KR101497930B1 - Composition for cell senescence comprising an inhibitor against heparan sulfate 2-O sulfotransferase 1 gene, or a protein encoded by the gene and method for inducing cell senescence using the same - Google Patents

Composition for cell senescence comprising an inhibitor against heparan sulfate 2-O sulfotransferase 1 gene, or a protein encoded by the gene and method for inducing cell senescence using the same Download PDF

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KR101497930B1
KR101497930B1 KR1020130069698A KR20130069698A KR101497930B1 KR 101497930 B1 KR101497930 B1 KR 101497930B1 KR 1020130069698 A KR1020130069698 A KR 1020130069698A KR 20130069698 A KR20130069698 A KR 20130069698A KR 101497930 B1 KR101497930 B1 KR 101497930B1
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이재선
정승희
김봉조
이형철
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Abstract

본 발명은 세포 노화 유도용 조성물 및 이를 이용한 세포 노화 유도 방법에 관한 것으로, 상기 조성물은 종양세포에서 HS2ST1 유전자를 억제하여 세포의 노화를 유도할 수 있으므로, 종양세포에서 항암 효과를 나타낼 수 있다.The present invention relates to a composition for inducing cell senescence and a method for inducing cell senescence using the same, and the composition can induce senescence of cells by inhibiting the HS2ST1 gene in tumor cells, and thus can exhibit an anticancer effect in tumor cells.

Description

ＨＳ2ＳＴ1 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제를 포함하는 세포 노화 유도용 조성물 및 이를 이용한 세포 노화 유도 방법{Composition for cell senescence comprising an inhibitor against heparan sulfate 2-O sulfotransferase 1 gene, or a protein encoded by the gene and method for inducing cell senescence using the same}[0001] The present invention relates to a composition for inducing cell senescence comprising an HS2ST1 gene or an inhibitor against a protein encoded by the gene, and a method for inducing cell senescence using the same. the gene and method for inducing cell senescence using the same}

본 발명은 HS2ST1 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제를 포함하는 세포 노화 유도용 조성물 및 이를 이용한 세포 노화 유도 방법에 관한 것이다.The present invention relates to a composition for inducing cell senescence comprising an inhibitor against the HS2ST1 gene or a protein encoded by the gene, and a method for inducing cell senescence using the same.

종양세포 조기 노화는 스트레스-유도 조기 노화라고도 하며, 다양한 자극에 의해 종양세포에서 발생하는 노화를 의미한다. 종양세포는, 일정한 횟수만큼만 분열하고 노화되는 정상세포와 달리, 암화 과정에서 복제성 노화의 특성이 변화되어 무한정 분열하는 세포들이므로, 종양세포는 세포 노화과정이 진행되지 않을 것이라는 개념이 일반적이었다. 그러나, 최근 종양세포도 다양한 자극에 의해 세포 노화가 급격히 유도됨이 알려지게 되었고 이를 스트레스-유도 조기 노화 (stress-induced premature senescence)라 한다(Sugrue et al., Proc. Natl. Acad.Sci.USA, 94:9648-9653, 1997; Mason et al., Oncogene, 23;9238-9246, 2004). 종양세포의 노화를 유도할 수 있는 스트레스원으로는 유전적 독성 화학물질 (예, etoposide, cyclophophamide), 방사선, 자외선 등이 보고되었다(Hemann and Narita, Genes & Dev., 21:1-5, 2007; Chang et al., Proc. Natl. Acad. Sci. USA, 99:389-394, 2002).Early aging of tumor cells is also called stress-induced premature aging, which means aging that occurs in tumor cells by various stimuli. Unlike normal cells, which divide and aging only a certain number of times, tumor cells are cells that change indefinitely due to the change of the characteristics of replication aging in the process of carcinogenesis. Therefore, the concept that tumor cells do not progress in cell aging process was common. Recently, however, it has become known that cell senescence is rapidly induced by various stimuli in tumor cells, which is called stress-induced premature senescence (Sugrue et al., Proc. Natl. Acad. Sci. USA , 94: 9648-9653, 1997; Mason et al., Oncogene, 23: 9238-9246, 2004). Genetic toxic chemicals (eg, etoposide, cyclophophamide), radiation, and ultraviolet light have been reported as sources of stress that can induce tumor cell senescence (Hemann and Narita, Genes & Dev., 21: 1-5, 2007 Chang et al., Proc Natl Acad Sci USA, 99: 389-394, 2002).

최근 스트레스 유도 조기노화를 이용한 종양세포의 노화를 통한 종양세포 증식 억제가 암 환자 치료를 위한 효율적인 기전으로 제시되었으며 (Roninson et al., Drug Resist Updates, 4:303-313, 2001; Campisi, Science, 309:886-887, 2005), 세포 노화 유도 기전 연구를 통한 암 치료의 효율성 증대가 필수적임이 제안되었다(Narita 및 Lowe, Nature Medicine, 11:920-922, 2005). 또한, 종양의 악성화가 저지된 암 환자의 조직 분석 결과, 종양세포 노화가 효율적으로 유도되었음이 보고되었고(Collado et al., Nature, 436:642, 2005), 종양 억제 단백질 p53이 작용해 세포 노화를 통해 종양조직이 효율적으로 제거됨이 종양모델 동물 실험에서 입증되었다(Wue et al., Nature, 445:656-660, 2007). 이는 종양 치료에 세포 노화가 유용하게 활용될 수 있음을 제시하는 것이다. 실질적으로 종양세포의 노화기전 활성화는 세포사멸 기전 활성화보다 낮은 용량의 항암제나 방사선 투여를 가능하게 함으로서 암치료 부작용을 개선할 수 있고, 세포사멸에 대한 저항성을 획득한 종양세포의 암 치료 저항성 극복에 이용될 수 있다(Rebbaa, Cencer Lett, 219:1-13, 2005).Recently, inhibition of tumor cell proliferation through aging of tumor cells using stress-induced premature aging has been suggested as an effective mechanism for the treatment of cancer patients (Roninson et al., Drug Resist Updates, 4: 303-313, 2001; Campisi, Science, 309: 886-887, 2005), it has been suggested that increasing the efficiency of cancer treatment through research on the induction of cell senescence is essential (Narita and Lowe, Nature Medicine, 11: 920-922, 2005). In addition, tissue analysis of cancer patients with inhibited tumor metastasis showed that tumor cell senescence was efficiently induced (Collado et al., Nature, 436: 642, 2005) (Wue et al., Nature, 445: 656-660, 2007). ≪ RTI ID = 0.0 > This suggests that cell senescence may be useful for tumor therapy. Actually, the activation of aging mechanism of tumor cells enables to reduce the side effects of cancer treatment by enabling the administration of lower dose of anti-cancer agent or radiation than the activation of cell death mechanism, and overcoming resistance to cancer treatment of tumor cells that have acquired resistance to apoptosis (Rebbaa, Cencer Lett, 219: 1-13, 2005).

한편 HS2ST1 (heparan sulfate 2-O sulfotransferase 1)은 NCBI(미국 국립생물공학정보센터; National Center for Biotechnology Information) Access No. NM_012262 (서열번호 1) 또는 NM_001134492 (서열번호 2)인 유전자로 종양세포 조기노화와 관련하여 알려진 바가 없다. HS2ST1 (heparan sulfate 2-O sulfotransferase 1) was deposited at the NCBI (National Center for Biotechnology Information) Access. It is known that NM_012262 (SEQ ID NO: 1) or NM_001134492 (SEQ ID NO: 2) is not related to early senescence of tumor cells.

Sugrue et al., Proc. Natl. Acad.Sci.USA, 94:9648-9653, 1997, Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53.Sugrue et al., Proc. Natl. Acad.Sci.USA, 94: 9648-9653, 1997, Wild-type p53 triggers a rapid senescence program in human tumor cells lacking functional p53. Mason et al., Oncogene, 23;9238-9246, 2004, Molecular signature of oncogenic ras-induced senescence.Mason et al., Oncogene, 23: 9238-9246, 2004, Molecular signature of oncogenic ras-induced senescence. Hemann and Narita, Genes & Dev., 21:1-5, 2007, Oncogenes and senescence: breaking down in the fast lane.Hemann and Narita, Genes & Dev., 21: 1-5, 2007, Oncogenes and senescence: breaking down in the fast lane. Chang et al., Proc. Natl. Acad. Sci. USA, 99:389-394, 2002, Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent.Chang et al., Proc. Natl. Acad. Sci. USA, 99: 389-394, 2002, Molecular determinants of terminal growth arrest induced in tumor cells by a chemotherapeutic agent. Roninson et al., Drug Resist Updates, 4:303-313, 2001, If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells.Roninson et al., Drug Resist Updates, 4: 303-313, 2001, If not apoptosis, then what? Treatment-induced senescence and mitotic catastrophe in tumor cells. Campisi, Science, 309:886-887, 2005, Suppressing cancer: the importance of being senescent.Campisi, Science, 309: 886-887, 2005, Suppressing cancer: the importance of being senescent. Narita 및 Lowe, Nature Medicine, 11:920-922, 2005, Senescence comes of age.Narita and Lowe, Nature Medicine, 11: 920-922, 2005, Senescence comes of age. Collado et al., Nature, 436:642, 2005, Tumour biology: senescence in premalignant tumours.Collado et al., Nature, 436: 642, 2005, Tumor biology: senescence in premalignant tumors. Wue et al., Nature, 445:656-660, 2007, Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas.Wue et al., Nature, 445: 656-660, 2007, Senescence and tumor clearance is triggered by p53 restoration in murine liver carcinomas. Rebbaa, Cencer Lett, 219:1-13, 2005, Targeting senescence pathways to reverse drug resistance in cancer.Rebbaa, Cencer Lett, 219: 1-13, 2005, Targeting senescence pathways to reverse drug resistance in cancer.

이에 본 발명자들은 종양세포에서 세포내 황산화 과정을 억제할 경우(예를 들어, HS2ST1 (heparan sulfate 2-O sulfotransferase 1) 유전자를 억제할 경우) 종양세포의 노화를 유도함을 발견하고 본 발명을 완성하였다.Accordingly, the inventors of the present invention discovered that when inhibiting the intracellular sulfation process (for example, suppressing the HS2ST1 (heparan sulfate 2-O sulfotransferase 1) gene) in tumor cells leads to aging of tumor cells, Respectively.

따라서 본 발명의 목적은 세포 노화 유도용 조성물을 제공하는 데에 있다. 또한 본 발명의 다른 목적은 세포 노화 유도용 조성물을 이용한 세포 노화 유도 방법을 제공하는 데에 있다.Accordingly, an object of the present invention is to provide a composition for inducing cell senescence. Another object of the present invention is to provide a method for inducing cell senescence using a composition for inducing cell senescence.

상기 목적을 달성하기 위하여, 본 발명은 HS2ST1 (heparan sulfate 2-O sulfotransferase 1) 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제를 포함하는 세포 노화 유도용 조성물을 제공한다.In order to achieve the above object, the present invention provides a composition for inducing cell senescence comprising an inhibitor against HS2ST1 (heparan sulfate 2-O sulfotransferase 1) gene or a protein encoded by the gene.

본 발명에서 상기 유전자는 mRNA(messenger Ribonucleic acid)일 수 있으며, 상기 억제제는 상기 mRNA를 억제할 수 있는 siRNA(small interfering RNA)일 수 있다.In the present invention, the gene may be messenger ribonucleic acid (mRNA), and the inhibitor may be siRNA (small interfering RNA) capable of inhibiting the mRNA.

본 발명에서 상기 HS2ST1 유전자는 서열번호 1의 염기서열 (NCBI Access No. NM_012262) 또는 서열번호 2의 염기서열 (NCBI Access No. NM_001134492)을 포함한다. 또한 상기 서열번호 1의 염기서열 또는 서열번호 2의 염기서열에서 하나 이상의 염기가 결실, 치환 또는 삽입된 염기서열을 포함한다.In the present invention, the HS2ST1 gene includes the nucleotide sequence of SEQ ID NO: 1 (NCBI Access No. NM_012262) or the nucleotide sequence of SEQ ID NO: 2 (NCBI Access No. NM_001134492). Also, a base sequence of SEQ ID NO: 1 or a base sequence of deletion, substitution or insertion of one or more bases in the nucleotide sequence of SEQ ID NO: 2 is included.

본 발명에서 상기 HS2ST1 유전자가 암호화하는 단백질은 서열번호 3의 폴리펩티드 또는 서열번호 4의 폴리펩티드를 포함한다. 또한 서열번호 3의 폴리펩티드 또는 서열번호 4의 폴리펩티드에서 하나 이상의 아미노산의 결실, 치환 또는 삽입된 아미노산 서열을 포함한다. In the present invention, the protein encoded by the HS2ST1 gene comprises the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4. Or a polypeptide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4, wherein the amino acid sequence is deleted, substituted or inserted.

본 발명에서 상기 HS2ST1 유전자의 서열은 예시일 뿐 이에 한정되는 것은 아님은 당업자에게 자명하다. 상기 서열들에 대해 실질적인 서열 동일성 또는 실질적인 서열 상동성을 지닌 서열 또한 본 발명의 범주에 포함된다. 여기서 사용된 "실질적인 서열 동일성" 또는 "실질적인 서열 상동성"이라는 용어는 서열이 또 다른 서열과의 실질적인 구조적 또는 기능적 동일성을 나타냄을 표현하기 위해 사용된다. 이러한 차이는 예를 들어 다른 종 간의 코돈 용법의 고유의 변이에 기인한다. 2 이상의 다른 서열 사이의 유의적인 양의 서열 중복 또는 유사성이 있는 경우 이들 서열의 길이 또는 구조가 다르더라도 유사한 물리적 특성을 지니는 경우 구조적 차이는 무시할만한 정도가 된다.In the present invention, the sequence of the HS2ST1 gene is illustrative but not limited to those skilled in the art. Sequences having substantial sequence identity or substantial sequence homology to the sequences are also within the scope of the present invention. As used herein, the term "substantial sequence identity" or "substantial sequence identity" is used to denote that the sequence represents substantial structural or functional identity with another sequence. These differences are due, for example, to inherent variations in the codon usage between different species. If there is a significant amount of sequence redundancy or similarity between two or more different sequences, the structural differences will be negligible if they have similar physical properties even if the length or structure of these sequences is different.

본 발명에서 상기 억제제는 HS2ST1 유전자 발현을 억제할 수 있는 siRNA일 수 있다. 즉, 상기 억제제는 HS2ST1 유전자에 대한 mRNA의 발현을 억제할 수 있는 siRNA일 수 있다. 상기 siRNA는 서열번호 5의 센스서열(5'-UGU AGU CUC UCC CAC AGA U-3') 및 이에 상보적인 서열번호 6의 안티센스 서열(5'-AUC UGU GGG AGA GAC UAC A-3')인 이중가닥 siRNA 일 수 있다. 상기 siRNA는 센스서열 및/또는 안티센스서열의 3' 말단에 티민 2 염기(dTdT)가 결합된 것일 수 있다.In the present invention, the inhibitor may be an siRNA capable of inhibiting HS2ST1 gene expression. That is, the inhibitor may be an siRNA capable of inhibiting the expression of mRNA for the HS2ST1 gene. The siRNA comprises a sense sequence (5'-UGU AGU CUC UCC CAC AGA U-3 ') of SEQ ID NO: 5 and an antisense sequence (5'-AUC UGU GGG AGA GAC UAC A-3' Double stranded siRNA. The siRNA may have a thymine base (dTdT) bound to the 3 'end of the sense sequence and / or the antisense sequence.

본 발명에서 상기 단백질에 대한 억제제는 항체일 수 있다. 상기 항체는 HS2ST1 유전자가 암호화하는 단백질에 특이적으로 결합할 수 있는 단일클론 항체, 다클론 항체, 및/또는 재조합 항체 등일 수 있으며, 시판되는 것을 구입하거나 공지의 방법에 의해 직접 제조할 수 있다.In the present invention, the inhibitor for the protein may be an antibody. The antibody may be a monoclonal antibody, a polyclonal antibody, and / or a recombinant antibody capable of specifically binding to a protein encoded by HS2ST1 gene, and may be commercially available or may be prepared directly by known methods.

본 발명에서 상기 세포는 암세포일 수 있다. 상기 암은 간암, 위암, 유방암, 결장암, 골암, 췌장암, 두부 또는 경부 암, 자궁암, 대장암, 폐암, 난소암, 직장암, 식도암, 소장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 전립선암, 방광암, 신장암, 수뇨관암, 신장세포암종, 신장골반암종, 골육종 및 중추신경계 종양으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. 바람직하게는 상기 세포는 유방암 세포일 수 있으나, 이에 한정되지 않는다.In the present invention, the cells may be cancer cells. The cancer may be selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectum cancer, esophageal cancer, small intestine cancer, The present invention can be any one or more selected from the group consisting of a tumor, a vaginal cancer, a vulvar carcinoma, a Hodgkin's disease, a prostate cancer, a bladder cancer, a renal cancer, a ureter cancer, a renal cell carcinoma, a kidney pelvic carcinoma, Preferably, the cell may be a breast cancer cell, but is not limited thereto.

본 발명의 세포 노화 유도용 조성물은 약학조성물일 수 있으며, 본 발명의 세포 노화 유도용 조성물 중 HS2ST1 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제에 의해 종양세포에 노화가 유도되어 항암작용을 나타낼 수 있다.The composition for inducing cell senescence of the present invention may be a pharmaceutical composition, and the HS2ST1 gene or an inhibitor for the protein encoded by the gene in the composition for inducing cell senescence of the present invention may induce aging of the tumor cells, have.

본 발명의 세포 노화 유도용 조성물은 생물학적 제제에 통상적으로 사용되는 담체, 희석제, 부형제 또는 둘 이상의 이들의 조합을 포함할 수 있다. 약제학적으로 허용 가능한 담체는 조성물을 생체내 전달에 적합한 것이면 특별히 제한되지 않으며, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상 을 혼합하여 이용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 환약, 캡슐, 과립 또는 정제로 제제화할 수 있다. 더 나아가 당 분야의 적정한 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.The composition for inducing cell senescence of the present invention may comprise a carrier, diluent, excipient or a combination of two or more thereof commonly used in biological preparations. The pharmaceutically acceptable carrier is not particularly limited as long as the composition is suitable for in vivo delivery, and may be a salt solution, a sterilized water, a Ringer's solution, a buffered saline solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, And if necessary, other conventional additives such as an antioxidant, a buffer, a bacteriostatic agent and the like may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate into main dosage forms such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules or tablets. Further, the pharmaceutical composition of the present invention can be suitably formulated according to each disease or ingredient, using appropriate methods in the art.

본 발명의 세포 노화 유도용 조성물은 특히, 종양세포의 노화를 유도하기 위하여 유전자 억제제, 예를 들어 siRNA 기준으로 성인 1일 1회 0.01ng/kg~100㎎/kg의 용량으로 투여할 수 있고, 단백질 억제제, 예를 들어 항체 기준으로 성인 1일 1회 2~10mg/kg 의 용량으로 투여할 수 있으며, 정맥내, 동맥내, 복강내, 근육내, 동맥내, 복강내, 흉골내, 경피, 비측내, 흡입, 국소, 직장, 경구, 안구내 또는 피내 경로를 통해 통상적인 방식으로 투여할 수 있다.The composition for inducing cell senescence of the present invention may be administered in an amount of 0.01 ng / kg to 100 mg / kg once a day for adults, in order to induce senescence of tumor cells, The compounds of the present invention may be administered in a dose of 2 to 10 mg / kg once a day for an adult, for example, on a protein inhibitor such as an antibody, and may be administered intravenously, intraarterially, intraperitoneally, intramuscularly, intraarterially, intraperitoneally, Intraperitoneal, intranasal, inhalation, topical, rectal, oral, intraocular, or intradermal routes.

본 발명의 세포 노화 유도용 조성물의 치료적으로 유효한 양은 여러 요소, 예를 들면 투여방법, 목적부위, 환자의 상태 등에 따라 달라질 수 있다. 따라서, 인체에 사용 시 투여량은 안전성 및 효율성을 함께 고려하여 적정량으로 결정되어야 한다. 동물실험을 통해 결정한 유효량으로부터 인간에 사용되는 양을 추정하는 것도 가능하다. The therapeutically effective amount of the composition for inducing cell senescence of the present invention may be varied depending on various factors, for example, an administration method, a target site, a patient's condition, and the like. Therefore, when used in the human body, the dosage should be determined in consideration of safety and efficacy. It is also possible to estimate the amount used in humans from the effective amount determined through animal experiments.

상기 다른 목적을 달성하기 위하여, 본 발명은 세포에 HS2ST1 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제를 포함하는 세포 노화 유도용 조성물을 처리하는 단계를 포함하는 세포의 노화 유도 방법을 제공한다.According to another aspect of the present invention, there is provided a method for inducing senescence of cells comprising the step of treating cells with an HS2ST1 gene or a composition for inducing cell senescence comprising an inhibitor for a protein encoded by the gene.

본 발명에서 상기 세포는 인간을 제외한 포유동물의 세포일 수 있다. 바람직하게는 상기 세포는 암세포일 수 있다. 상기 암은 간암, 위암, 유방암, 결장암, 골암, 췌장암, 두부 또는 경부 암, 자궁암, 대장암, 폐암, 난소암, 직장암, 식도암, 소장암, 항문부근암, 나팔관암종, 자궁내막암종, 자궁경부암종, 질암종, 음문암종, 호지킨병, 전립선암, 방광암, 신장암, 수뇨관암, 신장세포암종, 신장골반암종, 골육종 및 중추신경계 종양으로 구성된 군으로부터 선택되는 어느 하나 이상일 수 있다. 바람직하게는 상기 세포는 유방암세포일 수 있으나, 이에 한정되지 않는다.In the present invention, the cell may be a cell of a mammal other than a human. Preferably, the cell may be a cancer cell. The cancer may be selected from the group consisting of liver cancer, stomach cancer, breast cancer, colon cancer, bone cancer, pancreatic cancer, head or neck cancer, uterine cancer, colon cancer, lung cancer, ovarian cancer, rectum cancer, esophageal cancer, small intestine cancer, The present invention can be any one or more selected from the group consisting of a tumor, a vaginal cancer, a vulvar carcinoma, a Hodgkin's disease, a prostate cancer, a bladder cancer, a renal cancer, a ureter cancer, a renal cell carcinoma, a kidney pelvic carcinoma, Preferably, the cell may be a breast cancer cell, but is not limited thereto.

상기 다른 목적을 달성하기 위하여, 종양세포 노화 유도용 조성물의 제조를 위한 HS2ST1 (heparan sulfate 2-O sulfotransferase 1 유전자 또는 상기 유전자가 암호화하는 단백질에 대한 억제제의 용도를 제공한다.In order to accomplish the above object, there is provided use of HS2ST1 (heparan sulfate 2-O sulfotransferase 1 gene or an inhibitor for a protein encoded by the gene) for the preparation of a composition for inducing tumor cell senescence.

본 발명에 따른 세포 노화 유도용 조성물은 종양세포에서 HS2ST1 유전자를 억제하여 세포의 노화를 유도할 수 있으므로, 종양세포에서 항암 효과를 나타낼 수 있다.The composition for inducing cell senescence according to the present invention can induce cell senescence by inhibiting the HS2ST1 gene in tumor cells, and thus can exhibit an anticancer effect in tumor cells.

도 1은 인간 유방암 세포주 MCF7에 세포내 황산화 과정의 저해제인 차아염소산나트륨를 농도별 처리 또는 미처리(대조군)하고 노화-관련 베타갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 위상차 현미경 (ECLIPSE TE300, Nikon)을 사용하여 관찰한 결과를 보여주는 도이다.
도 2는 인간 유방암 세포주 MCF7에 세포내 황산화 과정의 저해제인 차아염소산나트륨를 농도별 처리 또는 미처리(대조군)하고 노화-관련 베타갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율을 나타낸 그래프이다.
도 3은 인간 유방암 세포주 MCF-7에 차아염소산나트륨을 농도별로 처리하고 24시간 배양한 후 p53 및 p21의 발현 정도를 확인한 웨스턴블럿팅 결과이다.
도 4는 황산화 과정의 저해제인 차아염소산나트륨(NaClO3)에 의한 세포내 황산화과정 중 하나인 헤파란 황산화(Heparan sulfate proteoglycan; HSPG)의 저해 효과를 보여주는 도이다.
도 5는 인간 섬유아 세포주 HDF의 계대횟수별 활성화된 노화-관련 베타-갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 위상차 현미경으로 관찰한 사진을 보여주는 도이다.
도 6은 인간 섬유아 세포주 HDF의 계대횟수별 활성화된 노화-관련 베타-갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율을 나타낸 그래프이다.
도 7은 인간 섬유아 세포주 HDF의 복제성 노화에 따른 헤파란 설페이트의 발현 감소를 공초점 현미경 (LSM-710, 칼 자이스 사)으로 630 X 배율로 관찰한 사진을 보여주는 도이다.
도 8은 인간 섬유아 세포주 HDF의 복제성 노화에 따른 헤파란 설페이트의 발현 감소를 확인한 웨스턴블럿팅 결과이다.
도 9는 HS2ST1 억제제 또는 HS3ST1 억제제에 의한 헤파란 황산화의 저해 효과를 보여주는 도이다.
도 10은 HS2ST1 억제제 또는 HS3ST1 억제제 처리에 의한 세포증식율 을 보여주는 도이다.
도 11은 인간 유방암 세포주 MCF-7에 siControl, siHS3ST1 또는 siHS2ST1를 처리하고 2 일 동안 배양한 후 Rb의 인산화 정도 및 p21 발현 정도를 확인한 웨스턴블럿팅 결과이다.
도 12는 인간 유방암 세포주 MCF7에 각각 siControl, siHS2ST1 siHS3ST1를 처리하고 노화-관련 베타갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 위상차 현미경 (ECLIPSE TE300, Nikon)을 사용하여 관찰한 결과를 보여주는 도이다.
도 13은 인간 유방암 세포주 MCF7에 각각 siControl, siHS2ST1 siHS3ST1를 처리하고 노화-관련 베타갈락토시다아제에 특이적인 염색시료를 이용한 염색법을 실시한 후 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율을 나타낸 그래프이다.FIG. 1 shows the results of immunohistochemical staining of the human breast cancer cell line MCF7 by using sodium hypochlorite as an inhibitor of intracellular sulfation process (control group) and staining method using a staining specimen specific for aging-related beta-galactosidase. ECLIPSE TE300, Nikon). ≪ / RTI >
FIG. 2 is a graph showing the effect of the sodium hypochlorite, which is an inhibitor of the intracellular sulfation process, on the human breast cancer cell line MCF7 by treatment with a concentration or an untreated control (control group) and staining using a staining specimen specific for aging-related beta-galactosidase, Beta-galactosidase-positive cells.
FIG. 3 is a Western blotting result in which the level of expression of p53 and p21 was confirmed after culturing human breast cancer cell line MCF-7 at a concentration of sodium hypochlorite for 24 hours.
Figure 4 shows the inhibitory effect of heparan sulfate proteoglycan (HSPG), which is one of the intracellular sulfation processes by sodium hypochlorite (NaClO 3 ), which is an inhibitor of sulfation process.
FIG. 5 is a photograph showing a phase contrast microscope image of a human fibroblast HDF after staining using a staining specimen specific to activated aging-related beta-galactosidase by passage number.
FIG. 6 shows the ratio of cells showing aging-related beta-galactosidase-positive cells after staining using a staining sample specific for activated aging-related beta-galactosidase by the number of passages of human fibroblast cell line HDF Fig.
FIG. 7 is a photograph showing a decrease in the expression of heparan sulfate by replication-age of human fibroblast HDF at 630 × magnification with a confocal microscope (LSM-710, Carl Zeiss).
FIG. 8 is a Western blotting result confirming a decrease in the expression of heparan sulfate by the replication-induced aging of the human fibroblast cell line HDF.
Figure 9 shows the inhibitory effect of heparan sulfation by HS2ST1 inhibitor or HS3ST1 inhibitor.
FIG. 10 shows the cell proliferation rate by treatment with HS2ST1 inhibitor or HS3ST1 inhibitor. FIG.
11 Western blot analysis confirmed the degree of phosphorylation of Rb and the expression level of p21 after treatment with human breast cancer cell line MCF-7, siControl, siHS3ST1 or siHS2ST1 for 2 days.
FIG. 12 shows the result of observing a human breast cancer cell line MCF7 using a staining method using siControl, siHS2ST1 siHS3ST1 and a staining sample specific for aging-related beta-galactosidase, using a phase contrast microscope (ECLIPSE TE300, Nikon) It is a figure that shows.
FIG. 13 shows the ratio of cells showing senescence-associated beta-galactosidase-positive cells after staining with a staining sample specific for aging-related beta-galactosidase and siControl, siHS2ST1 siHS3ST1 on human breast cancer cell line MCF7 Fig.

이하, 하기 실시예에 의해 본 발명을 보다 상세하게 설명한다. 그러나, 하기 실시예는 본 발명의 범위를 제한하는 것은 아니며, 이는 본 발명의 이해를 돕기 위한 것으로 해석되어야 할 것이다.Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples should not be construed as limiting the scope of the present invention, but should be construed to facilitate understanding of the present invention.

실시예Example 1.  One. 세포내Intracellular 황산화과정의Of the sulfation process 저해와 세포노화와의 유도와의 상관관계  Correlation between inhibition and induction of cell senescence 확Sho 인sign

1.1. 노화-관련 베타갈락토시다아제 활성 염색을 이용한 종양세포주의 1.1. Aging-associated beta-galactosidase activity. 황산Sulfuric acid 화과정의 저해에 의한 노화 유도 확인Confirmation of induction of aging by inhibition of the process

인간 유방암 세포주 MCF-7 (ATCC, USA)의 세포내 황산화과정의 저해와 세포노화와의 상관관계를 알아내기 위하여 노화-관련 베타갈락토시다아제 활성 염색을 실시하였다. 세포내 황산화과정의 저해제인 차아염소산나트륨 0 mM, 15 mM 또는 30 mM 처리한 인간 유방암 세포주 MCF7을 5% CO2 농도의 37℃ 항온 배양기에서 3일 동안 10% 우태아 혈청(fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium) 배양 배지에서 배양한 후 노화-관련 베타갈락토시다아제 활성 염색을 실시하였다. 상기 염색은 Dimri 등의 방법 (Dimri et al., Proc. Natl. Acad. Sci. USA, 92:9363-9367, 1995)에 따라 다음과 같이 수행하였다.Aging-related beta-galactosidase activity staining was performed to determine the inhibition of intracellular sulfation of the human breast cancer cell line MCF-7 (ATCC, USA) and its correlation with cell senescence. The inhibitor same procedure as set forth in 0 mM, 15 mM or 30 mM treatment of human breast cancer cell line MCF7 in in sulfation process cells 10% while at 37 ℃ constant temperature incubator of 5% CO 2 concentration of 3 days FBS (fetal bovine serum: FBS, Weljin Co., Ltd.) and a culture medium of Dulbeco's Modified Eagle's Medium (DMEM) containing 100 ug / ml of streptomycin, an antibiotic, and 100 units / ml penicillin (Gibco BRL), followed by aging-related beta-galactosidase activity And then stained. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995) as follows.

세포를 PBS로 2번 세척하고 3% 포름알데히드로 실온에서 3-5분간 고정하였다. 고정된 세포를 PBS로 다시 한 번 세척하고, β-갈락토시다아제 활성 염색 용액 (1 mg/ml의 X-Gal, 40 mM 시트르산 /소듐 포스페이트 (pH 6.0), 5 mM 포타슘 페로시아니드, 5 mM 포타슘 페리시아니드, 150 mM 염화클로라이드, 2 mM 마그네슘 클로라이드) 5 ml를 첨가하고, 37℃ 항온 배양기에서 12-16시간 반응시켰다. 반응을 진행하는 동안 빛이 들어가지 않도록 은박 호일로 배양접시를 싸서 배양하였다.The cells were washed twice with PBS and fixed with 3% formaldehyde at room temperature for 3-5 min. The immobilized cells were washed once more with PBS, and a solution of β-galactosidase active dye (1 mg / ml of X-Gal, 40 mM citric acid / sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM chloride chloride, 2 mM magnesium chloride) was added thereto, and the mixture was reacted for 12-16 hours at 37 ° C in a constant temperature incubator. During the reaction, the culture dish was wrapped with a thin foil foil to prevent light from entering.

인간 유방암 세포주 MCF7에 세포내 황산화 과정의 저해제인 차아염소산나트륨를 처리 또는 미처리(대조군)한 후 베타-갈락토시다아제 활성 정도는 위상차 현미경 (ECLIPSE TE300, Nikon)을 사용하여 관찰하고, 그 결과를 도 1에 나타내었다. 그 결과 대조군과 달리 황산화 과정의 저해제인 차아염소산나트륨을 처리한 후 3일 경과한 인간 유방암 세포주 MCF-7는 노화세포의 특징인 세포의 크기가 커지며 편평해진 것을 확인할 수 있다. 특히 차아염소산나트륨 처리 농도가 높아질수록 노화세포의 특징을 보이는 세포의 비중이 커짐을 확인할 수 있다.The activity of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon) after treatment or non-treatment (control) of sodium hypochlorite, which is an inhibitor of intracellular sulfation, in human breast cancer cell line MCF7. 1. As a result, unlike the control group, the human breast cancer cell line MCF-7, which has been treated three days after sodium hypochlorite, which is an inhibitor of the sulfation process, has a large cell size and flattened characteristic of aging cells. Especially, the higher the treatment concentration of sodium hypochlorite, the larger the proportion of cells showing the characteristics of aging cells.

또한, 현미경 (ECLIPSE TE300, Nikon)을 이용하여 염색된 세포의 갯수를 측정하여 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율에 관한 그래프를 도 2에 나타내었다. 특히 차아염소산나트륨 처리 농도가 높아질수록 노화-관련 베타-갈락토시다아제의 양성을 보이는 세포의 비율이 더 커짐을 확인할 수 있다.FIG. 2 is a graph showing the proportion of cells showing aging-related beta-galactosidase-positive cells by measuring the number of stained cells using a microscope (ECLIPSE TE300, Nikon). In particular, it can be seen that as the sodium hypochlorite treatment concentration is increased, the proportion of cells showing aging-related beta-galactosidase-positive is larger.

따라서, 상기 결과들로부터 종양세포에서 세포내 황산화과정을 억제할 경우 종양세포의 노화를 유도할 수 있음을 확인할 수 있다.Therefore, it can be confirmed from the above results that suppression of intracellular sulfation process in tumor cells can induce aging of tumor cells.

1.2. 1.2. 웨스턴Western 분석을 이용한 종양세포주의  Analysis of tumor cell lines using 황산화과정의Of the sulfation process 저해에 의한 노화 유도 확인 Confirmation of induction of aging by inhibition

차아염소산나트륨 0 mM, 15 mM 또는 30 mM을 처리하고 5% CO2 농도의 37℃ 항온 배양기에서 24시간 배양 배지에서 배양된 인간 유방암 세포주 MCF-7은 PBS (phosphate buffered saline)로 세척 후 세포 시료를 세포 용해 완충액 (50mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM PMSF, 50 mM NaF, 0.2 mM Na3VO4, 10 μg/ml 아프로티닌, 2 μg/ml 루펩틴)을 사용하여 단백질을 추출하고, 추출한 단백질은 11,000rpm으로 10분간 원심분리한 후 상등액만 취하여 브래드포드법 {bradford, M., Anal. Biochem. 72:248-254(1976)}을 이용하여 정량하였다. 20ug의 단백질 2X SDS 로딩 버퍼(60 mm Tris-Cl (pH6.8), 25% 글리세롤, 2% SDS, 14.4 mM 머캅토에탄올, 0.1% 브로모페놀 블루)를 가해 95℃에서 5분간 가열하고 8% 내지 10% SDS 폴리아크릴 아마이드 젤에서 80V로 2시간 전기영동하였다.The human breast cancer cell line MCF-7 cultured in a culture medium for 24 hours in a constant temperature incubator at a concentration of 0 mM, 15 mM, or 30 mM sodium hypochlorite at a concentration of 5% CO 2 was washed with PBS (phosphate buffered saline) Were incubated for 30 minutes in a cell lysis buffer (50 mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 μg / ml aprotinin, The extracted protein was centrifuged at 11,000 rpm for 10 minutes, and then the supernatant was taken out by the Bradford method {bradford, M., Anal. Biochem. 72: 248-254 (1976)). 20 g of protein 2X SDS loading buffer (60 mM Tris-Cl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) % To 10% SDS polyacrylamide gel at 80 V for 2 hours.

전기영동 후 분리된 단백질을 나이트로셀룰로오즈 멤브레인(Whatman 사)으로 전이시켰다. 단백질이 전이되어 있는 멤브레인은 5% 탈지분유가 함유된 PBS용액에 넣고 실온에서 1시간 동안 방치하여 블록킹(blocking)하고 1:500 ~ 1:1000으로 희석한 일차 항체들을 넣고 4℃에서 16시간동안 반응시켰다. 1차 항체는 다클론 항-p21 항체 (polyclonal anti-p21 Ab, Santa Cruz 사), 다클론 항-actin 항체 (polyclonal anti-actin Ab, Santa Cruz 사), 다클론 항-p53 항체 (polyclonal anti-p53 Ab, Leica 사) 를 사용하였고 2차 항체는 호스 래디쉬 퍼옥시다아제 복합 항-토끼 또는 항-쥐 항체 (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz 사)를 이용해 ECL (enhanced chemiluminescence) 시약 (Amersham 사)으로 세포노화여부를 확인하였다. 그 결과를 도 3에 나타내었다. 도 3은 인간 유방암 세포주 MCF-7에 차아염소산나트륨을 농도별로 처리하고 24시간 배양한 후 p53 및 p21의 발현 정도를 확인한 웨스턴블럿팅 결과이다. 도 3에서 액틴(Actin)은 모든 세포에서 동일한 수준으로 발현되는 유전자(하우스키핑 유전자, housekeeping gene)로 동량의 단백질에 대해 분석되었는지를 보여주는 기준으로 사용되었다. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Whatman). Protein-transferred membranes were blocked in PBS solution containing 5% skimmed milk, incubated at room temperature for 1 hour, and primary antibodies diluted 1: 500 ~ 1: 1000 were added and incubated at 4 ° C for 16 hours Lt; / RTI > Primary antibodies include polyclonal Anti-p21 antibody (polyclonal anti-p21 Ab, Santa Cruz), polyclonal anti-actin Ab (Santa Cruz), polyclonal anti-p53 Ab And secondary antibody was detected by ECL (enhanced chemiluminescence) reagent using horseradish peroxidase complex anti-rabbit or anti-mouse antibody (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz) (Amersham). The results are shown in Fig. FIG. 3 is a Western blotting result in which the level of expression of p53 and p21 was confirmed after culturing human breast cancer cell line MCF-7 at a concentration of sodium hypochlorite for 24 hours. In FIG. 3, actin was used as a reference to show that the same level of expression (the housekeeping gene) in all cells was analyzed for the same amount of protein.

웨스턴블럿팅 분석 결과를 통해, 차아염소산나트륨 처리에 의해 인간 유방암 세포주 MCF-7에서 노화 특이적 p53 및 p21의 발현이 증가하여 차아염소산나트륨에 의한 노화가 유도되었음을 확인할 수 있다. 특히 차아염소산나트륨 처리 농도가 높아질수록 노화 특이적 p53 및 p21의 발현 증가가 더 커져 차아염소산나트륨에 의한 노화 유도 비율이 커짐을 확인할 수 있다.Western blotting analysis showed that the treatment with sodium hypochlorite induced the senescence by sodium hypochlorite in the human breast cancer cell line MCF-7 by increasing the expression of aging-specific p53 and p21. Especially, as the concentration of sodium hypochlorite treatment increased, the increase in the expression of p53 and p21 specific to senescence was greater, and the rate of induction of senescence by sodium hypochlorite was increased.

따라서, 상기 결과들로부터 종양세포에서 세포내 황산화 과정을 억제할 경우 종양세포의 노화를 유도할 수 있음을 확인할 수 있다.Therefore, it can be confirmed from the above results that suppression of intracellular sulfation process in tumor cells can induce aging of tumor cells.

실시예Example 2.  2. 차아염소산나트륨의Of sodium hypochlorite 세포내Intracellular 황산화과정Sulfation process 중 하나인  One of 헤파란Heparan 황산화( Sulfation HeparanHeparan sulfatesulfate proteoglycanproteoglycan HSPGHSPG )의 저해 확인) Confirmation of inhibition

차아염소산나트륨 0 mM, 15 mM 또는 30 mM을 처리하고 5% CO2 농도의 37℃ 항온 배양기에서 24시간 10% 우태아 혈청(fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium) 배양 배지에서 배양하였다. 인간 유방암 세포주 MCF-7의 세포배양액을 걷어내고 PBS로 2회 세척하고 3.7 % 포름알데하이드에 20분 동안 상온에 방치하여 세포를 고정시켰다. 그 후 PBS로 2회 세척한 후 3% 소혈청알부민 (BSA)이 함유된 PBS 용액에 넣고 상온에서 30 분간 블로킹하고 1:100으로 희석한 항-헤파란 황산화 항체 (10E4, US biological 사)를 넣고 4℃에서 16시간동안 반응시켰다. PBS로 10분간 3회 세척후 2차 항체로 Alexa 555 복합 항-쥐 항체 (Molecular probe 사)를 1:200 으로 희석한 PBS 용액에 상온에서 30 분간 반응시켰다. PBS 로 10분간 2회 세척후 50 % 글리세롤로 마운팅 (mounting) 한 후 공초점 현미경 (LSM-710, 칼 자이스 사)으로 630 X 배율로 관찰하였다. 그 결과를 도 4에 나타내었다. Same procedure as set forth in 0 mM, 15 mM, or handle 30 mM and 10% for 24 hours at 37 ℃ constant temperature incubator of 5% CO 2 concentration of FBS (fetal bovine serum: FBS, ㈜ weljin) and antibiotic streptomycin 100 ug / ml and penicillin 100 units / ml (Gibco BRL) in a Dulbeco's Modified Eagle's Medium (DMEM) culture medium. Cell culture of human breast cancer cell line MCF-7 was removed, washed twice with PBS, and fixed in 3.7% formaldehyde for 20 minutes at room temperature. Heparan sulfate antibody (10E4, US biological) diluted 1: 100 was added to PBS solution containing 3% bovine serum albumin (BSA), and then blocked at room temperature for 30 minutes. And the mixture was reacted at 4 ° C for 16 hours. After washing three times for 10 minutes with PBS, Alexa 555 conjugated anti-mouse antibody (Molecular probe) was reacted as a secondary antibody in a 1: 200 diluted PBS solution at room temperature for 30 minutes. Washed twice with PBS for 10 minutes, mounted with 50% glycerol, and observed with a confocal microscope (LSM-710, Carl Zeiss) at 630 × magnification. The results are shown in Fig.

그 결과 대조군과 달리 황산화 과정의 저해제인 차아염소산나트륨을 처리한 후 24시간 경과한 인간 유방암 세포주 MCF-7에서 세포내 단백다당의 황산화과정 중 하나인 헤파란 황산화가 저해됨을 확인할 수 있다. 특히 차아염소산나트륨 처리 농도가 높아질수록 세포내 단백다당의 황산화과정 중 하나인 헤파란 황산화 저해 정도가 커짐을 확인할 수 있다.As a result, unlike the control group, heparan sulfate inhibition, which is one of the sulfation processes of intracellular protein polysaccharide, is inhibited in the human breast cancer cell line MCF-7 after 24 hours of treatment with sodium hypochlorite, which is an inhibitor of sulfation process. Especially, the higher the concentration of sodium hypochlorite treatment, the greater the degree of heparan sulfate inhibition, one of the sulfation processes of intracellular protein polysaccharides.

실시예Example 3.  3. 복제성Replicability 노화와  Aging and 단백다당의Protein-bound polysaccharide 헤파란Heparan 황산화Sulfation 과정의 연관성을  The relevance of the process 확인Confirm

3.1. 계대 횟수에 따른 3.1. Depending on the number of passages 복제성Replicability 노화 확인 Aging confirmation

복제성 노화를 연구하기 위하여, 인간 섬유아 세포주 HDF (ATCC, USA)를 100 mm 배양접시 (SPL 사) 상에서 10% 우태아 혈청 (fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM (Dulbeco's Modified Eagle's Medium) 배양 배지 안에서 5%의 CO2 농도 및 37℃ 온도가 항시 유지되는 세포배양기에서 배양하였다. 3~4일 후 HDF 세포가 배양접시에 90 % 이상 가득차면 배양액을 걷어내고 PBS (phosphate buffered saline, PBS (주) 웰진) 10 ml 로 남아있는 배양액을 씻어낸 후 트립신((주) 웰진)을 처리하여 배양접시에 붙어있는 HDF를 걷어내고 그 중 4분의 1의 세포만을 새로운 배양접시에 옮겨 위의 배양배지 안에서 5%의 CO2 농도 및 37℃ 온도가 항시 유지되는 세포배양기에서 배양하였다. 이러한 과정을 한번 거치면 계대횟수 1로 계산하여 10 번 계대배양하면 p10, 20번 계대배양하면 p20, 30번 계대배양하면 p30 으로 하여 각각 젊은 세포, 중간-늙은 세포, 늙은 세포로 구분하여 세포노화가 제대로 일어났는지 노화-관련 베타갈락토시다아제 활성 염색을 실시하였다. 상기 염색은 Dimri 등의 방법 (Dimri et al., Proc. Natl. Acad. Sci. USA, 92:9363-9367, 1995)에 따라 다음과 같이 수행하였다.Human fibroblast HDF (ATCC, USA) was cultured on a 100 mm culture dish (SPL) with 10% fetal bovine serum (FBS, Weljin) and 100 ug of streptomycin were cultured in / ml penicillin and 100 units / ml (Gibco BRL Co.) containing a DMEM (Dulbeco's Modified Eagle's medium ) cell culture incubator where the CO 2 concentration and 37 ℃ temperature of 5% in the medium maintained at all times. After 3 to 4 days, if the HDF cells are over 90% in the culture dish, the culture medium is removed and the remaining culture is washed with 10 ml of PBS (phosphate buffered saline, PBS, Weljin) And the HDF attached to the culture dish was removed. Only a quarter of the cells were transferred to a new culture dish and cultured in the above culture medium in a cell incubator maintained at a constant CO 2 concentration of 5% and a temperature of 37 ° C. When this process is performed once, the number of passages is calculated as 1, and when 10 passages are cultured, p20 is cultured by passage 20, p20 when 30 passages are cultured Aged-related beta-galactosidase activity staining was performed to distinguish whether the cell aging occurred properly by dividing into young cells, middle-aged cells, and old cells, respectively. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995) as follows.

세포를 PBS로 2번 세척하고 3% 포름알데히드로 실온에서 3-5분간 고정하였다. 고정된 세포를 PBS로 다시 한 번 세척하고, β-갈락토시다아제 활성 염색 용액 (1 mg/ml의 X-Gal, 40 mM 시트르산 /소듐 포스페이트 (pH 6.0), 5 mM 포타슘 페로시아니드, 5 mM 포타슘 페리시아니드, 150 mM 염화클로라이드, 2 mM 마그네슘 클로라이드) 1 ml를 첨가하고, 37℃ 항온 배양기에서 12-16시간 반응시켰다. 반응을 진행하는 동안 빛이 들어가지 않도록 은박 호일로 배양접시를 싸서 배양하였다.The cells were washed twice with PBS and fixed with 3% formaldehyde at room temperature for 3-5 min. The immobilized cells were washed once more with PBS, and a solution of β-galactosidase active dye (1 mg / ml of X-Gal, 40 mM citric acid / sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM chloride chloride, 2 mM magnesium chloride) was added thereto, followed by reaction at 37 ° C for 12-16 hours in a constant temperature incubator. During the reaction, the culture dish was wrapped with a thin foil foil to prevent light from entering.

베타-갈락토시다아제 활성 정도는 위상차 현미경 (ECLIPSE TE300, Nikon)을 사용하여 관찰하고, 그 결과를 도 5에 나타내었다. 또한, 현미경 (ECLIPSE TE300, Nikon)을 이용하여 염색된 세포의 갯수를 측정하여 전체 세포에 대한 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율에 관한 그래프를 도 6에 나타내었다. 도 6에서 p10 및 p20의 경우 검출할 수 있는 범위에 있지 않았다. 도 6에서 p10 및 p20의 경우 검출할 수 있는 범위에 있지 않았다. The degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon) and the results are shown in FIG. FIG. 6 is a graph showing the ratio of cells showing aging-related beta-galactosidase-positive cells to total cells by measuring the number of stained cells using a microscope (ECLIPSE TE300, Nikon). In the case of p10 and p20 in Fig. 6, it was not within the detectable range. In the case of p10 and p20 in Fig. 6, it was not within the detectable range.

도 5 및 6에 나타난 바와 같이, 계대 횟수가 증가될수록 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율이 증가함을 확인할 수 있다. As shown in FIGS. 5 and 6, it can be seen that as the number of passages increases, the proportion of cells showing aging-related beta-galactosidase-positive increases.

따라서, 계대 횟수가 증가될수록 복제성 노화가 진행됨을 확인할 수 있다.Therefore, it can be confirmed that the replication aging progresses as the number of passages increases.

3.2. 3.2. 복제성Replicability 노화에 따른  Aging 단백다당의Protein-bound polysaccharide 헤파란Heparan 황산화Sulfation 정도의 변화 확인 Confirm change of degree

복제성 노화와 단백다당의 헤파란 황산화 과정의 연관성을 확인하기 위하여, 항-헤파란 항체를 이용하여 염색하였다. 구체적으로, 인간 섬유아 세포주 HDF (ATCC, USA)를 100 mm 배양접시 (SPL 사) 상에서 10% 우태아 혈청 (fetal bovine serum: FBS, ㈜ 웰진) 과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM (Dulbeco's Modified Eagle's Medium) 배양 배지 안에서 5%의 CO2 농도 및 37℃ 온도가 항시 유지되는 세포배양기에서 배양하였다. 3~4일 후 HDF 세포가 배양접시에 90 % 이상 가득차면 배양액을 걷어내고 PBS (phosphate buffered saline, PBS (주) 웰진) 10 ml로 남아있는 배양액을 씻어낸 후 트립신((주) 웰진)을 처리하여 배양접시에 붙어있는 HDF를 걷어내고 그 중 4분의 1의 세포만을 새로운 배양접시에 옮겨 위의 배양배지 안에서 5%의 CO2 농도 및 37℃ 온도가 항시 유지되는 세포배양기에서 배양하였다. 이러한 과정을 한번 거치면 계대횟수 1로 계산하여 10 번 계대배양하면 p10, 20번 계대배양하면 p20, 30번 계대배양하면 p30 으로 하여 각각 젊은 세포, 중간-늙은 세포, 늙은 세포로 구분하였고 각각의 세포를 PBS로 2회 세척하고 3.7 % 포름알데하이드에 20분 동안 상온에 방치하여 세포를 고정시킨다. 그 후 PBS로 2회 세척한 후 3% 소혈청알부민 (BSA) 이 함유된 PBS 용액에 넣고 상온에서 30 분간 블로킹하고 1:100으로 희석한 항-헤파란 황산화 항체 (10E4, US biological 사)를 넣고 4℃에서 16시간동안 반응시켰다. PBS로 10분간 3회 세척 후 2차 항체로 Alexa 555 복합 항-쥐 항체 (Alexa 555 conjugated goat anti-mouse IgG, Molecular probe 사)를 1:200으로 희석한 PBS 용액에 상온에서 30 분간 반응시켰다. PBS로 10분간 2회 세척 후 50 % 글리세롤로 마운팅 (mounting)한 후 공초점 현미경 (LSM-710, 칼 자이스 사)으로 630 X 배율로 관찰하여 도 7에 나타내었다.In order to confirm the relationship between the replication aging and the heparan sulfation process of the protein polysaccharide, anti-heparan antibody was used for staining. Specifically, human fibroblast cell line HDF (ATCC, USA) was cultured on a 100 mm culture dish (SPL) with 10% fetal bovine serum (FBS, Weljin), 100 μg / ml of streptomycin as an antibiotic and 100 μg / ml of penicillin 100 Cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) culture medium containing 5% CO 2 and 37 ° C in a cell culture incubator with unit / ml (Gibco BRL). After 3 to 4 days, if the HDF cells are over 90% in the culture dish, the culture medium is removed and the remaining culture is washed with 10 ml of PBS (phosphate buffered saline, PBS, Weljin) And the HDF attached to the culture dish was removed. Only a quarter of the cells were transferred to a new culture dish and cultured in the above culture medium in a cell incubator maintained at a constant CO 2 concentration of 5% and a temperature of 37 ° C. When this process is performed once, it is divided into the young cell, the middle-aged cell, and the old cell, respectively, by counting the number of the passage number 1 and cultivating the 10th passage by p20, The cells are washed twice with PBS and fixed in 3.7% formaldehyde for 20 minutes at room temperature. Heparan sulfate antibody (10E4, US biological) diluted 1: 100 was added to PBS solution containing 3% bovine serum albumin (BSA), and then blocked at room temperature for 30 minutes. And the mixture was reacted at 4 ° C for 16 hours. After washing three times for 10 minutes with PBS, Alexa 555 conjugated goat anti-mouse IgG (Molecular Probe, Inc.) was reacted with 1: 200 diluted PBS solution as a secondary antibody for 30 minutes at room temperature. Washed twice with PBS for 10 minutes, mounted with 50% glycerol, observed with a confocal microscope (LSM-710, Carl Zeiss) at 630 X magnification and shown in FIG.

도 7과 동일 실험군의 세포를 PBS 로 세척 후 세포 시료를 세포 용해 완충액 (50mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM PMSF, 50 mM NaF, 0.2 mM Na3VO4, 10 ㎍/ml 아프로티닌, 2 ㎍/ml 루펩틴)을 사용하여 단백질을 추출하고, 추출한 단백질은 13,200rpm으로 20분간 원심분리한 후 상등액만 취하여 브래드포드법 {bradford, M., Anal. Biochem. 72:248-254(1976)}을 이용하여 정량하였다. 20㎍의 단백질 2X SDS 로딩 버퍼(60 mm Tris-Cl (pH6.8), 25% 글리세롤, 2% SDS, 14.4 mM 머캅토에탄올, 0.1% 브로모페놀 블루)를 가해 95℃에서 5분간 가열하고 8% 내지 10% SDS 폴리아크릴 아마이드 젤에서 80V로 2시간 전기영동하였다. 전기영동 후 분리된 단백질을 나이트로셀룰로오즈 멤브레인(Whatman 사)으로 전이시켰다. 단백질이 전이되어 있는 멤브레인은 5% 탈지분유가 함유된 PBS용액에 넣고 실온에서 1시간 동안 방치하여 블록킹(blocking)하고 1:500 ~ 1:1000으로 희석한 일차 항체들을 넣고 4℃에서 16시간동안 반응시켰다. 1차 항체는 단클론 항-헤파란 (monoclonal anti-HS Ab, US biology 사) 항체과 다클론 항-actin 항체 (polyclonal anti-actin Ab, Santa Cruz 사)를 사용하였고 2차 항체는 호스 래디쉬 퍼옥시다아제 복합 항-토끼 혹은 항-쥐 항체 (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz 사) 를 이용해 ECL (enhanced chemiluminescence) 시약 (Amersham 사)으로 세포내 단백다당의 헤파란 황산화 정도를 측정하였다. 도 8은 인간 섬유아 세포주 HDF의 복제성 노화에 따른 헤파란 설페이트의 발현 감소를 확인한 웨스턴블럿팅 결과이다.After washing to Figure 7 and the cells of the same experiment with PBS cells sample a cell lysis buffer (50mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM PMSF, 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 쨉 g / ml aprotinin, 2 쨉 g / ml rupeptin), and the extracted protein was centrifuged at 13,200 rpm for 20 minutes, and only the supernatant was taken out by the Bradford method {bradford, M., Anal. Biochem. 72: 248-254 (1976)). 20 μg of protein 2 × SDS loading buffer (60 mM Tris-Cl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) was added and heated at 95 ° C. for 5 minutes And electrophoresed at 80 V for 2 hours on 8% to 10% SDS polyacrylamide gels. After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Whatman). Protein-transferred membranes were blocked in PBS solution containing 5% skimmed milk, incubated at room temperature for 1 hour, and primary antibodies diluted 1: 500 ~ 1: 1000 were added and incubated at 4 ° C for 16 hours Lt; / RTI > The primary antibody was monoclonal anti-HS Ab antibody (US Biology) and polyclonal anti-actin antibody (Santa Cruz). The secondary antibody was horseradish peroxidase (Amersham) using a complex anti-rabbit or anti-mouse antibody (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz) The degree of sulfation was measured. FIG. 8 is a Western blotting result confirming a decrease in the expression of heparan sulfate by the replication-induced aging of the human fibroblast cell line HDF.

도 7 및 8에서 나타난 바와 같이, 복제성 노화가 진행될수록 세포내 헤파란의 황산화 정도가 감소됨을 확인하였다.As shown in FIGS. 7 and 8, it was confirmed that the degree of sulfation of heparan in the cells was decreased as the replication-induced aging progressed.

따라서, 상기 결과들로부터 종양세포에서 세포내 황산화 과정을 억제할 경우 종양세포의 노화를 유도할 수 있음을 확인할 수 있다.Therefore, it can be confirmed from the above results that suppression of intracellular sulfation process in tumor cells can induce aging of tumor cells.

실시예Example 4.  4. HS2ST1HS2ST1 억제제 및  Inhibitors and HS3ST1HS3ST1 억제제에 의한  By inhibitor 헤파란Heparan 황산화( Sulfation HeparanHeparan sulfate  sulfate proteoglycanproteoglycan ; ; HSPGHSPG )의 저해 확인) Confirmation of inhibition

단백다당의 헤파란 황산화 과정을 매개하는 효소들 1) HS3ST1 {(heparan sulfate 3-O sulfotransferase; NCBI Access No. NM_005114(서열번호 7)}, 2) HS2ST1 (heparan sulfate 2-O sulfotransferase; Access No. NM_012262(서열번호 1) 또는 NM_001134492(서열번호 2)}의 유전자 발현량 감소에 의한 효과를 확인하기 위해 종양세포주인 인간 유방암 세포주 MCF-7 (ATCC, USA)을 10% 우태아 혈청(fetal bovine serum: FBS, ㈜ 웰진)과 항생제 (Gibco BRL 사)가 포함된 DMEM 배양 배지 안에서 배양하고 작은 간섭 RNA의 주입 하루 전, 60 mm 배양접시로 계대배양한 후 2 μl의 RNAiMAX (invitrogen, Cat#13778-075)와 OptiMEM®I 배양배지 (Invitrogen, Cat#31985)를 섞고 HS3ST1 유전자에 대한 작은 간섭 RNA{siHS3ST1; 5'-CUG ACU ACA CCC AAG UGU U-3' (서열번호 8) 및 5'-AAC ACU UGG GUG UAG UCA G-3' (서열번호 9)의 염기서열로 이루어진 이중가닥 siRNA로, 각각의 서열 3' 말단에 티민 2 염기(dTdT)가 결합된 상태임)} 또는 HS2ST1 유전자에 대한 작은 간섭 RNA{siHS2ST1; 5'-UGU AGU CUC UCC CAC AGA U-3' (서열번호 5) 및 5'-AUC UGU GGG AGA GAC UAC A-3' (서열번호 6)로 이루어진 이중가닥 siRNA로, 각각의 서열 3' 말단에 티민 2 염기(dTdT)가 결합된 상태임}를 50 nM의 농도로 세포에 처리하였다. 6시간 후 배양배지를 10% 우태아 혈청 (fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM 배양 배지로 교환한 후, 5% CO2 농도의 37℃ 항온 배양기에서 4 일간 배양하였다.Heparan sulfate 2-O sulfotransferase (HS2ST1; Access No: NM05114 (SEQ ID NO: 7)), heparan sulfate 2-O sulfotransferase The human breast cancer cell line MCF-7 (ATCC, USA) was inoculated into 10% fetal bovine serum (FBS) to confirm the effect of decreasing the gene expression level of NM_012262 (SEQ ID NO: 1) or NM_001134492 The cells were cultured in a DMEM culture medium containing serum (FBS, Welch, Inc.) and antibiotics (Gibco BRL), subcultured in a 60 mm culture dish one day before the injection of small interference RNA, and then 2 μl of RNAiMAX (Invitrogen, Cat # 13778 (SEQ ID NO: 8) and 5'-CUG ACU ACA CCC AAG UGU U-3 '(SEQ ID NO: 8) for the HS3ST1 gene were mixed with OptiMEM®I culture medium (Invitrogen, Cat # 31985) AAC ACU UGG A double-stranded siRNA consisting of the nucleotide sequence of UGG GUG UAG UCA G-3 '(SEQ ID NO: 9) A second base (dTdT) is engaged state Im)} or small interfering RNA for the gene HS2ST1 {siHS2ST1; (SEQ ID NO: 5) and 5'-AUC UGU GGG AGA GAC UAC A-3 '(SEQ ID NO: 6), with the sequence of SEQ ID NO: (DTdT) was bound to the cells at a concentration of 50 nM. After 6 hours, the culture medium was replaced with DMEM culture medium containing 10% fetal bovine serum (FBS, Weljin) and 100 ug / ml of antibiotic streptomycin and 100 units / ml of penicillin (Gibco BRL) And then cultured for 4 days in a constant temperature incubator at 37 ° C with 5% CO 2 .

또한, 상기 인간 유방암 세포주 MCF-7 중 비특이적 작은 간섭 RNA{siControl; 5'-CCU ACG CCA CCA AUU UCG U-3'(서열번호 10)과 5'-ACG AAA UUG GUG GCG UAG G-3'(서열번호 11)의 염기서열로 이루어진 이중가닥 siRNA로, 각각의 서열 3' 말단에 티민 2염기(dTdT)가 결합된 상태임}를 주입한 세포주를 대조군으로 하였다.Also, among the human breast cancer cell line MCF-7, a small nonspecific interference RNA {siControl; Stranded siRNA consisting of the nucleotide sequence of 5'-CCU ACG CCA CCA AUU UCG U-3 '(SEQ ID NO: 10) and 5'-ACG AAA UUG GUG GCG UAG G-3' (SEQ ID NO: 11) (DTdT) was bound to the 3 'terminus} was used as a control.

siRNA는 모두 바이오니아(대한민국)에 의뢰하여 제작한 것을 사용하였다. 구체적으로, β-시아노에틸 포스포라미다이트(β-cyanoethyl phosphoramidite)를 이용하여 DNA 구조의 골격을 이루는 포스포디에스터 결합을 연결해가는 방법을 사용하여 siRNA를 합성하였다(참조: Sinha et al., Nucleic Acids Research, 12:4539-4557, 1984). 즉, RNA 합성기(Perseptive Biosystems 8909, PE Biosystems, USA)를 사용하여, 뉴클레오티드가 부착된 고형지지체 상에서, 차단제거(deblocking), 결합(coupling), 산화(oxidation) 및 캐핑(capping)으로 이루어지는 일련의 과정을 반복하여 원하는 길이의 RNA를 포함하는 반응물을 수득하였다. 이어, 전기 반응물을 Daisogel C18(Daiso, Japan)을 사용한 HPLC LC918(Japan Analytical Industry, Japan)에 적용하여, RNA를 분리하고 이를 MALDI-TOF 질량 흡광분석기(Shimadzu, Japan)에 적용하여, 합성하고자 하는 염기서열과 부합하는지 확인하였다. 그런 다음, 센스와 안티센스 RNA가닥을 결합시켜서, 목적하는 이중가닥 siRNA를 각각 제조하였다.All of the siRNAs used were those made by Biona (Korea). Specifically, siRNA was synthesized using a method of linking phosphodiester bonds forming the backbone of the DNA structure using? -Cyanoethyl phosphoramidite (see Sinha et al. Nucleic Acids Research, 12: 4539-4557, 1984). That is, using a RNA synthesizer (Perseptive Biosystems 8909, PE Biosystems, USA), a series of oligonucleotides consisting of deblocking, coupling, oxidation and capping on a nucleotide- The procedure was repeated to obtain a reaction product containing the desired length of RNA. Next, the reaction product was applied to HPLC LC918 (Japan Analytical Industry, Japan) using Daisogel C18 (Daiso, Japan), RNA was isolated and applied to a MALDI-TOF mass spectrometer (Shimadzu, Japan) Was confirmed to match the nucleotide sequence. The sense and antisense RNA strands were then combined to produce the desired double stranded siRNAs, respectively.

인간 유방암 세포주에 HS3ST1에 대한 특이적 작은 간섭 RNA (siHS3ST1), HS2ST1에 대한 특이적 작은 간섭 RNA (siHS2ST1) 또는 비특이적 작은 간섭 RNA (siControl)를 주입한 후 3 일 동안 5% CO2 농도의 37℃ 항온 배양기에서 배양한 후 세포배양액을 걷어내고 PBS 로 2회 세척하고 3.7 % 포름알데하이드에 20분 동안 상온에 방치하여 세포를 고정시킨다. 그 후 PBS 로 2회 세척한 후 3% 소혈청알부민 (BSA)이 함유된 PBS 용액에 넣고 상온에서 30 분간 블로킹하고 1:100 으로 희석한 항-헤파란 황산화 항체 (10E4, US biological 사)를 넣고 4℃에서 16시간동안 반응시켰다. PBS 로 10분간 3회 세척 후 2차 항체로 Alexa 555 복합 항-쥐 항체 (Molecular probe 사)를 1:200 으로 희석한 PBS 용액에 상온에서 30 분간 반응시켰다. PBS 로 10분간 2회 세척 후 50 % 글리세롤로 마운팅 (mounting) 한 후 공초점 현미경 (LSM-710, 칼 자이스 사)으로 630 X 배율로 관찰하였다. 그 결과를 도 9에 나타내었다.Specific for HS3ST1 the human breast cancer cell line ever small interfering RNA (siHS3ST1), after injecting a specific small interfering RNA (siHS2ST1) or non-specific small interfering RNA (siControl) for HS2ST1 3 5% CO 37 ℃ of concentrations during a single After culturing in a constant temperature incubator, the cell culture broth is washed, washed twice with PBS, and fixed in 3.7% formaldehyde for 20 minutes at room temperature to fix the cells. Heparan sulfate antibody (10E4, US biological) diluted 1: 100 was added to PBS solution containing 3% bovine serum albumin (BSA), and then blocked at room temperature for 30 minutes. And the mixture was reacted at 4 ° C for 16 hours. After washing three times for 10 minutes with PBS, Alexa 555 conjugated anti-mouse antibody (Molecular probe) was reacted as a secondary antibody in a 1: 200 diluted PBS solution at room temperature for 30 minutes. Washed twice with PBS for 10 minutes, mounted with 50% glycerol, and observed with a confocal microscope (LSM-710, Carl Zeiss) at 630 × magnification. The results are shown in Fig.

그 결과 대조군(siCON)과 달리 HS3ST1에 대한 특이적 작은 간섭 RNA 처리군(siHS3ST1), HS2ST1에 대한 특이적 작은 간섭 RNA 처리군(siHS2ST1)은 인간 유방암 세포주 MCF-7에서 세포내 단백다당의 황산화과정 중 하나인 헤파란 황산화가 저해됨을 확인할 수 있다. As a result, unlike the control group (siCON), the specific small interfering RNA treatment group (siHS3ST1) for HS3ST1 and the specific small interfering RNA treatment group (siHS2ST1) for HS2ST1 inhibited the sulfation of intracellular protein polysaccharide in the human breast cancer cell line MCF- One of the processes, heparan sulfate inhibition can be confirmed.

실시예Example 5.  5. HS2ST1HS2ST1 억제제 또는  Inhibitor or HS3ST1HS3ST1 억제제 처리에 의한  By inhibitor treatment 세포증식율Cell proliferation rate 변화 여부 확인 Check for changes

인간 유방암 세포주 MCF-7에 실시예 4와 같이 준비한 HS3ST1에 대한 특이적 작은 간섭 RNA (siHS3ST1), HS2ST1에 대한 특이적 작은 간섭 RNA (siHS2ST1) 또는 비특이적 작은 간섭 RNA (siControl)를 주입한 후 5% CO2 농도의 37℃ 항온 배양기에서 1~3 일 동안 10% 우태아 혈청(fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium) 배양 배지에서 배양한 후 도 10에 표기된 날짜별(0일, 1일, 2일, 3일)로 세포 상층액을 수확하고 트립신을 이용하여 세포를 떼어낸 후 PBS로 2차례 세척 후 0.4 % 트립판 블루 (NUNK 사) 와 1:1 로 혼합한 후 상온에서 5분 동안 방치 후 혈구계수기(MARIENFELD사) 상에서 트립판 블루에 염색되지 않은 세포의 개수를 세어 전체 세포 개수에 대한 백분율로 세포 증식율을 도 10에 나타내었다. 도 10에서 x축은 siHS3ST1, siHS2ST1 또는 siControl 처리한 후의 세포배양 일수를 나타내며, y축은 0일의 세포 개수를 기준으로 비교한 세포 증식율을 나타내다.A specific small interfering RNA (siHS3ST1) for HS3ST1, a specific small interfering RNA (siHS2ST1) for HS2ST1 or a nonspecific small interfering RNA (siControl) prepared as in Example 4 was injected into human breast cancer cell line MCF-7, (FBS, Weljin) and 100 μg / ml of antibiotic streptomycin and 100 units / ml of penicillin (Gibco BRL) for 1 to 3 days in a CO 2 -conjugated 37 ° C incubator After culturing in Dulbeco's Modified Eagle's Medium containing DMEM, the supernatant was harvested on days 0, 1, 2, and 3 as shown in FIG. 10, and cells were removed using trypsin After washing twice with PBS, the mixture was mixed with 0.4% trypan blue (NUNK) at a ratio of 1: 1, and the mixture was allowed to stand at room temperature for 5 minutes. After counting the number of untouched cells on the trypan blue on a hemocyte counter (MARIENFELD) The cell proliferation rate as a percentage of the cell number is shown in Fig. 10 Served. In FIG. 10, the x-axis represents the number of days of cell culture after siHS3ST1, siHS2ST1, or siControl treatment, and the y-axis represents the cell proliferation rate compared with the number of cells on day 0.

도 10에서 나타낸 바와 같이, siHS2ST1 처리군은 시간이 지남에 따라 대조군(siControl)군 및 siHS3ST1 처리군에 비해 종양세포(인간 유방암 세포주 MCF7)의 세포증식율이 낮아짐을 확인할 수 있다. 따라서, 상기 결과를 토대로 종양세포(예를 들어, 인간 유방암 세포주 MCF7)에서 HS2ST1유전자 발현을 억제시킬 경우, 종양세포의 노화가 유도되어 세포증식율이 낮아짐을 확인할 수 있다.As shown in FIG. 10, the siHS2ST1-treated group showed a lower cell proliferation rate of tumor cells (human breast cancer cell line MCF7) as compared with the control (siControl) group and the siHS3ST1-treated group over time. Therefore, when the expression of HS2ST1 gene is inhibited in tumor cells (for example, human breast cancer cell line MCF7) based on the above results, it can be confirmed that the cell proliferation rate is induced by inducing tumor cell senescence.

실시예Example 6.  6. HS2ST1HS2ST1 억제제 또는  Inhibitor or HS3ST1HS3ST1 억제제 처리에 의한 세포노화 유도 여부 확인 Confirmation of cell aging induced by inhibitor treatment

6.1. 6.1. 웨스턴Western 분석을 이용한 종양세포주의  Analysis of tumor cell lines using HS2ST1HS2ST1 억제제 또는  Inhibitor or HS3ST1HS3ST1 억제제에 의한 노화 유도 여부 확인 Determination of aging induction by inhibitor

실시예 4와 같이 준비한 HS3ST1에 대한 특이적 작은 간섭 RNA (siHS3ST1), HS2ST1에 대한 특이적 작은 간섭 RNA (siHS2ST1) 또는 비특이적 작은 간섭 RNA (siControl)를 주입한 후 5% CO2 농도의 37℃ 항온 배양기에서 2 일 동안 10% 우태아 혈청(fetal bovine serum: FBS, ㈜ 웰진)과 항생제인 스트렙토마이신 100 ug/ml과 페니실린 100 유니트/ml (Gibco BRL 사)이 포함된 DMEM(Dulbeco's Modified Eagle's Medium) 배양 배지에서 배양한 인간 유방암 세포주 MCF-7은 PBS (phosphate buffered saline)로 세척 후 세포 시료를 세포 용해 완충액 (50mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150mM NaCl, 1mM PMSF, 50 mM NaF, 0.2 mM Na3VO4, 10 μg/ml 아프로티닌, 2 μg/ml 루펩틴)을 사용하여 단백질을 추출하고, 추출한 단백질은 11,000rpm으로 10분간 원심분리한 후 상등액만 취하여 브래드포드법 {bradford, M., Anal. Biochem. 72:248-254(1976)} 을 이용하여 정량하였다. 20ug의 단백질 2X SDS 로딩 버퍼(60 mm Tris-Cl (pH6.8), 25% 글리세롤, 2% SDS, 14.4 mM 머캅토에탄올, 0.1% 브로모페놀 블루)를 가해 95℃에서 5분간 가열하고 8% 내지 10% SDS 폴리아크릴 아마이드 젤에서 80V로 2시간 전기영동하였다.Example 4 and prepared as specific for HS3ST1 ever small interfering RNA (siHS3ST1), specific small interfering RNA (siHS2ST1) or 37 ℃ of nonspecific after injection of a small interfering RNA (siControl) 5% CO 2 concentration of the HS2ST1 constant temperature (Dulbeco's Modified Eagle's Medium) containing 10% fetal bovine serum (FBS, Weljin) and 100 μg / ml of streptomycin, an antibiotic, and 100 units / ml penicillin (Gibco BRL) Human breast cancer cell line MCF-7 cultured in the culture medium was washed with PBS (phosphate buffered saline), and the cell samples were suspended in a cell lysis buffer (50 mM Tri-HCl, 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM PMSF, Protein was extracted using 50 mM NaF, 0.2 mM Na 3 VO 4 , 10 μg / ml aprotinin, 2 μg / ml rupeptin), and the extracted protein was centrifuged at 11,000 rpm for 10 minutes, Law {bradford, M., Anal. Biochem. 72: 248-254 (1976)). 20 g of protein 2X SDS loading buffer (60 mM Tris-Cl (pH 6.8), 25% glycerol, 2% SDS, 14.4 mM mercaptoethanol, 0.1% bromophenol blue) % To 10% SDS polyacrylamide gel at 80 V for 2 hours.

전기영동 후 분리된 단백질을 나이트로셀룰로오즈 멤브레인(Whatman 사)으로 전이시켰다. 단백질이 전이되어 있는 멤브레인은 5% 탈지분유가 함유된 PBS용액에 넣고 실온에서 1시간 동안 방치하여 블록킹(blocking)하고 1:500 ~ 1:1000으로 희석한 일차 항체들을 넣고 4℃에서 16시간동안 반응시켰다. 1차 항체는 다클론 항-p21 항체 (polyclonal anti-p21 Ab, Santa Cruz 사), 다클론 항-actin 항체 (polyclonal anti-actin Ab, Santa Cruz 사), pRb 의 세린 잔기 중 인산화된 아미노산 807/811 만을 특이적으로 인지하는 항 p-pRb 항체 (anti-phospho-pRb Ab, cell signaling 사) 를 사용하였고 2차 항체는 호스 래디쉬 퍼옥시다아제 복합 항-토끼 항체 (HRP conjugated goat anti-rabbit IgG, HRP conjugated goat anti-mouse IgG, Santa Cruz 사) 를 이용해 ECL (enhanced chemiluminescence) 시약 (Amersham 사)으로 세포노화여부를 확인하였다. 그 결과를 도 11에 나타내었다.After electrophoresis, the separated proteins were transferred to a nitrocellulose membrane (Whatman). Protein-transferred membranes were blocked in PBS solution containing 5% skimmed milk, incubated at room temperature for 1 hour, and primary antibodies diluted 1: 500 ~ 1: 1000 were added and incubated at 4 ° C for 16 hours Lt; / RTI > The primary antibodies were polyclonal anti-p21 Ab (Santa Cruz), polyclonal anti-actin Ab (Santa Cruz), phosphorylated amino acids 807 of the serine residue of pRb, (Anti-phospho-pRb Ab, cell signaling), which specifically recognizes only 811-specific anti-rabbit IgG antibody, and a secondary antibody, horseradish peroxidase complex anti-rabbit antibody (HRP conjugated goat anti- rabbit IgG, HRP-conjugated goat anti-mouse IgG, Santa Cruz), and the aging was confirmed by ECL (enhanced chemiluminescence) reagent (Amersham). The results are shown in Fig.

도 11은 인간 유방암 세포주 MCF-7에 siControl, siHS3ST1 또는 siHS2ST1를 처리하고 2 일 동안 배양한 후 Rb의 인산화 정도 및 p21 발현 정도를 확인한 웨스턴블럿팅 결과이다. 도 11에서 actin은 모든 세포에서 동일한 수준으로 발현되는 유전자(하우스키핑 유전자, housekeeping gene)로 동량의 단백질에 대해 분석되었는지를 보여주는 기준으로 사용되었다. FIG. 11 is a Western blotting result showing the degree of phosphorylation of Rb and the expression level of p21 after treatment with human breast cancer cell line MCF-7, siControl, siHS3ST1 or siHS2ST1 for 2 days. In FIG. 11, actin was used as a standard to show that the same level of expression (the housekeeping gene) in all cells was analyzed for the same amount of protein.

웨스턴블럿팅 분석 결과를 통해, siHS2ST1 처리군은 대조군(siControl)군 및 siHS3ST1 처리군에 비해 인간 유방암 세포주 MCF-7에서 노화 특이적 Rb의 인산화 감소와 노화 특이적 p21의 발현의 증가를 나타내어 노화가 더 많이 유도되었음을 확인할 수 있다. Western blotting analysis showed that the siHS2ST1-treated group showed a decrease in the phosphorylation of aging-specific Rb and an increase in the expression of p21 specific for senescence in the human breast cancer cell line MCF-7 compared to the siControl group and the siHS3ST1-treated group, It can be confirmed that it is induced more.

따라서 상기 결과로부터 종양세포에서 HS2ST1유전자 발현을 억제시킬 경우, 종양세포의 노화가 유도될 수 있음을 확인할 수 있다.Therefore, it can be confirmed that inhibition of HS2ST1 gene expression in tumor cells can induce aging of tumor cells.

6.2. 노화-관련 베타갈락토시다아제 활성 염색을 이용한 종양세포주의 HS2ST1 억제제 또는 6.2. HS2ST1 inhibitor of tumor cell lines using aging-related beta-galactosidase active staining or HS3ST1HS3ST1 억제제에 의한 노화 유도 여부 확인 Determination of aging induction by inhibitor

실시예 4와 같이 준비한 HS3ST1에 대한 특이적 작은 간섭 RNA (siHS3ST1), HS2ST1에 대한 특이적 작은 간섭 RNA (siHS2ST1) 또는 비특이적 작은 간섭 RNA (siControl)를 주입한 인간 유방암 세포 MCF7을 5% CO2 농도의 37℃ 항온 배양기에서 3일 동안 배양한 후 노화-관련 베타갈락토시다아제 활성 염색을 실시하였다. 상기 염색은 Dimri 등의 방법 (Dimri et al., Proc. Natl. Acad. Sci. USA, 92:9363-9367, 1995)에 따라 다음과 같이 수행하였다.Human breast cancer cell MCF7 infected with HSHCST1 specific HSC1ST1, HS2ST1 specific siH2ST1 or non-specific small interfering RNA (siControl) prepared as in Example 4 was inoculated with 5% CO 2 For 3 days, and then subjected to aging-related beta-galactosidase activity staining. The staining was performed according to the method of Dimri et al. (Dimri et al., Proc. Natl. Acad. Sci. USA, 92: 9363-9367, 1995) as follows.

세포를 PBS로 2번 세척하고 3% 포름알데히드로 실온에서 3-5분간 고정하였다. 고정된 세포를 PBS로 다시 한 번 세척하고, β-갈락토시다아제 활성 염색 용액 (1 mg/ml의 X-Gal, 40 mM 시트르산 /소듐 포스페이트 (pH 6.0), 5 mM 포타슘 페로시아니드, 5 mM 포타슘 페리시아니드, 150 mM 염화클로라이드, 2 mM 마그네슘 클로라이드) 5 ml를 첨가하고, 37℃ 항온 배양기에서 12-16시간 반응시켰다. 반응을 진행하는 동안 빛이 들어가지 않도록 은박 호일로 배양접시를 싸서 배양하였다.The cells were washed twice with PBS and fixed with 3% formaldehyde at room temperature for 3-5 min. The immobilized cells were washed once more with PBS, and a solution of β-galactosidase active dye (1 mg / ml of X-Gal, 40 mM citric acid / sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM chloride chloride, 2 mM magnesium chloride) was added thereto, and the mixture was reacted for 12-16 hours at 37 ° C in a constant temperature incubator. During the reaction, the culture dish was wrapped with a thin foil foil to prevent light from entering.

베타-갈락토시다아제 활성 정도는 위상차 현미경 (ECLIPSE TE300, Nikon)을 사용하여 관찰하고, 그 결과를 도 12에 나타내었다. 또한, 현미경 (ECLIPSE TE300, Nikon)을 이용하여 염색된 세포의 개수를 측정하여 노화-관련 베타-갈락토시다아제 양성을 보이는 세포의 비율에 관한 그래프를 도 13에 나타내었다.The degree of beta-galactosidase activity was observed using a phase contrast microscope (ECLIPSE TE300, Nikon), and the results are shown in Fig. FIG. 13 is a graph showing the proportion of cells showing aging-related beta-galactosidase-positive cells by measuring the number of stained cells using a microscope (ECLIPSE TE300, Nikon).

siControl을 처리한 대조군(siCON 또는 siControl) 및 siHS3ST1 처리군과 비교하여, siHS2ST1 처리군에서 염색된 세포수 즉, 노화-관련 베타-갈락토시다아제 양성 세포수가 증가함을 확인할 수 있다. 특히, HS2ST1에 대한 특이적 작은 간섭 RNA (siHS2ST1)를 처리한 siHS2ST1 처리군은 siControl을 처리한 대조군(siCON 또는 siControl) 및 siHS3ST1 처리군에 비해 노화현상이 현저하게 일어남을 확인할 수 있다.The number of cells stained in the siHS2ST1 treated group, that is, the number of senescence-associated beta-galactosidase-positive cells, can be confirmed as compared with the control group (siCON or siControl) treated with siControl and siHS3ST1 treated group. In particular, the siHS2ST1-treated group treated with the specific small interfering RNA (siHS2ST1) for HS2ST1 was significantly more aged than the siCONT treated group (siCON or siControl) and the siHS3ST1 treated group.

따라서, 상기 결과를 토대로 종양세포(예를 들어, 인간 유방암 세포주 MCF-7)에서 HS2ST1 유전자 발현을 억제할 경우, 종양세포의 노화가 유도되어 항암효과를 나타낼 수 있음을 확인할 수 있다.Therefore, it can be confirmed that when the expression of HS2ST1 gene is inhibited in tumor cells (for example, human breast cancer cell line MCF-7) based on the above-mentioned results, aging of tumor cells can be induced and antitumor effect can be obtained.

<110> Korea Institute of Radiological & Medical Sciences <120> Composition for cell senescence comprising an inhibitor against heparan sulfate 2-O sulfotransferase 1 gene, or a protein encoded by the gene and method for inducing cell senescence using the same <130> P12-095-KNUL <160> 11 <170> KopatentIn 2.0 <210> 1 <211> 6770 <212> DNA <213> Homo sapiens <220> <221> mRNA <222> (1)..(6770) <223> Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1), transcript variant 1, mRNA, Access No. NM_012262 <400> 1 ggaggggcgc gcggccgcga gcaaaggagg gagggaagga aggaagagag ggaggcgggc 60 aagcaggcgg gcgcgggggt cggggactga ggcagtagag ggaggcgaga gcccggcagc 120 cgcttcgcgc tgtttgctgc gcgggctttt ggagggggcg gccgtttagt cggctgagga 180 gaagcggaca ccagcggcgt tggtgatagc gcctggggga gggggactgg agaggcgaga 240 aggggggtcg ctgcggtggt tctctcgctg tcgctctctc tttgcctcgc tcccggctcg 300 gcgggctcct cccggcgtct ctctcgcctc cggggtcccg ctccccgccc cccgcggtat 360 gtcttgatcc cgagcagcgg gtttcatggg gctcctcagg attatgatgc cgcccaagtt 420 gcagctgctg gcggtggtgg ccttcgcggt ggcgatgctc ttcttggaaa accagatcca 480 gaaactggag gagtcccgct cgaagctaga aagggctatt gcaagacacg aagtccgaga 540 aattgagcag cgacatacaa tggatggccc tcggcaagat gccactttag atgaggaaga 600 ggacatggtg atcatttata acagagttcc caaaacggca agcacttcat ttaccaatat 660 cgcctatgac ctgtgtgcaa agaataaata ccatgtcctt catatcaaca ctaccaaaaa 720 taatccagtg atgtcattgc aagatcaggt gcgctttgta aagaatataa cttcctggaa 780 agagatgaaa ccaggatttt atcatggaca cgtttcttac ttggattttg caaaatttgg 840 tgtgaagaag aaaccaattt acattaatgt cataagggat cctattgaga ggctagtttc 900 ttattattac tttctgagat ttggagatga ttatagacca gggttacgga gacgaaaaca 960 aggagacaaa aagacctttg atgaatgtgt agcagaaggt ggctcagact gtgctccaga 1020 gaagctctgg cttcaaatcc cgttcttctg tggccatagc tccgaatgct ggaatgtggg 1080 aagcaggtgg gctatggatc aagccaagta taacctaatt aatgaatatt ttctggtggg 1140 agttactgaa gaacttgaag attttatcat gttattggag gcagcattgc cccggttttt 1200 caggggtgct actgaactct atcgcacagg aaagaaatct catcttagga aaaccacaga 1260 gaagaaactc cccactaaac aaaccattgc aaaactacag caatctgata tttggaaaat 1320 ggagaatgag ttctatgaat ttgcactaga gcagttccaa ttcatcagag cccatgccgt 1380 tcgagaaaaa gatggagacc tctacatcct cgcacaaaac tttttctatg aaaagattta 1440 ccctaagtcg aactgagtat aaggtgtgac tattggattc ttgaactaaa atttgaccct 1500 gtcttcacct ttgttctcag ctccacagtc tggattgctg acagtaggtg tatatgacaa 1560 tttgtattga gccaaattag gaaacagaca gtaacgtcaa ggaagtagat actggctggc 1620 attgtcagtg ttctaagttt caggcatttt tatttttcct ggctaaacgt tggtgaaagt 1680 tataacctcc tgcctgggag aaaatataca tcacctaaaa tgaacttatg gcaggtctaa 1740 tcaaaaggct aaatacaatt tcagaaaagg ttctgatact cttgtttttg ataaagcatt 1800 ttttcaacta accatgaatt aagatgagtc catttgcctc ttctgccttc actgagggtt 1860 tgggttatac acctctactg aattgtgtta ataactgttt ggcagtgtgt actttgtttt 1920 tgtgagtcat gtctcatgaa atttattgga atgtttaatc atatttgcta agaaatgttt 1980 ctgctgtagt tggatttgcc catatttatg taggtggttt taatttttta aatggtgatt 2040 agtgttaaaa atcaatttaa atcatgacta atatggtaaa aagataaagc atcaaagcag 2100 tatttctcat tcctgcctcc tcaatatcta atactgggaa gatacttcaa agaatattga 2160 gattgtctga agttttagtt aagattttca cacattaata tcaaaaaagt aagtttagta 2220 tttgtttctc catgggttat ttgtaaagct gtaaactgag atatcggtga ctccgtatta 2280 tgactccatt agtgagctgt ggtatgggta ggattttcct acttcttctg tacttttacc 2340 tgtagactat ttttactaag gtgctttata atgtgtttta aagcattgca tttacaaaac 2400 aaggaaaatg ctgtaaatat tgcatatttt atgtatttgg accaaaaggt tacaagtaat 2460 tagacaaaag tggttttgca ccaattttat gtcaagtaaa accatcagac ctactgttct 2520 tgtatttctc atttaacttt actgttaaga catcactgaa atgaacttca gtaagctttc 2580 aattttgata cacagttcat tattcataac ttgaggcagt aattacagtg gaatgagtac 2640 tggacaagga gtcaaaaaac ttgatttcag gtcctagctc tagcacttac agctgtgtga 2700 tcttgggcaa gtcacttaac ctctctttgc ctcaatttcc tcatcttgaa atgaggataa 2760 taatacctgc tgtacctacc tcacagggct gttgtgagga ttaaatgaga tggcatgtga 2820 aagcactttg aaaattgtaa agcgctatgt aaatgtaagg tattatagaa acatctttaa 2880 catatagttt cataccattc attttttaac aaagaaaggg aaaagtctgc ttgtaagctg 2940 gttgaaaaag ttaatcttga tataaatttg tgtttgataa atatcctctc agtgttttat 3000 cttccatgtt tcaacaacta ttgaaatatg aaatgcctgt gaactcttaa agcttcatga 3060 gcagctgctt gagttcagga agttcactgt tagaaatagg ctttgttagc tgactagggt 3120 cagggaaact tttctcttca aatttgaaag ctgtttctgt tttcatttta cattattatt 3180 cagaaatggt agctattcta tacctatggt ttaagtaaat atttctgaat aaggcttcac 3240 catactgtaa gcattttagg tagattgcct taaaggttat gggagggcat gagggaacac 3300 ttcttatgag aaaacattta taaacaaaag aaacatttat aaactaaaga aaaactaaaa 3360 gaatgacaga acaatcatct tagcaccctt tcctcacaat aatataaaaa tattaaaaga 3420 acataggcag gcttttttta aatttggctt ttttctttcc ttttttcaaa ttgactttta 3480 taggtatttc ctgaaagtgt atacaaatta tttcctcgcc caaaataaag caccacttca 3540 aggtgtggtt tgacattaca tgctaatgaa caaacccagt atgcaagtta ttcttgcacc 3600 acatgctcaa atcttcttga ggtgcattaa ctcttttagg taactagagc agtacttggt 3660 gaactagatc aggaggtcag taaactttct gtggaagggc cagagagtaa atattttagg 3720 ctttgcagcc catacggtct ctgtcacagc tagtcaaccc tgccatttta ccacaaaagc 3780 agcaatagac attatgtaaa caaatgagca cagttatgtt ccaataaaac tttatttaca 3840 aaaacagatg acatcccaga tgcagaccat gggcaaccaa ccattgcact ggccaaatca 3900 ttatttatgg agaaatcctc tttgtgtctc tactctagat gcctaaaaga gtttatatac 3960 ttctaaaagc tcctaactta tatccaaaga attgctttct gattcgtgta gtctctccca 4020 cagattcata aacttttatg acttatattg tttccaggtg ggcatggttt atttcccagt 4080 ttaacagttc agaatagggg catttatttt atcatatttt agggtgggtt aggagtatcc 4140 tttctggaga ctgagaaagg ggtgtattta attccatcag gtccagtaca gtactaggag 4200 tcataatact ttataatcaa ttaaataaat agaaccactg agacaataat gtattttttt 4260 aaagtggcaa atgtggtttt cttttttcag cctttgcgct ttttcagtat tttgaccata 4320 gggagataat ttttttataa tacaaaagta accacttgga attttaaaga taatgttatg 4380 tgtgtatgtg aaatatatat acatatatat atatatgttt atttcctaaa agaagaaaag 4440 atacctttct gttcaacttg tatcaactcc tcttttctaa ttgctgtgaa atggcaactg 4500 ttgataaatt attgtgattg ttttaaaatc taatgggaag taaaatatat tttgatttta 4560 cccagcttaa tctgtaaagt agcacttaaa tatatctgat agcaacactt aagatattgc 4620 atggggatta ctttcctatc atccatatgc atttgtgcaa cttcaaacat attgggtgct 4680 tctgaattcc tgatgattgg atttaagcta ttgaaaattg gataatttaa acttaatgat 4740 ttttataatt ttctgatctt aaaatttggt taatgcctat aatctgttgc tttttctcaa 4800 tatgtgtcct attggaaatt cctcaaatcg ttggtgccat cagtgattta caaacaatat 4860 tttgatattg cagatgactt gcttactgta tttgcattgt tagaaaacag tttgtagaca 4920 atgattcttt tttaataaaa tcaaataatt ctaaaagtgc tagagaattt aactaaaagc 4980 tggttcccaa atgcatagct ggcattttaa tttaaattca aatctacata gagaacatct 5040 gtgtaaatca tctaactgga tttttccatt ggtcattccc aaacacacct atggtcctag 5100 aatccttaag agaagcaccc tgtaaccttt tatgtggttt gcctttaaga ggcccaggtg 5160 cttctccttt atgatttgag ttggcctctt cataaattag tgctgtttac tttcagagga 5220 agcagagaag ttgctgttat gtttttgcat ccgtttaccc tatgcaaagt tgctgtatga 5280 tgccaactaa actactcttt aggcagcctt ctgaggagaa aagcaaccct gtttcaaatc 5340 cactgccaat tcagctcctc tggagtggag ctttctgatt tcttggagca ggaattttag 5400 agattgaaat gaatgatcat ttagtcagat ttatcctgta atttcatgca gctttgtggc 5460 ctttgcagta ctatttataa aatggaccct gatggtgatg aactctttag aacgcattac 5520 tgttaagcct gtgttgagac attgatgctg tctatctcat tttttagaca gtttttgtag 5580 ctttctattg agagtcaggt atgtgagcat ctctgaagca gtgttgaatg taattttcgg 5640 aaacatggat tgtgtatttt gacttttatt ttataaatac acagctcaac agtgcctttt 5700 ttttccctca tagtcctgtt ggaagatgct cactactttc tctcttctct ctccctgccc 5760 tcccccactc cattcagttg attcatttat gcaaattctg tttccaactt gaaaccattt 5820 tgtcacatct gttggagaga taatcactcc ttttccttaa cattctgcca gctttctgat 5880 gttgaagtgt ttcagttgac tacctgatgc aaaagctata aaataaacag tgggaagggg 5940 aaaaattggt gtcctgtttt aatattttct tttgtagcct tgacactgat ggacattttc 6000 caagctgact cagtgttcag tgtcaactta actctcagat agtgttgcca tcaagaaagc 6060 atgcaacatc attggtttct aatgatttta tggcttgtga caatatttta tctggactga 6120 catgcctctg ctgcttttgc tttgtacttc attgctggta ataaaatttc agatggaaaa 6180 cttacaaaat atatacttaa ttagaagaaa aaaatagaga aagggctatt agaattaaaa 6240 aaatttgaaa gtaacttaat ctaacattta tggcacagtt tggacatatc cataattttt 6300 tttgggaaca cacatttctg attttttttt tcccccttaa agaagaaagt ctcaattcca 6360 ttgattttca attcttagcc actggctcat tgctttgagc aatgcttgat tgattctatt 6420 tatattatat gatattgggt tgataaaata ccagttcaat gatgagtttt cttaacagaa 6480 tttggtttgt acttgcagtg gctgaacaaa gagcatggct tgagaatcaa agggatctgc 6540 atttagcaat gtgatgtcag taaatggaca taacaggatt gttgtaaagg ttgggcatga 6600 tgtatgcaaa gtactggcca gggtagacta ataactgatg gcatttatat gctgtgctgg 6660 aatattgtta ccaagctgat gtgccgttct caccctgcag aatactggtt ttgtcatttc 6720 ataaatgata tttttataaa tatttttgaa tgactgtaaa aaaaaaaaaa 6770 <210> 2 <211> 1623 <212> DNA <213> Homo sapiens <220> <221> mRNA <222> (1)..(1623) <223> Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1), transcript variant 2, mRNA, NCBI NM_001134492 <400> 2 ggaggggcgc gcggccgcga gcaaaggagg gagggaagga aggaagagag ggaggcgggc 60 aagcaggcgg gcgcgggggt cggggactga ggcagtagag ggaggcgaga gcccggcagc 120 cgcttcgcgc tgtttgctgc gcgggctttt ggagggggcg gccgtttagt cggctgagga 180 gaagcggaca ccagcggcgt tggtgatagc gcctggggga gggggactgg agaggcgaga 240 aggggggtcg ctgcggtggt tctctcgctg tcgctctctc tttgcctcgc tcccggctcg 300 gcgggctcct cccggcgtct ctctcgcctc cggggtcccg ctccccgccc cccgcggtat 360 gtcttgatcc cgagcagcgg gtttcatggg gctcctcagg attatgatgc cgcccaagtt 420 gcagctgctg gcggtggtgg ccttcgcggt ggcgatgctc ttcttggaaa accagatcca 480 gaaactggag gagtcccgct cgaagctaga aagggctatt gcaagacacg aagtccgaga 540 aattgagcag cgacatacaa tggatggccc tcggcaagat gccactttag atgaggaaga 600 ggacatggtg atcatttata acagagttcc caaaacggca agcacttcat ttaccaatat 660 cgcctatgac ctgtgtgcaa agaataaata ccatgtcctt catatcaaca ctaccaaaaa 720 taatccagtg atgtcattgc aagatcaggt gcgctttgta aagaatataa cttcctggaa 780 agagatgaaa ccaggatttt atcatggaca cgtttcttac ttggattttg caaaatttgg 840 tgtgaagaag aaaccaattt acattaatgt cataagggat cctattgaga ggctagtttc 900 ttattattac tttctgagat ttggagatga ttatagacca gggttacgga gacgaaaaca 960 aggagacaaa aagacctttg atgaatgtgt agcagaaggt ggctcagact gtgctccaga 1020 gaagctctgg cttcaaatcc cgttcttctg tggccatagc tccgaatgct ggtaggggag 1080 ataaagttgg ctcagattga ttatcatcct tattatctct gtaatctgtg tttcatttca 1140 caagggctag atatagggaa atcggtgaaa gactagacta aaaataacat gtaattcagt 1200 aatatctagt tttgcagtta cttttaaatg catttaaaag attcctcatg tagagtgata 1260 tcctaatatc cttgcattgt tttctgagat gccggttttt agtatttctt atttttggtg 1320 ttatgttttg ctgtattcca gcagagctct tagagactgg gggtgggggt gggtgtcata 1380 aatcttattt tgtccaaagc ttactgtttt agctattcat gttaaattaa gaaaaggctt 1440 agtgggttaa aattcacctg gttttactgt taaactgatt ttgactttaa gagaagccaa 1500 ggttatggct gtggtttagt ttgctagtaa atatcaagtg gaaaataaag atactttaat 1560 aagaactgta tttcctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620 aaa 1623 <210> 3 <211> 356 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(356) <223> Homo sapiens heparan sulfate 2-O-sulfotransferase 1(HS2ST1) isoform 1, protein <400> 3 Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Ser Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn 100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe 260 265 270 Arg Gly Ala Thr Glu Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg 275 280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu 290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr 340 345 350 Pro Lys Ser Asn 355 <210> 4 <211> 229 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE <222> (1)..(229) <223> Homo sapiens heparan sulfate 2-O-sulfotransferase 1(HS2ST1) isoform 2, protein <400> 4 Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala 1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln 20 25 30 Lys Leu Glu Glu Ser Arg Ser Lys Leu Glu Arg Ala Ile Ala Arg His 35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln 50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg 65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu 85 90 95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn 100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile 115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser 130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Phe 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln 180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp 195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His 210 215 220 Ser Ser Glu Cys Trp 225 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of siRNA for Homo sapiens heparan sulfate 2-O-sulfotransferase 1(HS2ST1) <400> 5 uguagucucu cccacagau 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense sequence of siRNA for Homo sapiens heparan sulfate 2-O-sulfotransferase 1(HS2ST1) <400> 6 aucuguggga gagacuaca 19 <210> 7 <211> 1965 <212> DNA <213> Homo sapiens <220> <221> mRNA <222> (1)..(1965) <223> Homo sapiens heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1), mRNA, NCBI Access No. NM_005114 <400> 7 aacgcccgcc tgcaaggtcc tctgcgccct ggcagccagg agtcgccgcc acgaccgccg 60 ggtctcagtg ggtgcctgcg ccttctcccc gcccgcctgc cccgggccat ccagaaactt 120 gctctacccg ccgcgggtgc tcggcagtgc tgcccatggc ccagcccagg agcctattta 180 gggcgccgga cgggctggac agaggcgcgg ctcagtaatt gaaggcctga aacgcccatg 240 tgccactgac taggaggctt ccctgctgcg gcacttcatg acccagcggc gcgcggccca 300 gtgaagccac cgtggtgtcc agcatggccg cgctgctcct gggcgcggtg ctgctggtgg 360 cccagcccca gctagtgcct tcccgccccg ccgagctagg ccagcaggag cttctgcgga 420 aagcggggac cctccaggat gacgtccgcg atggcgtggc cccaaacggc tctgcccagc 480 agttgccgca gaccatcatc atcggcgtgc gcaagggcgg cacgcgcgca ctgctggaga 540 tgctcagcct gcaccccgac gtggcggccg cggagaacga ggtccacttc ttcgactggg 600 aggagcatta cagccacggc ttgggctggt acctcagcca gatgcccttc tcctggccac 660 accagctcac agtggagaag acccccgcgt atttcacgtc gcccaaagtg cctgagcgag 720 tctacagcat gaacccgtcc atccggctgc tgctcatcct gcgagacccg tcggagcgcg 780 tgctatctga ctacacccaa gtgttctaca accacatgca gaagcacaag ccctacccgt 840 ccatcgagga gttcctggtg cgcgatggca ggctcaatgt ggactacaag gccctcaacc 900 gcagcctcta ccacgtgcac atgcagaact ggctgcgctt tttcccgctg cgccacatcc 960 acattgtgga cggcgaccgc ctcatcaggg accccttccc tgagatccaa aaggtcgaga 1020 ggttcctaaa gctgtcgccg cagatcaatg cttcgaactt ctactttaac aaaaccaagg 1080 gcttttactg cctgcgggac agcggccggg accgctgctt acatgagtcc aaaggccggg 1140 cgcaccccca agtcgatccc aaactactca ataaactgca cgaatatttt catgagccaa 1200 ataagaagtt cttcgagctt gttggcagaa catttgactg gcactgattt gcaataagct 1260 aagctcagaa actttcctac tgtaagttct ggtgtacatc tgaggggaaa aagaatttta 1320 aaaaagcatt taaggtataa tttatttgta aaatccataa agtacttctg tacagtatta 1380 gattcacaat tgccatatat actagttata tttttctact tgttaaatgg agggcatttt 1440 gtattgtttt tcatggttgt taacattgtg taatatgtct ctatatgaag gaactaaact 1500 atttcactga aaaaaaaaaa aagatttttt ctggagacgc tatgtttttt tgaatataat 1560 taacttgccc ccaactcaaa atagctgtct gtgttgcaat cattgcaaaa tctaaattct 1620 tttgttactt aaaaaaacat gtttttcatg gctattacaa tcctctctct cccctctcct 1680 ttctgcattg ctctttttaa tttttaatgt ctcattgtga tcatcagatt tatttttact 1740 tggattgtaa gatttatctc ctcggcgatt ccttgtcatt tttggaggtc acagttttac 1800 tcattcccat gtgtaaactt caagtaaaca aagcaatatt caggatggag aagcagttag 1860 tgactggcta gaggtctctt gggatccatc ttgacctcaa agaatatttg cacatgcttc 1920 tgatctcagc agttataata aagaggatta aacccttggc cttca 1965 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of siRNA for Homo sapiens heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) <400> 8 cugacuacac ccaaguguu 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense sequence of siRNA for Homo sapiens Homo sapiens heparan sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) <400> 9 aacacuuggg uguagucag 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of non-specific siRNA <400> 10 ccuacgccac caauuucgu 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> antisense sequence of non-specific siRNA <400> 11 acgaaauugg uggcguagg 19 <110> Korea Institute of Radiological & Medical Sciences <120> Composition for cell senescence comprising an inhibitor against          heparan sulfate 2-O sulfotransferase 1 gene, or a protein encoded          by the gene and method for inducing cell senescence using the          same <130> P12-095-KNUL <160> 11 <170> Kopatentin 2.0 <210> 1 <211> 6770 <212> DNA <213> Homo sapiens <220> <221> mRNA <222> (1) .. (6770) Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1),          transcript variant 1, mRNA, Access No. NM_012262 <400> 1 ggaggggcgc gcggccgcga gcaaaggagg gagggaagga aggaagagag ggaggcgggc 60 aagcaggcgg gcgcgggggt cggggactga ggcagtagag ggaggcgaga gcccggcagc 120 cgcttcgcgc tgtttgctgc gcgggctttt ggagggggcg gccgtttagt cggctgagga 180 gaagcggaca ccagcggcgt tggtgatagc gcctggggga gggggactgg agaggcgaga 240 aggggggtcg ctgcggtggt tctctcgctg tcgctctctc tttgcctcgc tcccggctcg 300 gcgggctcct cccggcgtct ctctcgcctc cggggtcccg ctccccgccc cccgcggtat 360 gtcttgatcc cgagcagcgg gtttcatggg gctcctcagg attatgatgc cgcccaagtt 420 gcagctgctg gcggtggtgg ccttcgcggt ggcgatgctc ttcttggaaa accagatcca 480 gaaactggag gagtcccgct cgaagctaga aagggctatt gcaagacacg aagtccgaga 540 aattgagcag cgacatacaa tggatggccc tcggcaagat gccactttag atgaggaaga 600 ggacatggtg atcatttata acagagttcc caaaacggca agcacttcat ttaccaatat 660 cgcctatgac ctgtgtgcaa agaataaata ccatgtcctt catatcaaca ctaccaaaaa 720 taatccagtg atgtcattgc aagatcaggt gcgctttgta aagaatataa cttcctggaa 780 agagatgaaa ccaggatttt atcatggaca cgtttcttac ttggattttg caaaatttgg 840 tgtgaagaag aaaccaattt acattaatgt cataagggat cctattgaga ggctagtttc 900 ttattattac tttctgagat ttggagatga ttatagacca gggttacgga gacgaaaaca 960 aggagacaaa aagacctttg atgaatgtgt agcagaaggt ggctcagact gtgctccaga 1020 gaagctctgg cttcaaatcc cgttcttctg tggccatagc tccgaatgct ggaatgtggg 1080 aagcaggtgg gctatggatc aagccaagta taacctaatt aatgaatatt ttctggtggg 1140 agttactgaa gaacttgaag attttatcat gttattggag gcagcattgc cccggttttt 1200 caggggtgct actgaactct atcgcacagg aaagaaatct catcttagga aaaccacaga 1260 gaagaaactc cccactaaac aaaccattgc aaaactacag caatctgata tttggaaaat 1320 ggagaatgag ttctatgaat ttgcactaga gcagttccaa ttcatcagag cccatgccgt 1380 tcgagaaaaa gatggagacc tctacatcct cgcacaaaac tttttctatg aaaagattta 1440 ccctaagtcg aactgagtat aaggtgtgac tattggattc ttgaactaaa atttgaccct 1500 gtcttcacct ttgttctcag ctccacagtc tggattgctg acagtaggtg tatatgacaa 1560 tttgtattga gccaaattag gaaacagaca gtaacgtcaa ggaagtagat actggctggc 1620 attgtcagtg ttctaagttt caggcatttt tatttttcct ggctaaacgt tggtgaaagt 1680 tataacctcc tgcctgggag aaaatataca tcacctaaaa tgaacttatg gcaggtctaa 1740 tcaaaaggct aaatacaatt tcagaaaagg ttctgatact cttgtttttg ataaagcatt 1800 ttttcaacta accatgaatt aagatgagtc catttgcctc ttctgccttc actgagggtt 1860 tgggttatac acctctactg aattgtgtta ataactgttt ggcagtgtgt actttgtttt 1920 tgtgagtcat gtctcatgaa atttattgga atgtttaatc atatttgcta agaaatgttt 1980 ctgctgtagt tggatttgcc catatttatg taggtggttt taatttttta aatggtgatt 2040 agtgttaaaa atcaatttaa atcatgacta atatggtaaa aagataaagc atcaaagcag 2100 tatttctcat tcctgcctcc tcaatatcta atactgggaa gatacttcaa agaatattga 2160 gattgtctga agttttagtt aagattttca cacattaata tcaaaaaagt aagtttagta 2220 tttgtttctc catgggttat ttgtaaagct gtaaactgag atatcggtga ctccgtatta 2280 tgactccatt agtgagctgt ggtatgggta ggattttcct acttcttctg tacttttacc 2340 tgtagactat ttttactaag gtgctttata atgtgtttta aagcattgca tttacaaaac 2400 aaggaaaatg ctgtaaatat tgcatatttt atgtatttgg accaaaaggt tacaagtaat 2460 tagacaaaag tggttttgca ccaattttat gtcaagtaaa accatcagac ctactgttct 2520 tgtatttctc atttaacttt actgttaaga catcactgaa atgaacttca gtaagctttc 2580 aattttgata cacagttcat tattcataac ttgaggcagt aattacagtg gaatgagtac 2640 tggacaagga gtcaaaaaac ttgatttcag gtcctagctc tagcacttac agctgtgtga 2700 tcttgggcaa gtcacttaac ctctctttgc ctcaatttcc tcatcttgaa atgaggataa 2760 taatacctgc tgtacctacc tcacagggct gttgtgagga ttaaatgaga tggcatgtga 2820 aagacactttg aaaattgtaa agcgctatgt aaatgtaagg tattatagaa acatctttaa 2880 catatagttt cataccattc attttttaac aaagaaaggg aaaagtctgc ttgtaagctg 2940 gttgaaaaag ttaatcttga tataaatttg tgtttgataa atatcctctc agtgttttat 3000 cttccatgtt tcaacaacta ttgaaatatg aaatgcctgt gaactcttaa agcttcatga 3060 gcagctgctt gagttcagga agttcactgt tagaaatagg ctttgttagc tgactagggt 3120 cagggaaact tttctcttca aatttgaaag ctgtttctgt tttcatttta cattattatt 3180 cagaaatggt agctattcta tacctatggt ttaagtaaat atttctgaat aaggcttcac 3240 catactgtaa gcattttagg tagattgcct taaaggttat gggagggcat gagggaacac 3300 ttcttatgag aaaacattta taaacaaaag aaacatttat aaactaaaga aaaactaaaa 3360 gaatgacaga acaatcatct tagcaccctt tcctcacaat aatataaaaa tattaaaaga 3420 acataggcag gcttttttta aatttggctt ttttctttcc ttttttcaaa ttgactttta 3480 taggtatttc ctgaaagtgt atacaaatta tttcctcgcc caaaataaag caccacttca 3540 aggtgtggtt tgacattaca tgctaatgaa caaacccagt atgcaagtta ttcttgcacc 3600 acatgctcaa atcttcttga ggtgcattaa ctcttttagg taactagagc agtacttggt 3660 gaactagatc aggaggtcag taaactttct gtggaagggc cagagagtaa atattttagg 3720 ctttgcagcc catacggtct ctgtcacagc tagtcaaccc tgccatttta ccacaaaagc 3780 agcaatagac attatgtaaa caaatgagca cagttatgtt ccaataaaac tttatttaca 3840 aaaacagatg acatcccaga tgcagaccat gggcaaccaa ccattgcact ggccaaatca 3900 ttatttatgg agaaatcctc tttgtgtctc tactctagat gcctaaaaga gtttatatac 3960 ttctaaaagc tcctaactta tatccaaaga attgctttct gattcgtgta gtctctccca 4020 cagattcata aacttttatg acttatattg tttccaggtg ggcatggttt atttcccagt 4080 ttaacagttc agaatagggg catttatttt atcatatttt agggtgggtt aggagtatcc 4140 tttctggaga ctgagaaagg ggtgtattta attccatcag gtccagtaca gtactaggag 4200 tcataatact ttataatcaa ttaaataaat agaaccactg agacaataat gtattttttt 4260 aaagtggcaa atgtggtttt cttttttcag cctttgcgct ttttcagtat tttgaccata 4320 gggagataat ttttttataa tacaaaagta accacttgga attttaaaga taatgttatg 4380 tgtgtatgtg aaatatatat acatatatat atatatgttt atttcctaaa agaagaaaag 4440 atacctttct gttcaacttg tatcaactcc tcttttctaa ttgctgtgaa atggcaactg 4500 ttgataaatt attgtgattg ttttaaaatc taatgggaag taaaatatat tttgatttta 4560 cccagcttaa tctgtaaagt agcacttaaa tatatctgat agcaacactt aagatattgc 4620 atggggatta ctttcctatc atccatatgc atttgtgcaa cttcaaacat attgggtgct 4680 tctgaattcc tgatgattgg atttaagcta ttgaaaattg gataatttaa acttaatgat 4740 ttttataatt ttctgatctt aaaatttggt taatgcctat aatctgttgc tttttctcaa 4800 tatgtgtcct attggaaatt cctcaaatcg ttggtgccat cagtgattta caaacaatat 4860 tttgatattg cagatgactt gcttactgta tttgcattgt tagaaaacag tttgtagaca 4920 atgattcttt tttaataaaa tcaaataatt ctaaaagtgc tagagaattt aactaaaagc 4980 tggttcccaa atgcatagct ggcattttaa tttaaattca aatctacata gagaacatct 5040 gtgtaaatca tctaactgga tttttccatt ggtcattccc aaacacacct atggtcctag 5100 aatccttaag agaagcaccc tgtaaccttt tatgtggttt gcctttaaga ggcccaggtg 5160 cttctccttt atgatttgag ttggcctctt cataaattag tgctgtttac tttcagagga 5220 agcagagaag ttgctgttat gtttttgcat ccgtttaccc tatgcaaagt tgctgtatga 5280 tgccaactaa actactcttt aggcagcctt ctgaggagaa aagcaaccct gtttcaaatc 5340 cactgccaat tcagctcctc tggagtggag ctttctgatt tcttggagca ggaattttag 5400 agattgaaat gaatgatcat ttagtcagat ttatcctgta atttcatgca gctttgtggc 5460 ctttgcagta ctatttataa aatggaccct gatggtgatg aactctttag aacgcattac 5520 tgttaagcct gtgttgagac attgatgctg tctatctcat tttttagaca gtttttgtag 5580 ctttctattg agagtcaggt atgtgagcat ctctgaagca gtgttgaatg taattttcgg 5640 aaacatggat tgtgtatttt gacttttatt ttataaatac acagctcaac agtgcctttt 5700 ttttccctca tagtcctgtt ggaagatgct cactactttc tctcttctct ctccctgccc 5760 tcccccactc cattcagttg attcatttat gcaaattctg tttccaactt gaaaccattt 5820 tgtcacatct gttggagaga taatcactcc ttttccttaa cattctgcca gctttctgat 5880 gttgaagtgt ttcagttgac tacctgatgc aaaagctata aaataaacag tgggaagggg 5940 aaaaattggt gtcctgtttt aatattttct tttgtagcct tgacactgat ggacattttc 6000 caagctgact cagtgttcag tgtcaactta actctcagat agtgttgcca tcaagaaagc 6060 atgcaacatc attggtttct aatgatttta tggcttgtga caatatttta tctggactga 6120 catgcctctg ctgcttttgc tttgtacttc attgctggta ataaaatttc agatggaaaa 6180 cttacaaaat atatacttaa ttagaagaaa aaaatagaga aagggctatt agaattaaaa 6240 aaatttgaaa gtaacttaat ctaacattta tggcacagtt tggacatatc cataattttt 6300 tttgggaaca cacatttctg attttttttt tcccccttaa agaagaaagt ctcaattcca 6360 ttgattttca attcttagcc actggctcat tgctttgagc aatgcttgat tgattctatt 6420 tatattatat gatattgggt tgataaaata ccagttcaat gatgagtttt cttaacagaa 6480 tttggtttgt acttgcagtg gctgaacaaa gagcatggct tgagaatcaa agggatctgc 6540 atttagcaat gtgatgtcag taaatggaca taacaggatt gttgtaaagg ttgggcatga 6600 tgtatgcaaa gtactggcca gggtagacta ataactgatg gcatttatat gctgtgctgg 6660 aatattgtta ccaagctgat gtgccgttct caccctgcag aatactggtt ttgtcatttc 6720 ataaatgata tttttataaa tatttttgaa tgactgtaaa aaaaaaaaaa 6770 <210> 2 <211> 1623 <212> DNA <213> Homo sapiens <220> <221> mRNA &Lt; 222 > (1) .. (1623) Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1),          transcript variant 2, mRNA, NCBI NM_001134492 <400> 2 ggaggggcgc gcggccgcga gcaaaggagg gagggaagga aggaagagag ggaggcgggc 60 aagcaggcgg gcgcgggggt cggggactga ggcagtagag ggaggcgaga gcccggcagc 120 cgcttcgcgc tgtttgctgc gcgggctttt ggagggggcg gccgtttagt cggctgagga 180 gaagcggaca ccagcggcgt tggtgatagc gcctggggga gggggactgg agaggcgaga 240 aggggggtcg ctgcggtggt tctctcgctg tcgctctctc tttgcctcgc tcccggctcg 300 gcgggctcct cccggcgtct ctctcgcctc cggggtcccg ctccccgccc cccgcggtat 360 gtcttgatcc cgagcagcgg gtttcatggg gctcctcagg attatgatgc cgcccaagtt 420 gcagctgctg gcggtggtgg ccttcgcggt ggcgatgctc ttcttggaaa accagatcca 480 gaaactggag gagtcccgct cgaagctaga aagggctatt gcaagacacg aagtccgaga 540 aattgagcag cgacatacaa tggatggccc tcggcaagat gccactttag atgaggaaga 600 ggacatggtg atcatttata acagagttcc caaaacggca agcacttcat ttaccaatat 660 cgcctatgac ctgtgtgcaa agaataaata ccatgtcctt catatcaaca ctaccaaaaa 720 taatccagtg atgtcattgc aagatcaggt gcgctttgta aagaatataa cttcctggaa 780 agagatgaaa ccaggatttt atcatggaca cgtttcttac ttggattttg caaaatttgg 840 tgtgaagaag aaaccaattt acattaatgt cataagggat cctattgaga ggctagtttc 900 ttattattac tttctgagat ttggagatga ttatagacca gggttacgga gacgaaaaca 960 aggagacaaa aagacctttg atgaatgtgt agcagaaggt ggctcagact gtgctccaga 1020 gaagctctgg cttcaaatcc cgttcttctg tggccatagc tccgaatgct ggtaggggag 1080 ataaagttgg ctcagattga ttatcatcct tattatctct gtaatctgtg tttcatttca 1140 caagggctag atatagggaa atcggtgaaa gactagacta aaaataacat gtaattcagt 1200 aatatctagt tttgcagtta cttttaaatg catttaaaag attcctcatg tagagtgata 1260 tcctaatatc cttgcattgt tttctgagat gccggttttt agtatttctt atttttggtg 1320 ttatgttttg ctgtattcca gcagagctct tagagactgg gggtgggggt gggtgtcata 1380 aatcttattt tgtccaaagc ttactgtttt agctattcat gttaaattaa gaaaaggctt 1440 agtgggttaa aattcacctg gttttactgt taaactgatt ttgactttaa gagaagccaa 1500 ggttatggct gtggtttagt ttgctagtaa atatcaagtg gaaaataaag atactttaat 1560 aagaactgta tttcctcaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1620 aaa 1623 <210> 3 <211> 356 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) .. (356) Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1)          isoform 1, protein <400> 3 Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala   1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln              20 25 30 Lys Leu Glu Glu Ser Ser Ser Lys Leu Glu Arg Ala Ile Ala Arg His          35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln      50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg  65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu                  85 90 95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn             100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile         115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser     130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Phe                 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln             180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp         195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His     210 215 220 Ser Ser Glu Cys Trp Asn Val Gly Ser Arg Trp Ala Met Asp Gln Ala 225 230 235 240 Lys Tyr Asn Leu Ile Asn Glu Tyr Phe Leu Val Gly Val Thr Glu Glu                 245 250 255 Leu Glu Asp Phe Ile Met Leu Leu Glu Ala Ala Leu Pro Arg Phe Phe             260 265 270 Arg Gly Ala Thr Glu Leu Tyr Arg Thr Gly Lys Lys Ser His Leu Arg         275 280 285 Lys Thr Thr Glu Lys Lys Leu Pro Thr Lys Gln Thr Ile Ala Lys Leu     290 295 300 Gln Gln Ser Asp Ile Trp Lys Met Glu Asn Glu Phe Tyr Glu Phe Ala 305 310 315 320 Leu Glu Gln Phe Gln Phe Ile Arg Ala His Ala Val Arg Glu Lys Asp                 325 330 335 Gly Asp Leu Tyr Ile Leu Ala Gln Asn Phe Phe Tyr Glu Lys Ile Tyr             340 345 350 Pro Lys Ser Asn         355 <210> 4 <211> 229 <212> PRT <213> Homo sapiens <220> <221> PEPTIDE &Lt; 222 > (1) Homo sapiens heparan sulfate 2-O-sulfotransferase 1 (HS2ST1)          isoform 2, protein <400> 4 Met Gly Leu Leu Arg Ile Met Met Pro Pro Lys Leu Gln Leu Leu Ala   1 5 10 15 Val Val Ala Phe Ala Val Ala Met Leu Phe Leu Glu Asn Gln Ile Gln              20 25 30 Lys Leu Glu Glu Ser Ser Ser Lys Leu Glu Arg Ala Ile Ala Arg His          35 40 45 Glu Val Arg Glu Ile Glu Gln Arg His Thr Met Asp Gly Pro Arg Gln      50 55 60 Asp Ala Thr Leu Asp Glu Glu Glu Asp Met Val Ile Ile Tyr Asn Arg  65 70 75 80 Val Pro Lys Thr Ala Ser Thr Ser Phe Thr Asn Ile Ala Tyr Asp Leu                  85 90 95 Cys Ala Lys Asn Lys Tyr His Val Leu His Ile Asn Thr Thr Lys Asn             100 105 110 Asn Pro Val Met Ser Leu Gln Asp Gln Val Arg Phe Val Lys Asn Ile         115 120 125 Thr Ser Trp Lys Glu Met Lys Pro Gly Phe Tyr His Gly His Val Ser     130 135 140 Tyr Leu Asp Phe Ala Lys Phe Gly Val Lys Lys Lys Pro Ile Tyr Ile 145 150 155 160 Asn Val Ile Arg Asp Pro Ile Glu Arg Leu Val Ser Tyr Tyr Tyr Phe                 165 170 175 Leu Arg Phe Gly Asp Asp Tyr Arg Pro Gly Leu Arg Arg Arg Lys Gln             180 185 190 Gly Asp Lys Lys Thr Phe Asp Glu Cys Val Ala Glu Gly Gly Ser Asp         195 200 205 Cys Ala Pro Glu Lys Leu Trp Leu Gln Ile Pro Phe Phe Cys Gly His     210 215 220 Ser Ser Glu Cys Trp 225 <210> 5 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of siRNA for Homo sapiens heparan sulfate          2-O-sulfotransferase 1 (HS2ST1) <400> 5 uguagucucu cccacagau 19 <210> 6 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Antisense sequence of siRNA for Homo sapiens heparan sulfate          2-O-sulfotransferase 1 (HS2ST1) <400> 6 aucuguggga gagacuaca 19 <210> 7 <211> 1965 <212> DNA <213> Homo sapiens <220> <221> mRNA <222> (1) .. (1965) <223> Homo sapiens heparan sulfate (glucosamine) 3-O-sulfotransferase 1          (HS3STl), mRNA, NCBI Access No. < / RTI > NM_005114 <400> 7 aacgcccgcc tgcaaggtcc tctgcgccct ggcagccagg agtcgccgcc acgaccgccg 60 ggtctcagtg ggtgcctgcg ccttctcccc gcccgcctgc cccgggccat ccagaaactt 120 gctctacccg ccgcgggtgc tcggcagtgc tgcccatggc ccagcccagg agcctattta 180 gggcgccgga cgggctggac agaggcgcgg ctcagtaatt gaaggcctga aacgcccatg 240 tgccactgac taggaggctt ccctgctgcg gcacttcatg acccagcggc gcgcggccca 300 gtgaagccac cgtggtgtcc agcatggccg cgctgctcct gggcgcggtg ctgctggtgg 360 cccagcccca gctagtgcct tcccgccccg ccgagctagg ccagcaggag cttctgcgga 420 aagcggggac cctccaggat gacgtccgcg atggcgtggc cccaaacggc tctgcccagc 480 agttgccgca gaccatcatc atcggcgtgc gcaagggcgg cacgcgcgca ctgctggaga 540 tgctcagcct gcaccccgac gtggcggccg cggagaacga ggtccacttc ttcgactggg 600 aggagcatta cagccacggc ttgggctggt acctcagcca gatgcccttc tcctggccac 660 accagctcac agtggagaag acccccgcgt atttcacgtc gcccaaagtg cctgagcgag 720 tctacagcat gaacccgtcc atccggctgc tgctcatcct gcgagacccg tcggagcgcg 780 tgctatctga ctacacccaa gtgttctaca accacatgca gaagcacaag ccctacccgt 840 ccatcgagga gttcctggtg cgcgatggca ggctcaatgt ggactacaag gccctcaacc 900 gcagcctcta ccacgtgcac atgcagaact ggctgcgctt tttcccgctg cgccacatcc 960 acattgtgga cggcgaccgc ctcatcaggg accccttccc tgagatccaa aaggtcgaga 1020 ggttcctaaa gctgtcgccg cagatcaatg cttcgaactt ctactttaac aaaaccaagg 1080 gcttttactg cctgcgggac agcggccggg accgctgctt acatgagtcc aaaggccggg 1140 cgcaccccca agtcgatccc aaactactca ataaactgca cgaatatttt catgagccaa 1200 ataagaagtt cttcgagctt gttggcagaa catttgactg gcactgattt gcaataagct 1260 aagctcagaa actttcctac tgtaagttct ggtgtacatc tgaggggaaa aagaatttta 1320 aaaaagcatt taaggtataa tttatttgta aaatccataa agtacttctg tacagtatta 1380 gattcacaat tgccatatat actagttata tttttctact tgttaaatgg agggcatttt 1440 gtattgtttt tcatggttgt taacattgtg taatatgtct ctatatgaag gaactaaact 1500 atttcactga aaaaaaaaaa aagatttttt ctggagacgc tatgtttttt tgaatataat 1560 taacttgccc ccaactcaaa atagctgtct gtgttgcaat cattgcaaaa tctaaattct 1620 gt; ttctgcattg ctctttttaa tttttaatgt ctcattgtga tcatcagatt tatttttact 1740 tggattgtaa gatttatctc ctcggcgatt ccttgtcatt tttggaggtc acagttttac 1800 tcattcccat gtgtaaactt caagtaaaca aagcaatatt caggatggag aagcagttag 1860 tgactggcta gaggtctctt gggatccatc ttgacctcaa agaatatttg cacatgcttc 1920 tgatctcagc agttataata aagaggatta aacccttggc cttca 1965 <210> 8 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of siRNA for Homo sapiens heparan sulfate          (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) <400> 8 cugacuacac ccaaguguu 19 <210> 9 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Antisense sequence of siRNA for Homo sapiens Homo sapiens heparan          sulfate (glucosamine) 3-O-sulfotransferase 1 (HS3ST1) <400> 9 aacacuuggg uguagucag 19 <210> 10 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> sense sequence of non-specific siRNA <400> 10 ccuacgccac caauuucgu 19 <210> 11 <211> 19 <212> RNA <213> Artificial Sequence <220> <223> Antisense sequence of non-specific siRNA <400> 11 acgaaauugg uggcguagg 19

Claims (8)

센스 서열이 서열번호 5, 안티센스 서열이 서열번호 6인 HS2ST1(heparan sulfate 2-O sulfotransferase 1)에 대한 siRNA를 포함하는 세포 노화 유도용 조성물.SiRNA for HS2ST1 (heparan sulfate 2-O sulfotransferase 1) having a sense sequence of SEQ ID NO: 5 and an antisense sequence of SEQ ID NO: 6. 삭제delete 삭제delete 삭제delete 제1항에 있어서, 상기 세포는 암세포인 것인 세포 노화 유도용 조성물.The composition for inducing cellular senescence according to claim 1, wherein the cell is a cancer cell. 제5항에 있어서, 상기 암세포는 유방암 세포인 세포 노화 유도용 조성물.6. The composition according to claim 5, wherein the cancer cells are breast cancer cells. 인간을 제외한 포유동물의 세포에 센스 서열이 서열번호 5, 안티센스 서열이 서열번호 6인 HS2ST1(heparan sulfate 2-O sulfotransferase 1)에 대한 siRNA를 포함하는 세포 노화 유도용 조성물을 처리하는 단계를 포함하는 인간을 제외한 포유동물 세포의 노화 유도 방법.Comprising treating siRNA for HS2ST1 (heparan sulfate 2-O sulfotransferase 1) whose sense sequence is SEQ ID NO: 5 and antisense sequence is SEQ ID NO: 6 to mammalian cells other than human, A method for inducing aging of mammalian cells other than human. 제7항에 있어서, 상기 세포는 암세포인 인간을 제외한 포유동물 세포의 노화 유도 방법.8. The method according to claim 7, wherein the cells are cancer cells.
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