ser Ala Gly 50 55 60 Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala Ala Cys Leu Ile Ala 65 70 75 80 Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly Lys Leu Asn Ser Val 85 90 95 Cys Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn Gly Pro Arg Pro Lys 100 105 110 Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg Pro Gly Leu Arg Thr Thr 115 120 125 Ile Leu Phe Leu Lys Leu Ala Leu Phe Ser Ala His Cys 130 135 140 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 forward primer <400> 8 aacccaagct tgccgccatg tgggtcctag gcatcg 36 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 reverse primer <400> 9 aacccggatc cgcagtgtgc cgagaagagg 30 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide_forward primer <400> 10 aagcccaagc ttatccagtg ctaccagtgt gaag 34 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide_reverse primer <400> 11 aacccggatc cctatcagca gtgtgccgag aag 33 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 sense strand <400> 12 cuuacgcuga guacuucga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 anti-sense strand <400> 13 ucgaaguacu cagcguaag 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 14 cccgagttca ttgtgaattg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 15 acaggacttg cggtacatga 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 16 catgaccaca gtccatgcca t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 17 aaggccatgc cagtgagctt c 21 <110> Korea Research Institute of Bioscience and Biotechnology <120> Use of LYPD1 for cancer diagnostic or therapeutic marker <130> KPA140674-KR-P1 <150> KR 10-2014-0105989 <151> 2014-08-14 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 2683 <212> DNA <213> Homo sapiens LYPD1 <400> 1 agggcggtgt caatgcaccc tccagcggtg cgcgcaggcg ggagaaggga gggcggcccg 60 ggcaagtgag acagttaagg cagtgtcccc accacacccc cacccagatt ggccacgccg 120 agctggttct tgacagaagg ccttcgcgga ggaagagggg gcacagctgc acaggacacc 180 ctacggagcc tgcgggcgtg gaactttgcc aggcgcacgg gaacgcgcgc ccttcctgtc 240 agcctcccgg ggcgccaggc tcccgcggcc cgcagcggga cagcctcagt tgtgtgggct 300 ggacccagtc gctggggtac cgaccagtcc tggaaggcgc agaggacgtg gagtggggag 360 gctgccttcc tatgtgcgaa gggccagccg ggcacgcagt cctcagaccc tagtccgcac 420 ccggcaggtc cccacggcac ctgctgcgcc ctcctcgccg ctcccccaac ctccccatct 480 cagaaaacta ccagttctct cccgcccccc ggcgcccctt tcccaggaac gtgcggaggc 540 gggagaagag gaagacagga agggggtggg gatgtgaagc gaccgtccca gccttccccg 600 cccgccaccc ccaccccaac tcggcagccg tcacgtgatg cctggagtgg gaggtgggga 660 gaaaaggcga gacttttgtg ggtgctcccg atcgccagta gttccttcag tctcagccgc 720 caactccgga ggcgcggtgc tcggcccggg agcgcgagcg ggaggagcag agacccgcag 780 ccgggagccc gagcgcgggc gatgcaggct ccgcgagcgg cacctgcggc tcctctaagc 840 tacgaccgtc gtctccgcgg cagcagcgcg ggccccagca gcctcggcag ccacagccgc 900 tgcagccggg gcagcctccg ctgctgtcgc ctcctctgat gcgcttgccc tctcccggcc 960 ccgggactcc gggagaatgt gggtcctagg catcgcggca actttttgcg gattgttctt 1020 gcttccaggc tttgcgctgc aaatccagtg ctaccagtgt gaagaattcc agctgaacaa 1080 cgactgctcc tcccccgagt tcattgtgaa ttgcacggtg aacgttcaag acatgtgtca 1140 gaaagaagtg atggagcaaa gtgccgggat catgtaccgc aagtcctgtg catcatcagc 1200 ggcctgtctc atcgcctctg ccgggtacca gtccttctgc tccccaggga aactgaactc 1260 agtttgcatc agctgctgca acacccctct ttgtaacggg ccaaggccca agaaaagggg 1320 aagttctgcc tcggccctca ggccagggct ccgcaccacc atcctgttcc tcaaattagc 1380 cctcttctcg gcacactgct gaagctgaag gagatgccac cccctcctgc attgttcttc 1440 cagccctcgc ccccaacccc ccacctccct gagtgagttt cttctgggtg tccttttatt 1500 ctgggtaggg agcgggagtc cgtgttctct tttgttcctg tgcaaataat gaaagagctc 1560 ggtaaagcat tctgaataaa ttcagcctga ctgaattttc agtatgtact tgaaggaagg 1620 aggtggagtg aaagttcacc cccatgtctg tgtaaccgga gtcaaggcca ggctggcaga 1680 gtcagtcctt agaagtcact gaggtgggca tctgcctttt gtaaagcctc cagtgtccat 1740 tccatccctg atgggggcat agtttgagac tgcagagtga gagtgacgtt ttcttagggc 1800 tggagggcca gttcccactc aaggctccct cgcttgacat tcaaacttca tgctcctgaa 1860 aaccattctc tgcagcagaa ttggctggtt tcgcgcctga gttgggctct agtgactcga 1920 gactcaatga ctgggactta gactggggct cggcctcgct ctgaaaagtg cttaagaaaa 1980 tcttctcagt tctccttgca gaggactggc gccgggacgc gaagagcaac gggcgctgca 2040 caaagcgggc gctgtcggtg gtggagtgcg catgtacgcg caggcgcttc tcgtggttgg 2100 cgtgctgcag cgacaggcgg cagcacagca cctgcacgaa cacccgccga aactgctgcg 2160 aggacaccgt gtacaggagc gggttgatga ccgagctgag gtagaaaaac gtctccgaga 2220 aggggaggag gatcatgtac gcccggaagt aggacctcgt ccagtcgtgc ttgggtttgg 2280 ccgcagccat gatcctccga atctggttgg gcatccagca tacggccaat gtcacaacaa 2340 tcagccctgg gcagacacga gcaggaggga gagacagaga aaagaaaaac acagcatgag 2400 aacacagtaa atgaataaaa ccataaaata tttagcccct ctgttctgtg cttactggcc 2460 aggaaatggt accaattttt cagtgttgga cttgacagct tcttttgcca caagcaagag 2520 agaatttaac actgtttcaa acccggggga gttggctgtg ttaaagaaag accattaaat 2580 gctttagaca gtgtatttat accagttgat gtctgttaat tttaaaaaaa tgttttcatt 2640 ggtgtttgtt tgcgtatcca gaaagcagtt catgttatcc ata 2683 <210> 2 <211> 19 <212> RNA <213> Homo sapiens <400> 2 gcacacugcu gaacugaag 19 <210> 3 <211> 19 <212> RNA <213> Homo sapiens <400> 3 gaacguucaa gacaugugu 19 <210> 4 <211> 19 <212> RNA <213> Homo sapiens <400> 4 ggagcaaagu gccgggauc 19 <210> 5 <211> 19 <212> RNA <213> Homo sapiens <400> 5 caaguccugu gcaucauca 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> integrin alpha5-specific siRNA <400> 6 ggaccaaggc agaaggcag 19 <210> 7 <211> 141 <212> PRT <213> Homo sapiens LYPD1 <400> 7 Met Trp Val Leu Gly Ile Ala Ala Thr Phe Cys Gly Leu Phe Leu Leu 1 5 10 15 Pro Gly Phe Ala Leu Gln Ile Gln Cys Tyr Gln Cys Glu Glu Phe Gln 20 25 30 Leu Asn Asn Asp Cys Ser Ser Pro Glu Phe Ile Val Asn Cys Thr Val 35 40 45 Asn Val Gln Asp Met Cys Gln Lys Glu Val Met Glu Gln Ser Ala Gly 50 55 60 Ile Met Tyr Arg Lys Ser Cys Ala Ser Ser Ala Ala Cys Leu Ile Ala 65 70 75 80 Ser Ala Gly Tyr Gln Ser Phe Cys Ser Pro Gly Lys Leu Asn Ser Val 85 90 95 Cys Ile Ser Cys Cys Asn Thr Pro Leu Cys Asn Gly Pro Arg Pro Lys 100 105 110 Lys Arg Gly Ser Ser Ala Ser Ala Leu Arg Pro Gly Leu Arg Thr Thr 115 120 125 Ile Leu Phe Leu Lys Leu Ala Leu Phe Ser Ala His Cys 130 135 140 <210> 8 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 forward primer <400> 8 aacccaagct tgccgccatg tgggtcctag gcatcg 36 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 reverse primer <400> 9 aacccggatc cgcagtgtgc cgagaagagg 30 <210> 10 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide forward primer <400> 10 aagcccaagc ttatccagtg ctaccagtgt gaag 34 <210> 11 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> LYPD1 except signal peptide_reverse primer <400> 11 aacccggatc cctatcagca gtgtgccgag aag 33 <210> 12 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 sense strand <400> 12 cuuacgcuga guacuucga 19 <210> 13 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> siGL3 anti-sense strand <400> 13 ucgaaguacu cagcguaag 19 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 14 cccgagttca ttgtgaattg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> LYPD1-specific primer <400> 15 acaggacttg cggtacatga 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 16 catgaccaca gtccatgcca t 21 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> glyceraldehyde 3-phosphate dehydrogenase-specific primer <400> 17 aaggccatgc cagtgagctt c 21
Claims (16)
A pharmaceutical composition for preventing or treating cancer, which comprises an inhibitor of expression of LYPD1 (LY6 / PLAUR domain containing 1) gene, an inhibitor of activity of LYPD1 protein, or a mixture thereof as an active ingredient.
The composition according to claim 1, wherein the LYPD1 gene expression inhibitor is selected from the group consisting of antisense oligonucleotides, siRNA, shRNA and microRNA of the LYPD1 gene.
3. The composition according to claim 2, wherein the LYPD1 gene expression inhibitor binds to at least one sequence selected from the group consisting of SEQ ID NOS: 2-5.
The composition of claim 1, wherein the activity inhibitor is an antibody that specifically binds to a LYPD1 protein.
A health functional food for preventing or ameliorating cancer, which comprises an inhibitor of expression of LYPD1 gene, an inhibitor of activity of LYPD1 protein, or a mixture thereof as an active ingredient.
A composition for diagnosing cancer, comprising an agent for measuring the level of LYPD1 gene mRNA or a protein thereof.
7. The composition for cancer diagnosis according to claim 6, wherein the agent for measuring the level of the gene mRNA comprises a primer or a probe specifically binding to the gene.
7. The composition according to claim 6, wherein the agent for measuring the level of the protein comprises an antibody specific to the protein.
7. The composition according to claim 6, wherein the cancer is colon cancer or prostate cancer.
A cancer diagnostic kit comprising the composition of claim 6.
[Claim 11] The cancer diagnostic kit according to claim 10, wherein the kit is an RT-PCR kit, a DNA chip kit or a protein chip kit.
(b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 정상 대조군 시료의 유전자의 mRNA 또는 이의 단백질의 수준보다 높은지 여부를 비교하는 단계를 포함하는, 암 진단을 위한 정보를 제공하는 방법.
(a) measuring the level of mRNA or a protein thereof of the LYPD1 gene from a separate sample of a subject suspected of having cancer; And
(b) comparing the level of the mRNA of the gene or the protein thereof to a level of the mRNA of the gene of the normal control sample or a protein thereof.
The method according to claim 12, wherein the step of measuring the mRNA level of the LYPD1 gene comprises the steps of: RT-PCR; competitive RT-PCR; real-time reverse transcriptase polymerase wherein the cancer is measured by quantitative RT-PCR, RNase protection method, Northern blotting or a gene chip.
13. The method according to claim 12, wherein the step of measuring the level of the mRNA of the LYPD1 gene uses a primer or a probe specifically binding to the LYPD1 gene.
13. The method according to claim 12, wherein the step of measuring the level of the protein of the LYPD1 gene uses an antibody that specifically binds to the protein.
(b) 상기 유전자의 mRNA 또는 이의 단백질의 수준이 상기 시료를 처리하지 않은 대조군 시료의 수준보다 낮은 경우 암 치료제로 선택하는 단계를 포함하는, 암 치료제의 스크리닝 방법. (a) treating a candidate substance for cancer treatment to a separate sample expressing the LYPD1 gene, and then measuring the level of the mRNA or the protein thereof of the LYPD1 gene; And
(b) selecting a cancer therapeutic agent when the level of the mRNA or the protein of the gene is lower than that of the control sample not treated with the sample.
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WO2019146729A1 (en) * | 2018-01-25 | 2019-08-01 | 学校法人東京女子医科大学 | Angiogenesis inhibitor and screening method for angiogeneis inhibitors |
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WO2018164141A1 (en) * | 2017-03-06 | 2018-09-13 | 学校法人東京女子医科大学 | Lypd1 inhibitor and method for producing biological tissue using same |
WO2019146729A1 (en) * | 2018-01-25 | 2019-08-01 | 学校法人東京女子医科大学 | Angiogenesis inhibitor and screening method for angiogeneis inhibitors |
JPWO2019146729A1 (en) * | 2018-01-25 | 2021-02-04 | 学校法人東京女子医科大学 | Screening method for angiogenesis inhibitor and angiogenesis inhibitor |
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