KR20210003502A - A composition for preventing, improving or treating for cancer - Google Patents
A composition for preventing, improving or treating for cancer Download PDFInfo
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- KR20210003502A KR20210003502A KR1020190079353A KR20190079353A KR20210003502A KR 20210003502 A KR20210003502 A KR 20210003502A KR 1020190079353 A KR1020190079353 A KR 1020190079353A KR 20190079353 A KR20190079353 A KR 20190079353A KR 20210003502 A KR20210003502 A KR 20210003502A
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Abstract
Description
본 발명은 암의 예방, 개선 또는 치료용 조성물에 관한 것이다.The present invention relates to a composition for preventing, improving or treating cancer.
암은 현재 전세계적으로 가장 많은 사망자를 내는 질병 중 하나로서, 평균 수명의 연장과 암 발생 연령이 낮아짐에 의해 암 발생률은 계속적으로 증가되고 있는 추세이다. 한국의 국립 암센터(National Cancer Center)에서 제공하는 2013년 통계 자료에 따르면, 2010년 암 등록 통계과에 등록된 우리 나라 암 발생 환자는 모두 202,053 명이며 계속적으로 증가되는 추세이다.Cancer is one of the most fatal diseases in the world at present, and the incidence of cancer continues to increase due to the prolongation of life expectancy and the decrease in the age of cancer. According to 2013 statistics provided by the National Cancer Center in Korea, the number of cancer-causing patients in Korea registered in the Department of Cancer Registration and Statistics in 2010 was 202,053, and the trend is increasing continuously.
상기 암이 고형 장기에 발생된 경우에는 전통적으로 외과적 절제술에 의해 치료하였다. 그러나, 환부가 너무 크거나, 고형 장기가 아닌 절제술이 불가능한 부분에 암이 발생된 경우 등에는 외과적 절제술이 불가능하여 경구 또는 주사제 등을 이용한 항암 화학요법(Chemotherapy)이 사용되었다. 상기 항암 화학요법에 사용되는 항암제는 주로 단분자 물질을 유기적 또는 무기적 방법을 이용하여 합성하여 사용하였다. 암이 주로 세포신호 전달체계에 포함된 인산 활성 인자 단백질의 과발현 등에 의해 세포신호 전달체계를 교란시키는 단백질에 효과적으로 결합하여, 단백질의 활성을 억제하는 용도로 개발되어 사용되었다. 그러나, 이러한 방식의 항암 화학요법은 머리카락과 같은 세포주기가 빠른 정상 세포의 성장을 저해하거나, 구강 건조, 구내염, 오심, 구토, 설사, 호중구 감소증 등과 같은 다양한 부작용을 야기할 수 있다는 문제점이 존재한다.When the cancer has occurred in solid organs, it has been traditionally treated by surgical resection. However, in cases where the affected area is too large or cancer occurs in a non-solid organ that cannot be resected, surgical resection is impossible, so chemotherapy using oral or injection drugs has been used. Anticancer agents used in the anticancer chemotherapy were mainly synthesized using monomolecular substances using organic or inorganic methods. Cancer has been developed and used for the purpose of inhibiting the activity of proteins by effectively binding to proteins that disturb the cellular signaling system by overexpression of phosphate activating factor proteins included in the cellular signaling system. However, this type of chemotherapy has a problem in that it can inhibit the growth of normal cells with fast cell cycles such as hair, or cause various side effects such as dry mouth, stomatitis, nausea, vomiting, diarrhea, and neutropenia. .
이와 같은 항암 화학요법의 문제점에 따라 다양한 방면으로 항암 화학요법에 사용되는 물질의 연구가 진행되고 있다. 일 예시로, siRNA(small interfering RNA)가 존재한다. 상기 siRNA는 리보핵산단백질과 RISC(RNA induced silencing complex)를 이뤄 siRNA에 상보적인 서열을 갖는 mRNA(messenger RNA)를 절단하도록 함으로써, mRNA로부터 단백질이 생성되는 것을 억제한다. 상기 siRNA 기반의 항암제는 단백질 생성 단계 이전의 mRNA를 차단한다는 점, 그리고 RNA와 세포 내재적인 RISC 시스템을 활용한다는 점에 있어서 상기 단분자 물질의 항암제보다 진보한 기술로 평가되고 있다. 그러나, siRNA 기반의 항암제는 비 표적 효과(off-target effect)와 같은 현상에 의해 나타나는 부작용이 존재한다는 기술적 한계점이 존재한다.In accordance with the problems of such anticancer chemotherapy, research on substances used in anticancer chemotherapy in various ways is underway. As an example, there is siRNA (small interfering RNA). The siRNA forms a ribonucleic acid protein and an RNA induced silencing complex (RISC) to cleave a messenger RNA (mRNA) having a sequence complementary to the siRNA, thereby inhibiting the protein from being produced from the mRNA. The siRNA-based anticancer agent is evaluated as a more advanced technology than the monomolecular anticancer agent in that it blocks the mRNA before the protein production stage, and that it utilizes the RNA and cell-intrinsic RISC system. However, there is a technical limitation that siRNA-based anticancer drugs have side effects caused by phenomena such as off-target effects.
한편, 이와 같은 현상의 대안으로 세포 분화, 증식, 세포사멸, 발생, 면역, 물질대사 및 줄기세포 유지 등과 같은 많은 생물학적 과정을 통제하는 miRNA에 대한 연구가 활발하게 진행되고 있다. 특히 miRNA는 펩타이드나 항체 치료제와 달리 개발 기간이 짧고, 확실한 질환 표적 유전자만 밝혀진다면 이론적으로 모두 치료제를 개발할 수 있다는 장점이 있다. 또한, miRNA는 일반적으로 1개가 아닌 복수의 표적 유전자의 발현을 동시에 조절함으로 매우 높은 특이성과 효능을 가지고 있다. 따라서 항암 화학요법에 사용되는 항암제가 갖는 상기 언급된 부작용들을 극복하기 위한 신규한 miRNA로 이루어진 항암제에 대한 연구가 절실히 필요한 상황이다.Meanwhile, as an alternative to this phenomenon, studies on miRNAs that control many biological processes such as cell differentiation, proliferation, apoptosis, development, immunity, metabolism, and stem cell maintenance are actively being conducted. In particular, unlike peptide or antibody therapeutics, miRNAs have a short development period, and theoretically all therapeutics can be developed if only certain disease target genes are identified. In addition, miRNAs generally have very high specificity and efficacy by simultaneously regulating the expression of multiple target genes rather than one. Therefore, there is an urgent need for research on anticancer drugs made of novel miRNAs to overcome the aforementioned side effects of anticancer drugs used in anticancer chemotherapy.
본 발명의 일 목적은 암의 예방, 개선 또는 치료용 조성물을 제공하는 것이다.One object of the present invention is to provide a composition for preventing, improving or treating cancer.
본 발명의 다른 목적은 암 치료제 스크리닝 방법을 제공하는 것이다.Another object of the present invention is to provide a method for screening a cancer therapeutic agent.
그러나 본 발명이 이루고자 하는 기술적 과제는 이상에서 언급한 과제에 제한되지 않으며, 언급되지 않은 또 다른 과제들은 아래의 기재로부터 당 업계에서 통상의 지식을 가진 자에게 명확하게 이해될 수 있을 것이다.However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned may be clearly understood by those of ordinary skill in the art from the following description.
본 발명의 일 구현 예에서는 암의 예방, 개선 또는 치료용 조성물을 제공한다.One embodiment of the present invention provides a composition for preventing, improving or treating cancer.
본 발명의 상기 조성물은 약학 조성물, 식품 조성물 및 화장료 조성물로 제공될 수 있다.The composition of the present invention may be provided as a pharmaceutical composition, a food composition, and a cosmetic composition.
본 발명의 상기 조성물은 miR-4516 또는 이의 모방체(mimic)를 유효성분으로 포함한다.The composition of the present invention contains miR-4516 or a mimic thereof as an active ingredient.
본 발명의 상기 miR-4516은 miRNA로서, 인간을 포함하는 동물, 예를 들면 원숭이, 침팬지, 돼지, 말, 소, 양, 개, 고양이, 생쥐, 토끼 등으로부터 유래된 것일 수 있고, 바람직하게는 인간으로부터 유래된 것일 수 있다.The miR-4516 of the present invention is a miRNA, and may be derived from animals including humans, for example, monkeys, chimpanzees, pigs, horses, cows, sheep, dogs, cats, mice, rabbits, etc., preferably It may be of human origin.
본 발명의 상기 miR-4516은 단일 가닥 또는 이중 가닥 형태로 존재할 수 있다. 성숙 miRNA 분자는 주로 단일 가닥으로 존재하지만, 전구체 miRNA 분자는 이중 가닥을 형성할 수 있는 부분적인 자가-상보적 인 구조(예를 들어 스템-루프 구조)를 포함할 수 있다. 또한, 본 발명의 miRNA는 RNA, PNA(peptide nucleic acids) 또는 LNA(locked nucleic acid) 같은 형태로 구성될 수 있다.The miR-4516 of the present invention may exist in a single-stranded or double-stranded form. Mature miRNA molecules exist primarily as single strands, but precursor miRNA molecules may contain partial self-complementary structures (eg stem-loop structures) capable of forming double strands. In addition, the miRNA of the present invention may be composed of RNA, peptide nucleic acids (PNA), or locked nucleic acid (LNA).
본 발명의 상기 miR-4516의 모방체는 전구체 miRNA로서, 이중 가닥으로 구성된 것일 수 있으나, 이에 제한되는 것은 아니다.The mimic of miR-4516 of the present invention is a precursor miRNA, and may be composed of double strands, but is not limited thereto.
본 발명의 상기 miR-4516이 단일 가닥인 경우에는 서열번호 1로 표시되는 염기 서열로 이루어진 것일 수 있고, 상기 miR-4516이 이중 가닥인 경우에는 서열번호 1과, 상기 서열번호 1과 상보적인 염기 서열인 서열번호 2가 상보적으로 결합되어 있는 형태로 이루어진 것일 수 있으나, 이에 제한되는 것은 아니다.When the miR-4516 of the present invention is single-stranded, it may consist of a nucleotide sequence represented by SEQ ID NO: 1, and when the miR-4516 is double-stranded, it is a base that is complementary to SEQ ID NO: 1 and SEQ ID NO: 1 The sequence may be formed in a form in which SEQ ID NO: 2 is complementarily linked, but is not limited thereto.
본 발명의 상기 "miRNA”는 다양한 유전자 발현을 조절하며 단백질을 암호화하지 않는 RNA 서열을 의미하며, mRNA가 단백질로 번역되는 단계를 억제하거나 mRNA 자체의 분해를 유도하여 전사 후 유전자 발현 수준을 조절한다.The "miRNA" of the present invention refers to an RNA sequence that controls the expression of various genes and does not encode a protein, and controls the level of gene expression after transcription by inhibiting the step of translating mRNA into a protein or inducing degradation of the mRNA itself. .
본 발명의 상기 암은 유방암, 방광암, 자궁경부암, 대장암, 폐암, 췌장암, 위암, 난소암, 혈액암, 간암, 전립선암 및 두경부암으로 이루어진 군으로부터 선택되는 적어도 하나인 것일 수 있고, 바람직하게는 유방암일 수 있으며, 더욱 바람직하게는 삼중음성유방암(Triple-negative breast cancer; TNBC)일 수 있으나, 이에 제한되는 것은 아니다.The cancer of the present invention may be at least one selected from the group consisting of breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, stomach cancer, ovarian cancer, hematologic cancer, liver cancer, prostate cancer, and head and neck cancer, and preferably May be breast cancer, more preferably triple-negative breast cancer (TNBC), but is not limited thereto.
본 발명의 상기 "삼중음성유방암"은 치료 표적 유전자에 해당하는 에스트로겐 수용체, 프로게스테론 수용체 및 HER2의 발현이 모두 음성을 나타내는 유방암의 종류로서, 삼중음성유방암은 비삼중음성유방암 환자와 비교하여 조직학적 분화도의 3등급 비율이 높고, 재발 시기가 빠르며, 예후가 매우 나쁘다. 특히, 상기 삼중음성유방암은 기존에 유방암 치료제로 사용되는 HER2 표적 항체 약물인 허셉틴과 같은 약물을 사용할 수 없다는 한계점이 존재한다. 따라서, 본 발명의 상기 miR-4516을 이용하는 경우에는 기존의 유방암 치료제로 치료가 어려운 삼중음성유방암을 매우 효과적으로 예방, 개선 또는 치료할 수 있다는 장점이 존재한다.The "triple-negative breast cancer" of the present invention is a type of breast cancer in which the expression of estrogen receptor, progesterone receptor, and HER2 corresponding to the therapeutic target gene is all negative, and triple-negative breast cancer is histological differentiation compared to non-triple-negative breast cancer patients The
본 발명의 상기 miR-4516은 FOSL1(FOS-like antigen 1) 유전자로부터 전사된 mRNA의 3'-UTR을 표적으로 하는 것일 수 있다. 본 발명의 상기 miR-4516은 FOSL1 유전자로부터 전사된 mRNA의 3'-UTR에 결합하여 FOSL1의 mRNA를 분해하거나 번역이 억제되도록 할 수 있다.The miR-4516 of the present invention may target the 3'-UTR of mRNA transcribed from the FOSL1 (FOS-like antigen 1) gene. The miR-4516 of the present invention may degrade FOSL1 mRNA by binding to 3'-UTR of mRNA transcribed from FOSL1 gene or inhibit translation.
본 발명의 상기 "FOSL1 유전자"는 유방암, 특히 비 TNBC에 비하여 TNBC에서 특이적으로 그 유전자 및 그에 의해 암호화되는 단백질의 발현 수준이 증가되어 있는 것으로서, 본 발명의 상기 miR-4516은 FOSL1의 mRNA의 3'-UTR을 표적하여 FOSL1의 mRNA 및 단백질의 발현을 매우 효과적으로 억제하여 암의 예방 또는 치료 효과가 발휘되도록 할 수 있다.The "FOSL1 gene" of the present invention is that the expression level of the gene and the protein encoded by it is increased specifically in TNBC compared to breast cancer, particularly non-TNBC, and the miR-4516 of the present invention is the mRNA of FOSL1. By targeting 3'-UTR, the expression of FOSL1 mRNA and protein can be very effectively inhibited, so that the effect of preventing or treating cancer can be exerted.
본 발명의 상기 “miRNA”는 상기 miR-4516과 기능적으로 동등한 작용을 할 수 있다면 miRNA를 구성하는 일부 염기서열이 결실, 치환 또는 삽입에 의해 변형되더라도 모두 포함될 수 있다. 예를 들면, 상기 miR-4516을 구성하는 염기 서열과 80%, 90%, 95%, 또는 99%의 상동성을 갖는 것을 포함할 수 있다.The "miRNA" of the present invention may be included in all of the nucleotide sequences constituting the miRNA, as long as it has a functional equivalent function to the miR-4516, even if it is modified by deletion, substitution, or insertion. For example, it may include those having 80%, 90%, 95%, or 99% homology with the base sequence constituting the miR-4516.
본 발명의 상기 “상동성”은 야생형(wild type) 유전자의 염기 서열과의 유사한 정도를 나타내기 위한 것으로서, 본 발명의 염기 서열과 상기와 같은 퍼센트 이상의 동일한 서열을 가지는 서열을 포함한다. 이러한 상동성은 두 서열을 육안으로 비교하여 결정할 수도 있으나, 비교대상이 되는 서열을 나란히 배열하여 상동성 정도를 분석해 주는 생물정보 알고리즘(bioinformatic algorithm)을 사용하여 결정할 수 있다. 상기 두 개의 염기 서열 사이의 상동성은 백분율로 표시될 수 있다. 유용한 자동화된 알고리즘은 Wisconsin Genetics Software Package(Genetics Computer Group, Madison, W, USA)의 GAP, BESTFIT, FASTA와 TFASTA 컴퓨터 소프트웨어 모듈에서 이용 가능하다. 상기 모듈에서 자동화된 배열 알고리즘은 Needleman & Wunsch와 Pearson & Lipman과 Smith & Waterman 서열 배열 알고리즘을 포함한다. 다른 유용한 배열에 대한 알고리즘과 상동성 결정은 FASTP, BLAST, BLAST2, PSIBLAST와 CLUSTAL W를 포함하는 소프트웨어에서 자동화될 수 있다.The "homology" of the present invention is intended to indicate the degree of similarity with the nucleotide sequence of a wild type gene, and includes a sequence having the same percentage or more of the same sequence as the nucleotide sequence of the present invention. Such homology can also be determined by visually comparing two sequences, but can be determined using a bioinformatic algorithm that analyzes the degree of homology by arranging the sequences to be compared side by side. The homology between the two base sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, W, USA). Alignment algorithms automated in the module include Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman sequence alignment algorithms. Algorithms and homology determinations for other useful arrangements can be automated in software including FASTP, BLAST, BLAST2, PSIBLAST and CLUSTAL W.
본 발명의 상기 miR-4516은 세포 내 전달을 위하여, 발현 벡터에 포함되어 제공될 수 있다. 본 발명에서 miRNA 핵산 분자는, 핵산 및 DEAE-덱스트란의 복합체, 핵산 및 핵 단백질의 복합체, 핵산 및 지질의 복합체 등의 다양한 형질전환 기술을 이용하여 세포 내로 도입될 수 있는데, 이를 위해 상기 세포 내로의 효율적인 도입을 가능하게 하는 전달체 내에 포함된 형태로 제공될 수 있다. 바람직하게, 상기 전달체는 벡터이며, 바이러스 벡터 및 비바이러스 벡터 모두 사용 가능하다. 바이러스 벡터(viral vector)로서 예를 들면, 렌티바이러스 (lentivirus), 레트로바이러스(retrovirus), 아데노바이러스(adenovirus), 허피스바이러스(herpes virus) 및 아비폭스바이러스(avipox virus) 벡터 등을 사용할 수 있으나, 이에 제한되는 것은 아니다.The miR-4516 of the present invention may be included in an expression vector and provided for intracellular delivery. In the present invention, miRNA nucleic acid molecules can be introduced into cells using various transformation techniques such as a complex of nucleic acids and DEAE-dextran, complex of nucleic acids and nuclear proteins, complex of nucleic acids and lipids, etc. It may be provided in a form contained in a delivery system that enables efficient introduction of. Preferably, the carrier is a vector, and both viral vectors and non-viral vectors can be used. As a viral vector, for example, lentivirus, retrovirus, adenovirus, herpes virus, and abipox virus vector, etc. can be used, It is not limited thereto.
본 발명의 상기 발현 벡터는, 형질전환된 세포의 선별을 용이하게 하기 위하여 선별 마커를 더 포함할 수 있다. 예를 들어, 약물 내성, 영양 요구성, 세포 독성제에 대한 내성 또는 표면 단백질의 발현과 같이 선택 가능한 표현형이 나타날 수 있는 마커, 즉 녹색 형광 단백질, 퓨로마이신, 네오마이신, 하이그로마이신, 히스티디놀 디하이드로게나제(hisD) 및 구아닌 포스포리보실트랜스퍼라제(Gpt) 등을 더 포함할 수 있으나, 이에 제한되는 것은 아니다.The expression vector of the present invention may further include a selection marker to facilitate selection of transformed cells. Markers that can exhibit a selectable phenotype, for example drug resistance, auxotrophic, resistance to cytotoxic agents, or expression of surface proteins, i.e. green fluorescent protein, puromycin, neomycin, hygromycin, histi Dinol dehydrogenase (hisD) and guanine phosphoribosyltransferase (Gpt) may further be included, but the present invention is not limited thereto.
본 발명의 상기 miR-4516은 세포에 도입된 형태로 제공될 수 있다. 상기 miR-4516을 세포 내로 도입하는 방법은 통상의 방법, 예를 들면 G-fectin, Mirus TrasIT-TKO 지질친화성 시약, 리포펙틴, 리포펙타민, 셀펙틴 (cellfectin), 양이온성 인지질 나노입자, 양이온성 고분자, 양이온성 미셀, 양이온성 에멀젼 또는 리포좀을 포함하는 전달 시약과 함께 세포 내로 도입되는 방법 등이 있으나, 이에 제한되는 것은 아니다.The miR-4516 of the present invention may be provided in a form introduced into a cell. The method of introducing miR-4516 into cells is a conventional method, for example, G-fectin, Mirus TrasIT-TKO lipophilic reagent, lipofectin, lipofectamine, cellfectin, cationic phospholipid nanoparticles, A method of introducing into a cell together with a delivery reagent including a cationic polymer, a cationic micelle, a cationic emulsion, or a liposome, and the like, but is not limited thereto.
본 발명의 상기 “세포”는 암 세포의 주위의 종양미세환경을 구성하는 세포라면 모두 포함될 수 있고, 예를 들면 섬유아세포일 수 있으나, 이에 제한되는 것은 아니다. The "cell" of the present invention may include any cells constituting the tumor microenvironment around cancer cells, and may be, for example, fibroblasts, but is not limited thereto.
본 발명의 상기 "예방"은 본 발명의 조성물을 이용하여 암으로 인해 발생한 증상을 차단하거나, 그 증상을 억제 또는 지연시키는 모든 행위라면 제한없이 포함할 수 있다. The "prevention" of the present invention may include, without limitation, any act of blocking or suppressing or delaying symptoms caused by cancer using the composition of the present invention.
본 발명의 상기 "개선" 또는 "치료"는 본 발명의 조성물을 이용하여 암으로 인해 발생한 증상이 호전되거나 이롭게 되는 모든 행위라면 제한없이 포함할 수 있다.The "improvement" or "treatment" of the present invention may include, without limitation, any action that improves or benefits the symptoms caused by cancer using the composition of the present invention.
본 발명의 상기 약학 조성물은 캡슐, 정제, 과립, 주사제, 연고제, 분말 또는 음료 형태임을 특징으로 할 수 있으며, 상기 약학 조성물은 인간을 대상으로 하는 것을 특징으로 할 수 있다. The pharmaceutical composition of the present invention may be characterized in that it is in the form of capsules, tablets, granules, injections, ointments, powders, or beverages, and the pharmaceutical composition may be characterized for human.
본 발명의 상기 약학 조성물은 이들로 한정되는 것은 아니지만, 각각 통상의 방법에 따라 산제, 과립제, 캡슐, 정제, 수성 현탁액 등의 경구형 제형, 외용제, 좌제 및 멸균 주사 용액의 형태로 제형화되어 사용될 수 있다. 본 발명의 약학 조성물은 약학적으로 허용 가능한 담체를 포함할 수 있다. 약학적으로 허용되는 담체는 경구 투여 시에는 결합제, 활탁제, 붕해제, 부형제, 가용화제, 분산제, 안정화제, 현탁화제, 색소, 향료 등이 사용될 수 있으며, 주사제의 경우에는 완충제, 보존제, 무통화제, 가용화제, 등장제, 안정화제 등이 혼합되어 사용될 수 있으며, 국소투여용의 경우에는 기제, 부형제, 윤활제, 보존제 등이 사용될 수 있다. 본 발명의 약학 조성물의 제형은 상술한 바와 같은 약제학적으로 허용되는 담체와 혼합하여 다양하게 제조될 수 있다. 예를 들어, 경구 투여시에는 정제, 트로키, 캡슐, 엘릭서(elixir), 서스펜션, 시럽, 웨이퍼 등의 형태로 제조할 수 있으며, 주사제의 경우에는 단위 투약 앰플 또는 다수회 투약 형태로 제조할 수 있다. 기타, 용액, 현탁액, 정제, 캡슐, 서방형 제제 등으로 제형화할 수 있다.The pharmaceutical composition of the present invention is not limited thereto, but is formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. I can. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers can be used as binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, flavoring agents, etc. for oral administration, and buffers, preservatives, painlessness, etc. for injections. An agent, a solubilizing agent, an isotonic agent, a stabilizer, and the like may be mixed and used, and in the case of topical administration, a base agent, an excipient, a lubricant, a preservative, and the like may be used. The formulation of the pharmaceutical composition of the present invention can be variously prepared by mixing with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixir, suspension, syrup, wafers, etc., and in the case of injections, it can be prepared in unit dosage ampoules or multiple dosage forms. have. Others, solutions, suspensions, tablets, capsules, can be formulated as sustained-release preparations.
본 발명의 상기 제제화에 적합한 담체, 부형제 및 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말디톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 또는 광물유 등이 사용될 수 있다. 또한, 충진제, 항 응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등을 추가로 포함할 수 있다.Examples of suitable carriers, excipients and diluents for the formulation of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium Silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may additionally be included.
본 발명의 상기 약학 조성물의 투여 경로는 이들로 한정되는 것은 아니지만 구강, 정맥내, 근육내, 동맥내, 골수내, 경막내, 심장내, 경피, 피하, 복강내, 비강내, 장관, 국소, 설하 또는 직장이 포함된다. 경구 또는 비경구 투하가 바람직하다. The route of administration of the pharmaceutical composition of the present invention is not limited thereto, but oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Includes sublingual or rectal. Oral or parenteral administration is preferred.
본 발명의 상기 "비경구"란, 피하, 피내, 정맥내, 근육내, 관절내, 활액낭내, 흉골내, 경막내, 병소내 및 두개골내 주사 또는 주입기술을 포함한다. 본 발명의 약학 조성물은 또한 직장 투여를 위한 좌제의 형태로 투여될 수 있다.The "parenteral" of the present invention includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of suppositories for rectal administration.
본 발명의 상기 약학 조성물은 사용된 특정 화합물의 활성, 연령, 체중, 일반적인 건강, 성별, 정식, 투여 시간, 투여 경로, 배출율, 약물 배합 및 예방 또는 치료될 특정 질환의 중증을 포함한 여러 요인에 따라 다양하게 변할 수 있고, 상기 약학 조성물의 투여량은 환자의 상태, 체중, 질병의 정도, 약무 형태, 투여 경로 및 기간에 따라 다르지만 당업자에 의해 적절하게 선택될 수 있고, 1일 0.0001 내지 50 mg/kg 또는 0.001 내지 50 mg/kg으로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. 본 발명에 따른 의약 조성물은 환제, 당의정, 캡슐, 액제, 겔, 시럽, 슬러리, 현탁제로 제형화될 수 있다.The pharmaceutical composition of the present invention depends on a number of factors including the activity of the specific compound used, age, weight, general health, sex, formulation, time of administration, route of administration, excretion rate, drug formulation and the severity of the specific disease to be prevented or treated. It may vary in various ways, and the dosage of the pharmaceutical composition varies depending on the patient's condition, weight, degree of disease, drug form, route and duration of administration, but may be appropriately selected by those skilled in the art, and 0.0001 to 50 mg/day It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be divided several times. The above dosage does not in any way limit the scope of the present invention. The pharmaceutical composition according to the present invention may be formulated as a pill, dragee, capsule, liquid, gel, syrup, slurry, or suspension.
본 발명의 상기 식품 조성물은 각종 식품류, 예를 들어, 음료, 껌, 차, 비타민 복합제, 분말, 과립, 정제, 캡슐, 과자, 떡, 빵 등의 형태로 제조될 수 있다. The food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, and bread.
본 발명의 상기 유효성분이 식품 조성물에 포함될 때 그 양은 전체 중량의 0.1 내지 50%의 비율로 첨가할 수 있으나, 이에 제한되는 것은 아니다.When the active ingredient of the present invention is included in the food composition, the amount may be added in a proportion of 0.1 to 50% of the total weight, but is not limited thereto.
본 발명의 상기 식품 조성물이 음료 형태로 제조되는 경우 지시된 비율로 상기 식품 조성물을 포함하는 것 외에 특별한 제한점은 없으며, 통상의 음료와 같이 다양한 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 구체적으로, 천연 탄수화물로서 포도당 등의 모노사카라이드, 과당 등의 디사카라이드, 슈크로스 등의 및 폴리사카라이드, 덱스트린, 시클로덱스트린 등과 같은 통상적인 당 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜 등을 포함할 수 있다. 상기 향미제로서는 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진등) 및 합성 향미제(사카린, 아스파르탐 등) 등일 수 있다. When the food composition of the present invention is prepared in the form of a beverage, there is no particular limitation other than including the food composition in the indicated ratio, and may contain various flavoring agents or natural carbohydrates, etc. as an additional component like a normal beverage. . Specifically, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and common sugars such as sucrose and polysaccharides, dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol are used. Can include. The flavoring agent may be a natural flavoring agent (taumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and a synthetic flavoring agent (saccharin, aspartame, etc.).
본 발명의 상기 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 더 포함할 수 있다.The food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavors and natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, It may further include a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like.
본 발명의 상기 성분은 독립적 또는 조합하여 사용할 수 있다. 상기 첨가제의 비율은 본 발명의 핵심적인 요소에 해당하지 아니하지만, 본 발명의 식품 조성물 100 중량부 당 0.1 내지 약 50 중량부의 범위에서 선택될 수 있으나, 이에 제한되는 것은 아니다.The above components of the present invention may be used independently or in combination. Although the ratio of the additive does not correspond to the core element of the present invention, it may be selected from 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention, but is not limited thereto.
본 발명의 상기 화장료 조성물은 화장수, 영양로션, 영양에센스, 마사지 크림, 미용목욕물첨가제, 바디로션, 바디밀크, 배스오일, 베이비오일, 베이비파우더, 샤워겔, 샤워크림, 선스크린로션, 선스크린크림, 선탠크림, 스킨로션, 스킨크림, 자외선차단용 화장품, 클렌징밀크, 탈모제화장용, 페이스 및 바디로션, 페이스 및 바디크림, 피부미백크림, 핸드로션, 헤어로션, 화장용 크림, 쟈스민오일, 목욕비누, 물비누, 미용비누, 샴푸, 손세정제(핸드클리너), 약용비누비의료용, 크림비누, 페이셜 워시, 전신 세정제, 두피 세정제, 헤어린스, 화장비누, 치아 미백용 겔, 치약 등의 형태로 제조될 수 있다. 본 발명의 조성물은 화장료 조성물의 제조에 통상적으로 사용하는 용매나, 적절한 담체, 부형제 또는 희석제를 더 포함할 수 있다.The cosmetic composition of the present invention is a lotion, nutritional lotion, nutritional essence, massage cream, beauty bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream , Suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory makeup, face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, bath Manufactured in the form of soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap, medical use, cream soap, facial wash, body cleaner, scalp cleaner, hair rinse, makeup soap, tooth whitening gel, toothpaste, etc. Can be. The composition of the present invention may further include a solvent commonly used in the manufacture of cosmetic compositions, or an appropriate carrier, excipient, or diluent.
본 발명의 상기 화장료 조성물 내에 더 추가될 수 있는 용매의 종류는 특별히 한정하지 않으나, 예를 들어, 물, 식염수, DMSO 또는 이들의 조합을 사용할 수 있다. 또한, 담체, 부형제 또는 희석제로는 정제수, 오일, 왁스, 지방산, 지방산 알콜, 지방산 에스테르, 계면활성제, 흡습제(humectant), 증점제, 항산화제, 점도 안정화제, 킬레이팅제, 완충제, 저급 알콜 등이 포함되지만, 이에 제한되는 것은 아니다. 또한, 필요에 따라 미백제, 보습제, 비타민, 자외선 차단제, 향수, 염료, 항생제, 항박테리아제, 항진균제를 포함할 수 있다. The type of solvent that can be further added to the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used. In addition, as carriers, excipients or diluents, purified water, oil, wax, fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, etc. Included, but not limited to. In addition, whitening agents, moisturizing agents, vitamins, sunscreen agents, perfumes, dyes, antibiotics, antibacterial agents, and antifungal agents may be included as needed.
본 발명의 상기 오일로서는 수소화 식물성유, 피마자유, 면실유, 올리브유, 야자인유, 호호바유, 아보카도유가 이용될 수 있으며, 왁스로는 밀랍, 경랍, 카르나우바, 칸델릴라, 몬탄, 세레신, 액체 파라핀, 라놀린이 이용될 수 있다.Hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm seed oil, jojoba oil, and avocado oil may be used as the oil of the present invention, and waxes include beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin , Lanolin can be used.
본 발명의 상기 지방산으로는 스테아르산, 리놀레산, 리놀렌산, 올레산이 이용될 수 있고, 지방산 알콜로는 세틸 알콜, 옥틸 도데칸올, 올레일 알콜, 판텐올, 라놀린 알콜, 스테아릴 알콜, 헥사데칸올이 이용될 수 있으며 지방산 에스테르로는 이소프로필 미리스테이트, 이소프로필 팔미테이트, 부틸 스테아레이트가 이용될 수 있다. 계면 활성제로는 당업계에 알려진 양이온 계면활성제, 음이온 계면활성제 및 비 이온성 계면활성제가 사용가능하며 가능한 한 천연물 유래의 계면활성제가 바람직하다. 그 외에도 화장품 분야에서 널리 알려진 흡습제, 증점제, 항산화제 등을 포함할 수 있으며, 이들의 종류와 양은 당업계에 공지된 바에 따른다.Stearic acid, linoleic acid, linolenic acid, and oleic acid may be used as the fatty acid of the present invention, and as fatty acid alcohols, cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, hexadecanol As the fatty acid ester, isopropyl myristate, isopropyl palmitate, and butyl stearate may be used. As the surfactant, cationic surfactants, anionic surfactants, and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible. In addition, hygroscopic agents, thickeners, antioxidants, and the like, which are widely known in the cosmetic field, may be included, and the types and amounts thereof are as known in the art.
본 발명의 다른 구현 예에서는 암 치료제의 스크리닝 방법을 제공한다.Another embodiment of the present invention provides a method for screening a cancer therapeutic agent.
본 발명의 상기 스크리닝 방법은 암 환자로부터 분리된 생물학적 시료에 목적하는 후보물질을 처리하는 단계; 및 상기 생물학적 시료에서 miR-4516이 존재하는 수준을 확인하는 단계;를 포함한다.The screening method of the present invention comprises the steps of treating a target candidate material in a biological sample isolated from a cancer patient; And determining the level of miR-4516 present in the biological sample.
본 발명의 상기 생물학적 시료에서 miR-4516이 존재하는 수준이 후보물질을 처리하기 이전에 비하여 증가된 경우, 상기 후보물질을 암 치료제로 선별하는 단계를 더 포함할 수 있다.When the level of miR-4516 present in the biological sample of the present invention is increased compared to prior to treatment of the candidate substance, the step of selecting the candidate substance as a cancer treatment may be further included.
본 발명의 상기 스크리닝 방법에서 암, 삼중음성유방암 및 miR-4516에 대한 내용은 상기 조성물에 기재된 바와 동일하여 명세서의 과도한 복잡성을 피하기 위해 생략한다.In the screening method of the present invention, the information on cancer, triple negative breast cancer and miR-4516 is the same as described in the composition, and thus is omitted to avoid excessive complexity of the specification.
본 발명의 상기 생물학적 시료는 개체로부터 얻어지거나 개체로부터 유래된 임의의 물질, 생물학적 체액, 조직 또는 세포를 의미하는 것으로, 예를 들면, 전혈(whole blood), 백혈구(leukocytes), 말초혈액 단핵 세포(peripheral blood mononuclear cells), 백혈구 연층(buffy coat), 혈장(plasma) 및 혈청(serum)을 포함하는 혈액, 객담(sputum), 눈물(tears), 점액(mucus), 세비액(nasal washes), 비강 흡인물(nasal aspirate), 호흡(breath), 소변(urine), 정액(semen), 침(saliva), 복강 세척액(peritoneal washings), 골반 내 유체액(pelvic fluids), 낭종액(cystic fluid), 뇌척수막 액(meningeal fluid), 양수(amniotic fluid), 선액(glandular fluid), 췌장액(pancreatic fluid), 림프액(lymph fluid), 흉수(pleural fluid), 유두 흡인물(nipple aspirate), 기관지 흡인물(bronchial aspirate), 활액(synovial fluid), 관절 흡인물(joint aspirate), 기관 분비물(organ secretions), 세포(cell), 세포 추출물(cell extract) 또는 뇌척수액(cerebrospinal fluid)을 포함할 수 있으나, 이에 제한되는 것은 아니다.The biological sample of the present invention refers to any substance, biological body fluid, tissue or cell obtained from or derived from an individual, for example, whole blood, leukocytes, peripheral blood mononuclear cells ( peripheral blood mononuclear cells), leukocyte buffy coat, blood including plasma and serum, sputum, tears, mucus, nasal washes, nasal cavity Nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, Meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate aspirate), synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid, but are limited thereto. It is not.
본 발명의 상기 후보물질은 암의 증상을 예방, 개선 또는 치료하기 위한 활성, 즉 miR-4516의 발현 수준을 증대시킬 수 있는 활성이 존재하는지 여부에 대해 테스트하기 위한 약제를 의미하며, 단백질, 올리고 펩타이드, 유기 분자, 다당류, 폴리뉴클레오티드 및 광범위한 화합물 등의 임의의 분자를 포함한다. 이러한 후보물질은 천연물질뿐만 아니라, 합성 물질도 모두 포함하는 것일 수 있다.The candidate substance of the present invention refers to a drug for testing whether there is an activity for preventing, ameliorating or treating cancer symptoms, that is, an activity capable of increasing the expression level of miR-4516, protein, oligo Includes any molecule such as peptides, organic molecules, polysaccharides, polynucleotides and a wide range of compounds. These candidate substances may include not only natural substances but also synthetic substances.
본 발명의 상기 miR-4516이 존재하는 수준의 측정은 상기 miR-4516 존재하는 수준을 측정할 수 있는 제제를 이용하여, RT-PCR, 정량 실시간 PCR(quantified real time PCR), 경쟁적 RT-PCR(Competitive RT-PCR), 실시간 RT-PCR(real time quantitative RT-PCR), RNase 보호 분석법(RPA; RNase protection assay), 노던 블럿 분석(Northern blot assay), DNA 칩 분석법 등의 방법을 통해 진단하는 데에 사용되는 것일 수 있으나, 이에 제한되는 것은 아니다.Measurement of the level of the present miR-4516 of the present invention, using a formulation capable of measuring the level of the presence of the miR-4516, RT-PCR, quantitative real time PCR (quantified real time PCR), competitive RT-PCR ( Competitive RT-PCR), real time quantitative RT-PCR (RT-PCR), RNase protection assay (RPA), Northern blot assay, DNA chip analysis, etc. It may be used for, but is not limited thereto.
본 발명의 상기 miR-4516이 존재하는 수준을 측정할 수 있는 제제는 상기 miR-4516에 특이적인 프라이머 또는 프로브일 수 있으나, 이에 제한되는 것은 아니다.The agent capable of measuring the level of the miR-4516 present of the present invention may be a primer or probe specific for miR-4516, but is not limited thereto.
본 발명의 상기 “프라이머”는 짧은 자유 3말단 수산화기(free 3'hydroxyl group)를 가지는 염기 서열로 상보적인 주형 가닥(template)와 염기쌍(base pair)을 형성할 수 있고, 주형 가닥 복사를 위한 시작 지점으로서 기능을 하는 짧은 염기 서열을 의미한다. 상기 프라이머는 적절한 완충 용액 및 온도에서 중합반응(즉, DNA 폴리머레이즈 또는 역전사효소)을 위한 시약 및 상이한 4가지 뉴클레오사이드 트리포스페이트의 존재 하에서 DNA 합성이 개시될 수 있다.The "primer" of the present invention is a base sequence having a short free 3'hydroxyl group and can form a complementary template strand and a base pair, and the start for template strand copying It refers to a short nucleotide sequence that functions as a point. The primers can initiate DNA synthesis in the presence of a reagent for polymerization (ie, DNA polymerase or reverse transcriptase) and four different nucleoside triphosphates at an appropriate buffer solution and temperature.
본 발명의 상기 “프로브”는 상기 유전자와 특이적으로 결합을 이룰 수 있는 염기에 해당하는 RNA 또는 DNA 등의 핵산 단편을 의미하고, 이와 같은 프로브는 특정 유전자와 그로부터 전사되는 mRNA의 존재 유무, 유전자로부터 전사되는 mRNA의 발현 수준(발현량)을 확인할 수 있도록 라벨링되어 있을 수 있다. 상기 프로브는 올리고뉴클레오타이드(Oligonucleotide) 프로브, 단쇄 DNA(Single strand DNA) 프로브, 이중쇄 DNA(Double strand DNA)프로브, RNA 프로브 등의 형태로 제작될 수 있으나, 이에 제한되는 것은 아니다.The "probe" of the present invention refers to a nucleic acid fragment such as RNA or DNA that corresponds to a base capable of specifically binding to the gene, and such a probe is the presence or absence of a specific gene and mRNA transcribed therefrom, a gene It may be labeled to confirm the expression level (expression amount) of the mRNA transcribed from. The probe may be manufactured in the form of an oligonucleotide probe, a single strand DNA probe, a double strand DNA probe, an RNA probe, etc., but is not limited thereto.
본 발명의 상기 프라이머 또는 프로브는 서열번호 1로 표시되는 염기 서열을 기반으로 하여 통상의 방법을 참조해 당업자에 의해 쉽게 제조될 수 있다.The primer or probe of the present invention can be easily prepared by a person skilled in the art by referring to a conventional method based on the nucleotide sequence represented by SEQ ID NO: 1.
[서열목록][Sequence List]
서열번호 1: miR-4516 SEQ ID NO: 1: miR-4516
GGGAGAAGGGUCGGGGCGGGAGAAGGGUCGGGGC
서열번호 2: miR-4516 SEQ ID NO: 2: miR-4516
3'- CCCGACCCUUCUCCCUU-5'3'- CCCGACCCUUCUCCCUU-5'
서열번호 3: miR-132-3p forward primerSEQ ID NO: 3: miR-132-3p forward primer
TAACAGTCTACAGCCATGGTTAACAGTCTACAGCCATGGT
서열번호 4: miR-132-3p reverse primerSEQ ID NO: 4: miR-132-3p reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 5: miR-199b-5p forward primerSEQ ID NO: 5: miR-199b-5p forward primer
CCCAGTGTTTAGACTATCTGCCCAGTGTTTAGACTATCTG
서열번호 6: miR-199b-5p reverse primerSEQ ID NO: 6: miR-199b-5p reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 7: miR-29b-3p forward primerSEQ ID NO: 7: miR-29b-3p forward primer
TAGCACCATTTGAAATCAGTTAGCACCATTTGAAATCAGT
서열번호 8: miR-29b-3p reverse primerSEQ ID NO: 8: miR-29b-3p reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 9: miR-1253 forward primerSEQ ID NO: 9: miR-1253 forward primer
AGAGAAGAAGATCAGCCTGCAGAGAAGAAGATCAGCCTGC
서열번호 10: miR-1253 reverse primerSEQ ID NO: 10: miR-1253 reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 11: miR-1915 forward primerSEQ ID NO: 11: miR-1915 forward primer
CCCCAGGGCGACGCGGCGGGCCCCAGGGCGACGCGGCGGG
서열번호 12: miR-1915 reverse primerSEQ ID NO: 12: miR-1915 reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 13: miR-144-3p forward primerSEQ ID NO: 13: miR-144-3p forward primer
TACAGTATAGATGATGTACTTACAGTATAGATGATGTACT
서열번호 14: miR-144-3p reverse primerSEQ ID NO: 14: miR-144-3p reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 15: miR-320e forward primerSEQ ID NO: 15: miR-320e forward primer
AAAGCTGGGTTGAGAAGGAAAGCTGGGTTGAGAAGG
서열번호 16: miR-320e reverse primerSEQ ID NO: 16: miR-320e reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 17: miR-4516 forward primerSEQ ID NO: 17: miR-4516 forward primer
GGGAGAAGGGTCGGGGCGGGAGAAGGGTCGGGGC
서열번호 18: miR-4516 reverse primerSEQ ID NO: 18: miR-4516 reverse primer
AACGAGACGACGACAGACTTTAACGAGACGACGACAGACTTT
서열번호 19: FOSL1 forward primerSEQ ID NO: 19: FOSL1 forward primer
AGCTGCAGAAGCAGAAGGAGAGCTGCAGAAGCAGAAGGAG
서열번호 20: FOSL1 reverse primerSEQ ID NO: 20: FOSL1 reverse primer
GGAGTTAGGGAGGGTGTGGTGGAGTTAGGGAGGGTGTGGT
서열번호 21: PAX5 forward primerSEQ ID NO: 21: PAX5 forward primer
GGAGGAGTGAATCAGCTTGGGGAGGAGTGAATCAGCTTGG
서열번호 22: PAX5 reverse primerSEQ ID NO: 22: PAX5 reverse primer
GGCTTGATGCTTCCTGTCTCGGCTTGATGCTTCCTGTCTC
서열번호 23: TCL1A forward primerSEQ ID NO: 23: TCL1A forward primer
GCCTGGGAGAAGTTCGTGTAGCCTGGGAGAAGTTCGTGTA
서열번호 24: TCL1A reverse primerSEQ ID NO: 24: TCL1A reverse primer
ACTAAGCGCCAGAAACTGGAACTAAGCGCCAGAAACTGGA
본 발명에 따른 miR-4516 또는 이의 모방체(mimic)를 유효성분으로 포함하는 조성물은 FOSL1(FOS-like antigen 1) 유전자로부터 전사된 mRNA의 3'UTR에 특이적으로 결합하여, 상기 FOSL1의 mRNA의 번역을 억제함으로써 암 세포의 성장을 효과적으로 저해할 수 있어, 암의 예방, 개선 또는 치료 용도로 사용될 수 있다. 나아가, 후보물질에 의하여 miR-4516의 발현 수준의 변화를 확인함으로써 암의 치료제를 효과적으로 선별해낼 수 있다.The composition comprising miR-4516 or a mimic thereof according to the present invention as an active ingredient specifically binds to the 3'UTR of the mRNA transcribed from the FOSL1 (FOS-like antigen 1) gene, and the mRNA of the FOSL1 By inhibiting the translation of can effectively inhibit the growth of cancer cells, it can be used for prevention, improvement or treatment of cancer. Furthermore, by confirming the change in the expression level of miR-4516 by the candidate substance, it is possible to effectively screen a therapeutic agent for cancer.
도 1은 본 발명의 일 실시예에 따른 NF(normal fibroblast) 세포주 또는 CAF(cancer-associated fibroblast) 세포주의 엑소좀에서 miRNA 발현 수준 변화를 실시간 중합효소 연쇄반응을 통해 확인한 결과를 나타낸 것이다.
도 2는 본 발명의 일 실시예에 따른 miR-4516의 과발현에 의한 유방암 세포주의 증식 감소 효과를 CCK-8을 이용한 세포 성장율 분석을 통해 확인한 결과를 나타낸 것이다.
도 3은 본 발명의 일 실시예에 따른 miR-4516이 과발현되도록 형질전환된 NF 세포주 또는 CAF 세포주와 유방암 세포주(MCF7 또는 MDA-MB-231)의 공배양에 따른 세포 증식 억제 효과를 형광염색을 통해 확인한 결과를 나타낸 것이다.
도 4는 본 발명의 일 실시예에 따른 miR-4516와 결합 활성이 있는 표적 유전자를 확인하기 위해 루시퍼라제 활성을 측정한 결과를 나타낸 것이다.
도 5의 A 및 B는 본 발명의 일 실시예에 따른 miR-4516이 과발현된 유방암 세포주에서 FOSL1, PAX5 또는 TCL1A의 mRNA 발현 수준 변화를 실시간 중합효소 연쇄반응을 통해 확인하고(A), 단백질의 발현 수준 변화를 웨스턴 블롯 분석을 통해 확인한 결과(B)를 나타낸 것이다.
도 6의 A 내지 C는 본 발명의 일 실시예에 따른 miR-4516의 과발현에 의해 억제된 FOSL1의 번역 억제에 의해 삼중음성유방암(TNBC) 세포주에서, 실시간 중합효소 연쇄반응을 통해 유전자의 발현 수준(A), 웨스턴 블롯 분석을 통해 단백질의 발현 수준(B) 및 CCK-8을 이용한 분석을 통해 증식 억제 효과(C)를 확인한 결과를 나타낸 것이다.
도 7은 본 발명의 일 실시예에 따른 miR-4516 모방체(mimic)의 처리에 의한 TNBC 세포주의 증식 억제 효과를 CCK-8을 이용한 분석을 통해 확인한 결과를 나타낸 것이다.
도 8은 본 발명의 일 실시예에 따른 CAF 세포주와 유방암 세포주의 공배양 시에도 miR-4516 모방체의 처리에 의해 TNBC 세포주의 증식 억제 효과를 CCK-8을 이용한 분석을 통해 확인한 결과를 나타낸 것이다.
도 9는 본 발명의 일 실시예에 따른 miR-4516의 과발현된 CAF 세포주와 유방암 세포주의 공배양에 따라 유방암 세포주의 증식 감소 효과가 GW4869의 처리에 억제되는 것을 CCK-8을 이용한 세포 성장율 분석을 통해 확인한 결과를 나타낸 것이다.
도 10은 본 발명의 일 실시예에 따른 miR-4516 모방체의 처리에 의해 비 TNBC 세포주와 TNBC 세포주에서의 증식 억제 효과를 비교한 결과를 나타낸 것이다.
도 11은 본 발명의 일 실시예에 따른 유방암 환자의 조직에서 miR-4516의 mRNA 발현 수준과(A), FOSL1의 단백질의 발현 수준을 조직면역염색을 통해 확인한 결과(B)를 나타낸 것이다. 1 shows the results of confirming changes in miRNA expression levels in exosomes of a normal fibroblast (NF) cell line or a cancer-associated fibroblast (CAF) cell line according to an embodiment of the present invention through real-time polymerase chain reaction.
2 shows the results of confirming the effect of reducing the proliferation of breast cancer cell lines by overexpression of miR-4516 according to an embodiment of the present invention through cell growth rate analysis using CCK-8.
3 is a fluorescence staining for the effect of inhibiting cell proliferation according to co-culture of an NF cell line or CAF cell line and a breast cancer cell line (MCF7 or MDA-MB-231) transformed to overexpress miR-4516 according to an embodiment of the present invention. It shows the results confirmed through.
4 shows the results of measuring luciferase activity in order to identify a target gene having miR-4516-binding activity according to an embodiment of the present invention.
5A and 5B show changes in the mRNA expression level of FOSL1, PAX5 or TCL1A in a breast cancer cell line overexpressing miR-4516 according to an embodiment of the present invention through real-time polymerase chain reaction (A), and The change in the expression level was confirmed through Western blot analysis, and the result (B) is shown.
6A to 6C are the expression levels of genes through real-time polymerase chain reaction in triple negative breast cancer (TNBC) cell lines by translational inhibition of FOSL1 suppressed by overexpression of miR-4516 according to an embodiment of the present invention. (A), the expression level of the protein through Western blot analysis (B) and the results of confirming the proliferation inhibitory effect (C) through analysis using CCK-8 are shown.
7 shows the result of confirming the proliferation inhibitory effect of the TNBC cell line by treatment with miR-4516 mimic according to an embodiment of the present invention through analysis using CCK-8.
FIG. 8 shows the results of confirming the proliferation inhibitory effect of the TNBC cell line by treatment with the miR-4516 mimic even when co-cultured with the CAF cell line and the breast cancer cell line according to an embodiment of the present invention through analysis using CCK-8. .
9 is a cell growth rate analysis using CCK-8 that the effect of reducing the proliferation of the breast cancer cell line according to the co-culture of the overexpressed CAF cell line of miR-4516 and the breast cancer cell line according to an embodiment of the present invention is inhibited by treatment with GW4869 It shows the results confirmed through.
10 shows the results of comparing the proliferation inhibitory effect in non-TNBC cell lines and TNBC cell lines by treatment with miR-4516 mimic according to an embodiment of the present invention.
11 shows the result of confirming the mRNA expression level of miR-4516 and the expression level of the protein of FOSL1 in the tissue of a breast cancer patient according to an embodiment of the present invention (A) through tissue immunostaining (B).
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로서, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for describing the present invention in more detail, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
[준비예 1] [Preparation Example 1] 활성화된 섬유아세포 유래 엑소좀 분리Separation of exosomes derived from activated fibroblasts
NF(normal fibroblast) 세포 및 CAF(cancer-associated fibroblast) 세포를 혈청이 포함되지 않은 배지에서 2일 동안 배양한 뒤 상등액을 수집하였다. 그런 다음, 상기 상등액을 3,000 g에서 20분간 원심분리하고, 0.22 μm 필터(Nalgene??, 와트만)를 이용하여 상기 원심분리가 완료된 상등액을 여과하였다. 이후, 9 kD 분자량 컷오프 농축기(9 kD molecular weight cutoff concentrators; Pierce, 락포드)를 이용하여 상기 여과액을 10 ml로 농축하였다.Normal fibroblast (NF) cells and cancer-associated fibroblast (CAF) cells were cultured in a medium containing no serum for 2 days, and then the supernatant was collected. Then, the supernatant was centrifuged at 3,000 g for 20 minutes, and the supernatant was filtered using a 0.22 μm filter (Nalgene??, Whatman). Thereafter, the filtrate was concentrated to 10 ml using 9 kD molecular weight cutoff concentrators (Pierce, Rockford).
상기 농축액에 ExoQuick-TC ?? (System Bioscience, 마운틴 뷰)를 첨가하고 4 ℃에서 하룻밤 동안 배양한 뒤에 3,500 g에서 30분간 원심분리하여 펠렛을 수득하였다. 상기 펠렛을 인산 완충 식염수(Phosphate buffered saline; PBS)를 이용하여 세척하고, 1 % 페니실린/스트렙토 마이신이 포함된 DMEM/F12 배지에 희석하였다.ExoQuick-TC ?? in the concentrate (System Bioscience, Mountain View) was added and incubated overnight at 4° C., followed by centrifugation at 3,500 g for 30 minutes to obtain a pellet. The pellet was washed with phosphate buffered saline (PBS), and diluted in DMEM/F12 medium containing 1% penicillin/streptomycin.
[준비예 2] [Preparation Example 2] 세포주의 배양 방법Cell line culture method
10 % 우태아 혈청(Fetal bovine serum; FBS)와 항생제(100 U/ml의 페니실린 및 10mg/ml의 스트렙토마이신)가 포함된 RPMI 배양 배지를 이용하여 37 ℃, 5 % CO2의 조건에서 삼중음성유방암(Triple-negative breast cancer; TNBC) 세포주 및 비 TNBC 세포주를 배양하였다.Triple negative in RPMI culture medium containing 10% Fetal bovine serum (FBS) and antibiotics (100 U/ml penicillin and 10 mg/ml streptomycin) at 37°C and 5% CO 2 Triple-negative breast cancer (TNBC) cell lines and non-TNBC cell lines were cultured.
[실시예 1] [Example 1] 엑소좀에서 miRNA 발현 수준 변화 확인Confirmation of changes in miRNA expression level in exosomes
트리졸을 이용하여 상기 준비예 1의 엑소좀(NF 세포주 또는 CAF 세포주)에서 전체 RNA를 추출하고, TOPscriptTM cDNA 합성 키트(엔지노믹스)를 이용하여 전체 RNA로부터 cDNA를 합성하였다. 이때, miRNA를 증폭하기 위해, cDNA 합성 이전에 대장균 폴리(A) 중합효소 I을 이용하여 전체 RNA를 폴리아데닐화 하였다. 그런 다음, 하기 표 1에 기재된 유전자에 따른 프라이머, 25 ng의 cDNA, 파워 SYBR® 녹색 PCR 마스터 믹스(Power SYBR® Green PCR Master Mix)를 전체 볼륨이 25 ㎕가 되도록 혼합한 뒤, 베르티 써모사이클러(Veriti Thermocycler; Applied Biosystems®)를 이용하여 실시간 중합효소연쇄반응을 수행하고, 델타-델타 Ct 방법을 통해 miRNA의 상대적인 발현 수준을 확인하여 그 결과를 도 1에 나타내었다.Total RNA was extracted from the exosomes (NF cell line or CAF cell line) of Preparation Example 1 using Trizol, and cDNA was synthesized from the total RNA using TOPscript TM cDNA synthesis kit (ENGINEOMICS). At this time, in order to amplify miRNA, the total RNA was polyadenylate using E. coli poly(A) polymerase I before cDNA synthesis. Then, primers according to the genes shown in Table 1, 25 ng of cDNA, and Power SYBR® Green PCR Master Mix were mixed so that the total volume was 25 μl, and then Berti Thermosai Real-time polymerase chain reaction was performed using a Cler (Veriti Thermocycler; Applied Biosystems®), and the relative expression level of miRNA was confirmed through the delta-delta Ct method, and the results are shown in FIG. 1.
도 1에서 보는 바와 같이, NF 세포로부터 분리된 엑소좀에 비하여, CAF 세포로부터 분리된 엑소좀에서 miR-199b-5p, miR-29b-3p 및 miR-4516이 통계적으로 유의하게 발현 수준이 감소되었고, 특히 miR-4516의 경우에는 2배 이상 발현 수준이 감소된 것을 확인하였다.As shown in FIG. 1, compared to exosomes isolated from NF cells, miR-199b-5p, miR-29b-3p and miR-4516 were statistically significantly decreased in expression levels in exosomes isolated from CAF cells. In particular, in the case of miR-4516, it was confirmed that the expression level was reduced by 2 times or more.
상기 결과를 통해 본 발명에 따른 miR-4516은 암의 진행을 억제할 수 있는 정상 섬유아세포에 비하여, 암 진행에 기여할 수 있는 종양미세환경 세포인 CAF 세포에서 특이적으로 그 발현 수준이 감소되어 있는 것을 알 수 있다.Through the above results, the expression level of miR-4516 according to the present invention is specifically reduced in CAF cells, which are tumor microenvironment cells that can contribute to cancer progression, compared to normal fibroblasts that can inhibit the progression of cancer. Can be seen.
[[ 실시예Example 2] 2] miRmiR -4516의 과발현에 의한 유방암 세포주의 증식 감소 효과 확인(1)Confirmation of the effect of reducing the proliferation of breast cancer cell lines by overexpression of -4516 (1)
리포펙타민®(라이프 테크놀로지)을 사용하여 제조사에서 제공하는 프로토콜에 따라, 상기 준비예 2에 기재되어 있는 MCF7 또는 MDA-MB-231 세포주에 서열번호 1로 표시되는 염기 서열로 이루어진 miR-4516이 삽입된 pENTRTM/H1/TO 벡터(인비트로젠)를 형질전환 하였다. 이때, 대조군으로 상기 세포주 각각에 상기 miR-4516이 삽입되어 있지 않은 pENTRTM/H1/TO 벡터만을 형질전환 하였다.Using Lipofectamine ® (Life Technology) according to the protocol provided by the manufacturer, miR-4516 consisting of the nucleotide sequence represented by SEQ ID NO: 1 in the MCF7 or MDA-MB-231 cell line described in Preparation Example 2 above was The inserted pENTR TM /H1/TO vector (Invitrogen) was transformed. At this time, as a control, only the pENTR TM /H1/TO vector to which the miR-4516 was not inserted into each of the cell lines was transformed.
상기 형질전환이 완료된 세포주들을 각각 상기 준비예 2의 배양 배지에 희석한 뒤, 100 ㎕의 상기 세포 희석액을 96웰 플레이트에 분주하였다. 그런 다음, 상기 플레이트의 각 웰에 10 ㎕의 CCK-8(도진도 분자 테크놀로지)를 추가로 첨가하고 37 ℃, 5 % CO2의 조건에서 한시간 동안 배양한 뒤, 450 nm에서 흡광도를 측정하여, 그 결과를 도 2에 나타내었다.Each of the transformed cell lines was diluted in the culture medium of Preparation Example 2, and then 100 µl of the cell dilution was dispensed into a 96-well plate. Then, 10 μl of CCK-8 (Dojindo Molecular Technology) was additionally added to each well of the plate and incubated for an hour at 37° C. and 5% CO 2 , and absorbance was measured at 450 nm, The results are shown in FIG. 2.
도 2에서 보는 바와 같이, 대조군 벡터가 형질전환된 경우(Control)와 비교하여, MCF7 세포주 및 MDA-MB-231 세포주 모두에서 miR-4516이 과발현되도록 형질전환 되었을 때(miR-4516) 세포주의 성장이 현저하게 감소되었다. 나아가, miR-4516이 과발현되도록 형질전환한지 3일째에, MCF7 세포주의 상대적 성장율이 2에 해당하는 반면, MDA-MB-231 세포주(MDA)의 상대적 성장율은 1.5임을 확인하였다.As shown in Figure 2, compared to the case in which the control vector was transformed (Control), when the cell line was transformed to overexpress miR-4516 in both the MCF7 cell line and the MDA-MB-231 cell line (miR-4516) growth of the cell line Was significantly reduced. Furthermore, it was confirmed that on the 3rd day after transformation so that miR-4516 is overexpressed, the relative growth rate of the MCF7 cell line corresponds to 2, while the relative growth rate of the MDA-MB-231 cell line (MDA) is 1.5.
상기 결과를 통해 본 발명에 따른 miR-4516의 과발현은 암의 증식을 억제할 수 있고, 특히 저침윤성 세포주와 비교하여 고침윤성 TNBC 세포주에서 더욱 효과적으로 암의 증식을 억제할 수 있음을 알 수 있다.From the above results, it can be seen that the overexpression of miR-4516 according to the present invention can inhibit the proliferation of cancer, and in particular, it can be more effectively inhibited the proliferation of cancer in the highly invasive TNBC cell line compared to the low invasive cell line.
[실시예 3] [Example 3] miR-4516의 과발현에 의한 암 세포주의 증식 감소 효과 확인(2)Confirmation of the effect of reducing the proliferation of cancer cell lines by overexpression of miR-4516 (2)
상기 실시예 2에 기재되어 있는 형질전환 방식과 동일한 방법으로 CAF 세포주에서 miR-4516의 과발현을 유도하였다. 그런 다음, miR-4516이 과발현되도록 형질전환된 NF 세포주 또는 CAF 세포주를 6웰 플레이트에 분주하고, CellTracker 녹색 CMFDA 염료(인비트로젠)가 포함된 무 혈청 DMEM/F12 배지를 첨가하여 상기 세포주를 염색하였다. 상기 염색된 세포주가 포함되어 있는 플레이트에 붉은색 염료로 염색된 MCF7 세포주 또는 MDA-MB-231 세포주를 추가로 분주한 뒤, 37 ℃, 5 % CO2의 조건에서 3일 동안 배양하고, 형광현미경을 통해 세포주를 확인하여 그 결과를 도 3에 나타내었다.The overexpression of miR-4516 was induced in CAF cell lines in the same manner as in the transformation method described in Example 2 above. Then, the NF cell line or CAF cell line transformed to overexpress miR-4516 was dispensed into a 6-well plate, and serum-free DMEM/F12 medium containing CellTracker green CMFDA dye (Invitrogen) was added to stain the cell line. I did. After additionally dispensing the MCF7 cell line or the MDA-MB-231 cell line stained with red dye to the plate containing the stained cell line, incubated for 3 days at 37° C. and 5% CO 2 , and under a fluorescence microscope The cell line was confirmed through and the results are shown in FIG. 3.
도 3에서 보는 바와 같이, 대조군 벡터가 형질전환된 CAF 세포주와 공배양된 MCF7 및 MDA-MB-231 세포주와 비교하여, miR-4516이 포함된 벡터가 형질전환된 CAF 세포주와 공배양된 MCF7 및 MDA-MB-231 세포주의 증식이 현저하게 억제되었다. 또한, miR-4516이 포함된 벡터가 형질전환된 CAF 세포주에 의한 증식 억제는 MCF7(MCF7/CAF)에 비하여 MDA-MB-231 세포주(MDA/CAF)에서 더욱 높은 수준으로 나타나는 것을 확인하였다.As shown in FIG. 3, compared with the control vector-transformed CAF cell line and the MCF7 and MDA-MB-231 cell lines, the vector containing miR-4516 was co-cultured with the transformed CAF cell line and Proliferation of the MDA-MB-231 cell line was remarkably suppressed. In addition, it was confirmed that the inhibition of proliferation by the CAF cell line transformed with the vector containing miR-4516 was higher in the MDA-MB-231 cell line (MDA/CAF) than in MCF7 (MCF7/CAF).
상기 결과를 통해 본 발명에 따른 miR-4516은 CAF 세포주에서 과발현됨으로써 암의 증식을 억제할 수 있고, 특히 저침윤성 세포주에 비하여 고침윤성 TNBC 세포주에서 더욱 효과적으로 암의 증식을 억제할 수 있음을 알 수 있다.From the above results, it can be seen that miR-4516 according to the present invention can inhibit the proliferation of cancer by being overexpressed in the CAF cell line, and in particular, it can be more effectively inhibited in the high invasive TNBC cell line than in the low invasive cell line. have.
[실시예 4] [Example 4] miR-4516의 표적 mRNA와의 결합 확인Confirmation of binding of miR-4516 to target mRNA
miR-4516가 표적할 것으로 예상되는 FOSL1(FOS-like antigen 1), PAX5(paired box 5) 또는 TCL1A(T Cell Leukemia/Lymphoma 1A) 유전자로부터 전사된 mRNA의 3'UTR과의 결합 활성을 확인하였다.The binding activity of the mRNA transcribed from FOSL1 (FOS-like antigen 1), PAX5 (paired box 5) or TCL1A (T Cell Leukemia/Lymphoma 1A) genes predicted to be targeted by miR-4516 with 3'UTR was confirmed. .
구체적으로, 상기 FOSL1, PAX5 또는 TCL1A의 mRNA의 3'UTR(비 번역 영역)의 염기 서열을 pGL3 컨트롤 벡터에 삽입하였다. 그런 다음, 상기 벡터에 삽입되어 있는 miR-4516이 결합할 것으로 예상되는 3'UTR의 염기 서열에 돌연변이를 유도하기 위해 중첩 확장 중합효소연쇄반응(overlap extension PCR)을 수행하였다. 이후, 상기 실시예 2와 동일한 방법으로 HEK293T 세포주에 pGL3 컨트롤 벡터(정상 3'UTR 또는 돌연변이가 유도된 3'UTR)과 miR-4516이 삽입된 벡터 또는 pRL-TK 벡터를 공동 형질전환하고 2일 동안 배양하였다. 상기 공동 형질전환된 세포로부터 수득된 세포 용해물을 이용하여 통상의 방법에 따라 루시퍼라제 활성을 측정하여, 그 결과를 도 4에 나타내었다.Specifically, the nucleotide sequence of the 3'UTR (non-translated region) of the mRNA of FOSL1, PAX5 or TCL1A was inserted into the pGL3 control vector. Then, overlap extension PCR was performed to induce a mutation in the nucleotide sequence of 3'UTR, which is expected to bind to miR-4516 inserted in the vector. Thereafter, in the same manner as in Example 2, a pGL3 control vector (normal 3'UTR or mutant induced 3'UTR) and a miR-4516-inserted vector or pRL-TK vector were co-transformed in the HEK293T cell line, and 2 days During incubation. Luciferase activity was measured according to a conventional method using the cell lysate obtained from the co-transformed cells, and the results are shown in FIG. 4.
도 4에서 보는 바와 같이, 정상 3'UTR을 갖는 FOSL1, PAX5 및 TCL1A의 mRNA의 경우 miR-4516이 과발현되었을 때 루시퍼라제의 활성이 현저하게 억제되었다. 반면, 돌연변이 3'UTR을 갖는 FOSL1, PAX5 및 TCL1A mRNA의 경우에는 miR-4516이 과발현되어도 루시퍼라제 활성의 변화가 없는 것을 확인하였다.As shown in FIG. 4, in the case of the mRNA of FOSL1, PAX5, and TCL1A having normal 3'UTR, the activity of luciferase was remarkably suppressed when miR-4516 was overexpressed. On the other hand, in the case of FOSL1, PAX5, and TCL1A mRNA having mutant 3'UTR, it was confirmed that there was no change in luciferase activity even when miR-4516 was overexpressed.
상기 결과를 통해 본 발명에 따른 miR-4516은 FOSL1, PAX5 및 TCL1A mRNA의 3'UTR 부위에 특이적으로 결합함으로써 상기 mRNA가 분해되도록 유도할 수 있고, 나아가 암이 진행되는데 관여하는 FOSL1과 같은 유전자로부터 전사된 mRNA를 분해함으로써 암의 진행을 매우 효과적으로 억제할 수 있음을 알 수 있다.Through the above results, miR-4516 according to the present invention specifically binds to the 3'UTR region of FOSL1, PAX5 and TCL1A mRNA, thereby inducing the mRNA to be degraded, and furthermore, a gene such as FOSL1 that is involved in cancer progression. It can be seen that the progression of cancer can be very effectively inhibited by degrading the mRNA transcribed from.
[실시예 5] [Example 5] miR-4516에 의한 FOSL1 유전자의 번역 억제 효과 확인Confirmation of the effect of miR-4516 to inhibit translation of the FOSL1 gene
상기 실시예 2와 동일한 방법으로 MCF7 세포주 또는 MDA-MB-231 세포주에 miR-4516의 과발현을 유도하기 위해 miR-4516이 삽입된 pENTRTM/H1/TO 벡터를 형질전환 하였다. 이때, 대조군으로는 pENTRTM/H1/TO 벡터만을 형질전환 하였다.In the same manner as in Example 2, in order to induce overexpression of miR-4516 in the MCF7 cell line or the MDA-MB-231 cell line, the pENTR TM /H1/TO vector inserted with miR-4516 was transformed. At this time, only the pENTR TM /H1 /TO vector was transformed as a control.
상기 형질전환이 완료된 MCF7 세포주 및 MDA-MB-231 세포주로부터 전체 RNA를 분리한 뒤, 하기 표 2에 기재되어 있는 FOSL1, PAX5 또는 TCL1A에 특이적인 프라이머를 이용하여 상기 실시예 1과 동일한 방법으로 실시간 중합효소연쇄반응을 수행하여, 그 결과를 도 5의 A에 나타내었다.After separating total RNA from the transformed MCF7 cell line and MDA-MB-231 cell line, real-time in the same manner as in Example 1 using primers specific for FOSL1, PAX5, or TCL1A described in Table 2 below. Polymerase chain reaction was performed, and the results are shown in FIG. 5A.
또한, 상기 형질전환이 완료된 MCF7 세포주 및 MDA-MB-231 세포주를 50 ㎕의 PRO-PREP 단백질 추출 용액(iNtRON 바이오테크놀로지)에 녹이고 30 게이지 바늘로 충분히 균질화 한 뒤, 4 ℃에서 30분 동안 배양하였다. 그런 다음, 15,000 g에서 원심분리하여 단백질 추출물을 수득하였다. 브래드포드(Bradford) 방법을 통해 정량화된 상기 단백질 추출물을 20 ㎍만큼 10% 폴리 아크릴 아미드 겔에 전기영동하고, PVDF 막(Millipore)에 옮긴 뒤에 FOSL1(Abcam), PAX5(Abcam), TCL1A(Abcam) 및 GAPDH (SantaCruz)에 특이적인 항체를 이용하여 웨스턴 블롯 분석을 수행하여 그 결과를 도 5의 B에 나타내었다.In addition, the transformed MCF7 cell line and MDA-MB-231 cell line were dissolved in 50 μl of PRO-PREP protein extraction solution (iNtRON Biotechnology), sufficiently homogenized with a 30 gauge needle, and incubated at 4° C. for 30 minutes. . Then, it was centrifuged at 15,000 g to obtain a protein extract. The protein extract quantified through the Bradford method was electrophoresed on a 10% polyacrylamide gel by 20 μg, transferred to a PVDF membrane (Millipore), and then FOSL1 (Abcam), PAX5 (Abcam), TCL1A (Abcam) And GAPDH (SantaCruz) specific antibody was used to perform Western blot analysis, and the results are shown in FIG. 5B.
도 5의 A 및 B에서 보는 바와 같이, MCF7 세포주 및 MDA-MB-231 세포주 모두에서 miR-4516이 과발현 되었을 때, FOSL1, PAX5 및 TCL1A의 mRNA 및 단백질이 존재하는 수준이 감소되었다. 특히, FOSL1의 mRNA 및 단백질의 경우에는 MCF7 세포주에 비하여 MDA-MB-231 세포주에서 특히 높은 수준으로 존재하였으나, miR-4516의 과발현에 의하여 그 mRNA 및 단백질이 존재하는 수준이 현저하게 감소되었다.5A and 5B, when miR-4516 was overexpressed in both the MCF7 cell line and the MDA-MB-231 cell line, the levels of the mRNA and protein of FOSL1, PAX5, and TCL1A were reduced. In particular, the mRNA and protein of FOSL1 were present at a particularly high level in the MDA-MB-231 cell line compared to the MCF7 cell line, but the levels of the mRNA and protein were significantly reduced by overexpression of miR-4516.
상기 결과를 통해 본 발명에 따른 miR-4516은 비 TNBC인 MCF 세포주에 비하여, TNBC인 MDA-MB-231 세포주에 높은 수준으로 존재하는 FOSL1의 번역을 현저하게 억제함으로써 TNBC 세포주의 성장을 억제할 수 있음을 알 수 있다.Through the above results, miR-4516 according to the present invention can suppress the growth of TNBC cell lines by remarkably inhibiting the translation of FOSL1 present at a high level in the MDA-MB-231 cell line, which is TNBC, compared to the non-TNBC MCF cell line. You can see that there is.
[[ 실시예Example 6] 6] miRmiR -4516에 의한 By -4516 FOSL1FOSL1 유전자의 번역 억제에 따른 Due to inhibition of gene translation TNBCTNBC 세포주의 증식 억제 효과 확인 Confirmation of cell line proliferation inhibitory effect
BT-20 세포주 또는 Hs578T 세포주에 상기 실시예 2와 동일한 방법으로 miR-4516이 삽입된 pENTRTM/H1/TO 벡터를 형질전환 하였다. 이때, 대조군으로는 pENTRTM/H1/TO 벡터만을 형질전환 하였다. 그런 다음, 상기 실시예 5와 동일한 방법으로 상기 세포주들에서 FOSL1의 단백질 및 mRNA의 발현 수준을 확인하여 그 결과를 도 6의 A 및 B에 나타내었다. 또한, 상기 실시예 2와 동일한 방법으로 CCK-8을 이용하여 상기 세포주들의 성장율을 측정하여, 그 결과를 도 6의 C에 나타내었다.The BT-20 cell line or the Hs578T cell line was transformed with a pENTR TM /H1/TO vector into which miR-4516 was inserted in the same manner as in Example 2. At this time, only the pENTR TM /H1 /TO vector was transformed as a control. Then, the expression levels of FOSL1 protein and mRNA were checked in the cell lines in the same manner as in Example 5, and the results are shown in FIGS. 6A and 6B. In addition, the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. 6C.
도 6의 A 및 B에서 보는 바와 같이, TNBC 세포주인 BT-20 세포주 및 Hs578T 세포주에서 FOSL1의 단백질이 높은 수준으로 나타났으며, miR-4516이 과발현되었을 때, 그 FOSL1의 단백질 및 mRNA가 존재하는 수준이 현저하게 감소되었다.6A and 6B, the protein of FOSL1 appeared at high levels in the BT-20 cell line and the Hs578T cell line, which are TNBC cell lines, and when miR-4516 was overexpressed, the protein and mRNA of the FOSL1 were present. The level was significantly reduced.
도 6의 C에서 보는 바와 같이, BT-20 세포주 또는 Hs578T 세포주에 miR-4516이 과발현된 경우, miR-4516이 과발현되지 않은 경우와 비교하여 0.2 정도 감소된 것을 확인하였다.As shown in C of FIG. 6, when miR-4516 was overexpressed in BT-20 cell line or Hs578T cell line, it was confirmed that miR-4516 was reduced by about 0.2 compared to the case where miR-4516 was not overexpressed.
상기 결과를 통해 본 발명에 따른 miR-4516은 비 TNBC 세포주와 비교하여, TNBC 세포주에서 특이적으로 발현이 증가되어 있는 FOSL1의 mRNA 분해를 유도함으로써 TNBC 세포주의 성장을 효과적으로 억제할 수 있음을 알 수 있다.From the above results, it can be seen that miR-4516 according to the present invention can effectively inhibit the growth of TNBC cell lines by inducing mRNA degradation of FOSL1, which is specifically expressed in TNBC cell lines, compared to non-TNBC cell lines. have.
[[ 실시예Example 7] 7] miRmiR -4516 -4516 모방체(mimic)에On the mimic 의한 by TNBCTNBC 세포주의 증식 억제 효과 확인(1) Confirmation of cell line proliferation inhibitory effect (1)
TNBC 세포주(TNBC)인 MDA-MB-231 세포주, BT-20 세포주 또는 Hs578T 세포주를 6웰 플레이트에 분주하였다. 상기 각각의 세포주에 서열번호 1 및 서열번호 2로 표시되는 염기 서열이 이중 가닥으로 구성되어 있는 miR-4516 모방체를 10 nM의 농도로 처리하고, 상기 실시예 2와 동일한 방법으로 CCK-8을 이용하여 상기 세포주들의 성장율을 측정하여, 그 결과를 도 7에 나타내었다. 여기서, 대조군으로는 miR-4516 모방체를 대신하여 10 nM 농도의 대조군 miRNA를 처리하였다.The TNBC cell line (TNBC), the MDA-MB-231 cell line, the BT-20 cell line, or the Hs578T cell line, was dispensed into a 6-well plate. In each of the cell lines, miR-4516 mimics consisting of double-stranded nucleotide sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2 were treated at a concentration of 10 nM, and CCK-8 was used in the same manner as in Example 2. By measuring the growth rate of the cell lines, the results are shown in FIG. 7. Here, as a control, a control miRNA at a concentration of 10 nM was treated instead of the miR-4516 mimic.
센스: 3'- GGGAGAAGGGUCGGGGC-5'(서열번호 1)Sense: 3'- GGGAGAAGGGUCGGGGC-5' (SEQ ID NO: 1)
안티센스: 3'- CCCGACCCUUCUCCCUU-5'(서열번호 2)Antisense: 3'- CCCGACCCUUCUCCCUU-5' (SEQ ID NO: 2)
도 7에서 보는 바와 같이, 대조군 miRNA를 처리한 경우와 비교하여 TNBC 세포주들에 miR-4516 모방체를 처리하였을 때, MDA-MB-231 세포주의 경우 12.1, BT-20 세포주의 경우 9.8 및 Hs578T 세포주의 경우 8.5 성장율이 감소하였다.As shown in FIG. 7, when the miR-4516 mimic was treated in the TNBC cell lines compared to the case where the control miRNA was treated, the MDA-MB-231 cell line was 12.1, the BT-20 cell line was 9.8, and the Hs578T cell line. In the case of 8.5, the growth rate decreased.
상기 결과를 통해 본 발명에 따른 miR-4516 모방체는 TNBC 세포주에서 miR-4516과 동일하게 세포주의 성장을 저해하는 효과를 발휘하는 것을 알 수 있다.From the above results, it can be seen that the miR-4516 mimic according to the present invention exhibits an effect of inhibiting the growth of the cell line in the same manner as miR-4516 in the TNBC cell line.
[실시예 8] [Example 8] miR-4516 모방체에 의한 TNBC 세포주의 증식 억제 효과 확인(2)Confirmation of the effect of miR-4516 mimic on the inhibition of proliferation of TNBC cell lines (2)
실제 환자의 종양미세환경에서도 miR-4516 모방체에 의한 TNBC 세포주의 증식 억제 효과가 유도되는지 여부를 확인하기 위해, 상기 실시예 7과 동일한 조건으로 MDA-MB-231 세포주, BT-20 세포주 또는 Hs578T 세포주에 miR-4516 모방체를 처리하고, 상기 각각의 세포주들의 웰에 CAF 세포주가 분주되어 있는 트렌스웰 인설트(Transwell insert)를 삽입하였다. 그런 다음, 1 % 페니실린/스트렙토 마이신 및 10 % FBS가 포함된 DMEM 또는 DMEM/F12 배지에서 1 일 동안 배양하고, 상기 실시예 2와 동일한 방법으로 CCK-8을 이용하여 상기 세포주들의 성장율을 측정하여, 그 결과를 도 8에 나타내었다.In order to confirm whether the proliferation inhibitory effect of the miR-4516 mimic is induced in the tumor microenvironment of the actual patient, MDA-MB-231 cell line, BT-20 cell line or Hs578T under the same conditions as in Example 7 above. The cell line was treated with miR-4516 mimic, and a Transwell insert in which the CAF cell line was dispensed was inserted into the wells of each of the cell lines. Then, incubate for 1 day in DMEM or DMEM/F12 medium containing 1% penicillin/streptomycin and 10% FBS, and measure the growth rate of the cell lines using CCK-8 in the same manner as in Example 2. , The results are shown in FIG. 8.
도 8에서 보는 바와 같이, CAF 세포주와 공배양된 경우에도 miR-4516 모방체가 처리되었을 때, 대조군 miRNA를 처리한 경우와 비교하여 TNBC 세포주의 성장율이 감소되는 것을 확인하였다.As shown in FIG. 8, it was confirmed that the growth rate of the TNBC cell line decreased when the miR-4516 mimic was treated even when co-cultured with the CAF cell line compared to the case where the control miRNA was treated.
상기 결과를 통해 본 발명에 따른 miR-4516 모방체는 종양미세환경과 유사한 CAF 세포주와 공배양된 TNBC 세포주에서도 매우 효과적으로 세포 성장을 저해할 수 있음을 알 수 있다.From the above results, it can be seen that the miR-4516 mimic according to the present invention can very effectively inhibit cell growth even in a TNBC cell line co-cultured with a CAF cell line similar to a tumor microenvironment.
[실시예 9] [Example 9] miR-4516 모방체에 의한 TNBC 세포주의 증식 억제 효과 확인(3)Confirmation of the effect of miR-4516 mimic on the proliferation of TNBC cell lines (3)
상기 실시예 2와 동일한 방법으로 miR-4516이 삽입된 pENTRTM/H1/TO 벡터가 형질전환된 CAF 세포주를 트렌스웰 인설트(Transwell insert)에 GW4869(Cas no. 6823-69-4)를 처리하고 24시간 뒤에, MDA-MB-231 세포주, BT-20 세포주 또는 Hs578T 세포주가 분주된 6웰 플레이트 위에 삽입하여 상기 실시예 8과 동일한 방법으로 공배양 하였다. 그런 다음, 상기 실시예 2와 동일한 방법으로 CCK-8을 이용하여 상기 세포주들의 성장율을 측정하여, 그 결과를 도 9에 나타내었다.In the same manner as in Example 2 above, the CAF cell line transformed with the miR-4516-inserted pENTR TM /H1/TO vector was treated with GW4869 (Cas no. 6823-69-4) in a Transwell insert. After 24 hours, the MDA-MB-231 cell line, BT-20 cell line, or Hs578T cell line was inserted onto a 6-well plate dispensed and co-cultured in the same manner as in Example 8. Then, the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. 9.
도 9에서 보는 바와 같이, miR-4516이 과발현된 CAF 세포주에 엑소좀 억제제인 GW4869가 처리되었을 때에는 TNBC 세포주들의 성장율이 각각 10.3(MDA-MB-231 세포주), 7.8(BT-20 세포주) 및 9.1(Hs578T 세포주) 만큼 GW4869를 처리하지 않은 경우에 비하여 증가되었다.As shown in Figure 9, when the CAF cell line overexpressing miR-4516 was treated with the exosome inhibitor GW4869, the growth rates of the TNBC cell lines were 10.3 (MDA-MB-231 cell line), 7.8 (BT-20 cell line), and 9.1, respectively. (Hs578T cell line) increased as compared to the case not treated with GW4869.
상기 결과를 통해 본 발명에 따른 miR-4516은 CAF 세포주의 엑소좀을 통해 방출되어 TNBC 세포주의 성장을 매우 효과적으로 억제할 수 있음을 알 수 있다.From the above results, it can be seen that miR-4516 according to the present invention is released through the exosomes of the CAF cell line and can very effectively inhibit the growth of the TNBC cell line.
[실시예 10] [Example 10] miR-4516 모방체에 의한 TNBC 세포주의 증식 억제 효과 확인(4)Confirmation of the proliferation inhibitory effect of TNBC cell line by miR-4516 mimic (4)
비 TNBC 세포주인 BT-474 세포주, MCF 7 세포주 및 SK-BR-3 세포주와, TNBC 세포주인 MDA-MB-231 세포주, BT-20 세포주 및 Hs578T 세포주에서 상기 실시예 7과 동일한 방법으로 miR-4516 모방체를 0 nM, 4 nM, 10 nM, 50 nM 또는 100 nM을 처리한 뒤, 상기 실시예 2와 동일한 방법으로 CCK-8을 이용하여 상기 세포주들의 성장율을 측정하여, 그 결과를 도 10에 나타내었다.In the non-TNBC cell line BT-474 cell line, MCF 7 cell line and SK-BR-3 cell line, and TNBC cell line MDA-MB-231 cell line, BT-20 cell line, and Hs578T cell line in the same manner as in Example 7 miR-4516 After the mimic was treated with 0 nM, 4 nM, 10 nM, 50 nM or 100 nM, the growth rate of the cell lines was measured using CCK-8 in the same manner as in Example 2, and the results are shown in FIG. Indicated.
도 10에서 보는 바와 같이, 비 TNBC 세포주들에서는 암 세포주의 성장 억제 효과가 거의 나타나지 않았으며, MCF7 세포주의 경우에는 100 nM을 처리하였을 때 오히려 성장이 증가되었다. 그러나, TNBC 세포주들에서는 농도에 의존적으로 암 세포주의 성장이 매우 효과적으로 억제되었다.As shown in FIG. 10, in the non-TNBC cell lines, the growth inhibitory effect of the cancer cell line hardly appeared, and in the case of the MCF7 cell line, when 100 nM was treated, the growth was rather increased. However, in TNBC cell lines, the growth of cancer cell lines was very effectively inhibited in a concentration-dependent manner.
상기 결과를 통해, 본 발명에 따른 miR-4516은 비 TNBC 세포주가 아닌, TNBC 세포주의 증식을 매우 효과적으로 억제할 수 있음을 알 수 있다.From the above results, it can be seen that miR-4516 according to the present invention can very effectively inhibit the proliferation of TNBC cell lines, not non-TNBC cell lines.
[[ 실시예Example 11] 11] miRmiR -4516 및 -4516 and FOSL1의FOSL1 mRNA와mRNA and 환자 생존율과의 Patient survival rate 상관 관계correlation 분석 결과 Analysis
miR-4516 및 FOSL1의 mRNA 발현 수준과 환자의 생존율을 분석하기 위해, 비 TNBC 또는 TNBC 환자 조직으로부터 상기 실시예 1과 동일한 방법으로 전체 RNA를 추출한 뒤, miR-4516 및 FOSL1에 특이적인 프라이머를 이용하여 실시간 중합효소 연쇄반응을 수행하였다. 그런 다음, miR-4516 및 FOSL1의 mRNA 발현 수준의 상관관계에 대해 Windows 용 Prism 6 (GraphPad Software, Inc., La Jolla, CA, USA)를 통해 분석하였다. 또한, 4 ㎛로 절편화된 상기 환자의 조직을 실란이 코팅된 슬라이드에 올려놓고, 탈 파라핀화 한 뒤에 0.3 %(v/v) 과산화수소가 포함된 인산완충식염수 및 pH 6.5, 10 mM 소듐 시트레이트 완충액을 넣은 뒤 700 W 전자레인지에서 15분 동안 반응시켰다. 이후, 상기 슬라이드에 1 % (w/v)의 소 혈청 알부민이 포함되어 있는 완충액을 이용하여 30분 동안 블로킹 하고, FOSL1에 특이적인 항체와 반응시킨 뒤에 시각화 하여, 그 결과를 도 11의 A 및 B에 나타내었다.In order to analyze the mRNA expression level of miR-4516 and FOSL1 and the survival rate of the patient, total RNA was extracted from non-TNBC or TNBC patient tissue in the same manner as in Example 1, and then primers specific for miR-4516 and FOSL1 were used. Thus, real-time polymerase chain reaction was performed. Then, the correlation of the mRNA expression levels of miR-4516 and FOSL1 was analyzed by
도 11의 A에서 보는 바와 같이, 유방암 환자의 조직에서 miR-4516의 발현 수준이 증가될수록 FOSL1의 mRNA 발현 수준은 감소되는, 즉 음의 상관관계인 것을 확인하였다.As shown in FIG. 11A, it was confirmed that as the expression level of miR-4516 in breast cancer patients increased, the mRNA expression level of FOSL1 decreased, that is, a negative correlation.
또한, 도 11의 B에서 보는 바와 같이, 비 TNBC 환자에 비하여, TNBC 환자의 조직에서는 miR-4516의 발현 수준이 감소되어 있었으며, FOSL1의 단백질의 발현 수준이 증가되어 있는 것을 확인하였다.In addition, as shown in FIG. 11B, compared to the non-TNBC patients, it was confirmed that the expression level of miR-4516 was decreased in the tissues of the TNBC patient, and the expression level of the protein of FOSL1 was increased.
상기 결과를 통해 본 발명에 따른 miR-4516이 표적하는 FOSL1의 mRNA 발현 수준은 TNBC에서 특이적으로 증가되어 있으며, 이와 같은 현상은 miR-4516이 감소되어 있어 나타나는 것임을 알 수 있다.From the above results, it can be seen that the mRNA expression level of FOSL1 targeted by miR-4516 according to the present invention is specifically increased in TNBC, and this phenomenon is caused by a decrease in miR-4516.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As described above, a specific part of the present invention has been described in detail, and it is obvious that this specific technology is only a preferred embodiment for those of ordinary skill in the art, and the scope of the present invention is not limited thereto. Therefore, it will be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.
<110> Industry-Academic Cooperation Foundation, Yonsei University <120> A composition for preventing, improving or treating for cancer <130> PDPB194090 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> miR-4516 <400> 1 gggagaaggg ucggggc 17 <210> 2 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> miR-4516 <400> 2 cccgacccuu cucccuu 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-132-3p forward primer <400> 3 taacagtcta cagccatggt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-132-3p reverse primer <400> 4 aacgagacga cgacagactt t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-199b-5p forward primer <400> 5 cccagtgttt agactatctg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-199b-5p reverse primer <400> 6 aacgagacga cgacagactt t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-29b-3p forward primer <400> 7 tagcaccatt tgaaatcagt 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-29b-3p reverse primer <400> 8 aacgagacga cgacagactt t 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-1253 forward primer <400> 9 agagaagaag atcagcctgc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-1253 reverse primer <400> 10 aacgagacga cgacagactt t 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-1915 forward primer <400> 11 ccccagggcg acgcggcggg 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-1915 reverse primer <400> 12 aacgagacga cgacagactt t 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-144-3p forward primer <400> 13 tacagtatag atgatgtact 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-144-3p reverse primer <400> 14 aacgagacga cgacagactt t 21 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> miR-320e forward primer <400> 15 aaagctgggt tgagaagg 18 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-320e reverse primer <400> 16 aacgagacga cgacagactt t 21 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> miR-4516 forward primer <400> 17 gggagaaggg tcggggc 17 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-4516 reverse primer <400> 18 aacgagacga cgacagactt t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOSL1 forward primer <400> 19 agctgcagaa gcagaaggag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOSL1 reverse primer <400> 20 ggagttaggg agggtgtggt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX5 forward primer <400> 21 ggaggagtga atcagcttgg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX5 reverse primer <400> 22 ggcttgatgc ttcctgtctc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TCL1A forward primer <400> 23 gcctgggaga agttcgtgta 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TCL1A reverse primer <400> 24 actaagcgcc agaaactgga 20 <110> Industry-Academic Cooperation Foundation, Yonsei University <120> A composition for preventing, improving or treating for cancer <130> PDPB194090 <160> 24 <170> KoPatentIn 3.0 <210> 1 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> miR-4516 <400> 1 gggagaaggg ucggggc 17 <210> 2 <211> 17 <212> RNA <213> Artificial Sequence <220> <223> miR-4516 <400> 2 cccgacccuu cucccuu 17 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-132-3p forward primer <400> 3 taacagtcta cagccatggt 20 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-132-3p reverse primer <400> 4 aacgagacga cgacagactt t 21 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-199b-5p forward primer <400> 5 cccagtgttt agactatctg 20 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-199b-5p reverse primer <400> 6 aacgagacga cgacagactt t 21 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-29b-3p forward primer <400> 7 tagcaccatt tgaaatcagt 20 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-29b-3p reverse primer <400> 8 aacgagacga cgacagactt t 21 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-1253 forward primer <400> 9 agagaagaag atcagcctgc 20 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-1253 reverse primer <400> 10 aacgagacga cgacagactt t 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-1915 forward primer <400> 11 ccccagggcg acgcggcggg 20 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-1915 reverse primer <400> 12 aacgagacga cgacagactt t 21 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> miR-144-3p forward primer <400> 13 tacagtatag atgatgtact 20 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-144-3p reverse primer <400> 14 aacgagacga cgacagactt t 21 <210> 15 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> miR-320e forward primer <400> 15 aaagctgggt tgagaagg 18 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-320e reverse primer <400> 16 aacgagacga cgacagactt t 21 <210> 17 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> miR-4516 forward primer <400> 17 gggagaaggg tcggggc 17 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> miR-4516 reverse primer <400> 18 aacgagacga cgacagactt t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOSL1 forward primer <400> 19 agctgcagaa gcagaaggag 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FOSL1 reverse primer <400> 20 ggagttaggg agggtgtggt 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX5 forward primer <400> 21 ggaggagtga atcagcttgg 20 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> PAX5 reverse primer <400> 22 ggcttgatgc ttcctgtctc 20 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TCL1A forward primer <400> 23 gcctgggaga agttcgtgta 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TCL1A reverse primer <400> 24 actaagcgcc agaaactgga 20
Claims (12)
상기 miR-4516 또는 이의 모방체는 FOSL1(FOS-like antigen 1) 유전자로부터 전사된 mRNA의 3'-UTR을 표적으로 하는 것인, 약학 조성물.The method of claim 1,
The miR-4516 or a mimic thereof targets the 3'-UTR of mRNA transcribed from the FOSL1 (FOS-like antigen 1) gene.
상기 miR-4516 또는 이의 모방체는 발현 벡터에 포함되거나, 세포에 도입된 형태인 것인, 약학 조성물.The method of claim 1,
The miR-4516 or a mimic thereof is contained in an expression vector or introduced into a cell, the pharmaceutical composition.
상기 세포는 섬유아세포인 것인, 약학 조성물.The method of claim 3,
Wherein the cells are fibroblasts, pharmaceutical composition.
상기 암은 유방암, 방광암, 자궁경부암, 대장암, 폐암, 췌장암, 위암, 난소암, 혈액암, 간암, 전립선암 및 두경부암으로 이루어진 군으로부터 선택되는 적어도 하나인 것인, 약학 조성물.The method of claim 1,
The cancer is at least one selected from the group consisting of breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, gastric cancer, ovarian cancer, blood cancer, liver cancer, prostate cancer, and head and neck cancer.
상기 유방암은 삼중음성유방암(Triple-negative breast cancer; TNBC)인 것인, 약학 조성물.The method of claim 5,
The breast cancer is triple-negative breast cancer (TNBC), the pharmaceutical composition.
상기 생물학적 시료에서 miR-4516이 존재하는 수준을 확인하는 단계;를 포함하는 암 치료제의 스크리닝 방법.Treating a desired candidate substance in a biological sample isolated from a cancer patient; And
Checking the level of miR-4516 present in the biological sample; screening method for a cancer treatment comprising a.
상기 생물학적 시료에서 miR-4516이 존재하는 수준이 후보물질을 처리하기 이전에 비하여 증가된 경우, 상기 후보물질을 암 치료제로 선별하는 단계를 더 포함하는, 암 치료제의 스크리닝 방법.The method of claim 9,
When the level of miR-4516 present in the biological sample is increased compared to prior to treatment of the candidate substance, the method further comprising selecting the candidate substance as a cancer treatment agent.
상기 암은 유방암, 방광암, 자궁경부암, 대장암, 폐암, 췌장암, 위암, 난소암, 혈액암, 간암, 전립선암 및 두경부암으로 이루어진 군으로부터 선택되는 적어도 하나인 것인, 암 치료제의 스크리닝 방법.The method of claim 9,
The cancer is at least one selected from the group consisting of breast cancer, bladder cancer, cervical cancer, colon cancer, lung cancer, pancreatic cancer, gastric cancer, ovarian cancer, hematologic cancer, liver cancer, prostate cancer, and head and neck cancer.
상기 유방암은 삼중음성유방암(Triple-negative breast cancer; TNBC)인 것인, 암 치료제의 스크리닝 방법.The method of claim 11,
The breast cancer is triple-negative breast cancer (TNBC), the method of screening for cancer treatment.
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