보고서 정보
주관연구기관 |
국립암센터 National Cancer Center |
연구책임자 |
전경희
|
참여연구자 |
최일주
,
신지영
,
김석준
,
정택진
|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2008-12 |
과제시작연도 |
2009 |
주관부처 |
보건복지가족부 |
사업 관리 기관 |
보건복지가족부 |
등록번호 |
TRKO201100007019 |
과제고유번호 |
1355061065 |
DB 구축일자 |
2013-04-18
|
키워드 |
위암.갈렉틴.세포주기.세포사멸.Gastric cancer.galectins.cell-cycle.apoptosis.
|
초록
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◆ 연구목표
<최종목표>
위암의 발암 및 전이 과정 중에서 galectin-2와 galectin-3의 발현 및 역할을 연구하여 위암의 발생과 전이과정을 규명하고 예후를 예측하여 치료하는 데에 적용함
◆ 연구내용 및 방법
1. galectin-3에 의한 위암 발암, 진행 및 위암세포의 항암제 저항성 관련 기전 연구
① siRNA 전략 및 유전자 과발현 등의 방법으로 galectin-3의 발현을 인위적으로 변화시킨 세포주를 확립함
② 위암세포주 중에서 galectin-3의 발현 정도를 알아내어 발현이 높
◆ 연구목표
<최종목표>
위암의 발암 및 전이 과정 중에서 galectin-2와 galectin-3의 발현 및 역할을 연구하여 위암의 발생과 전이과정을 규명하고 예후를 예측하여 치료하는 데에 적용함
◆ 연구내용 및 방법
1. galectin-3에 의한 위암 발암, 진행 및 위암세포의 항암제 저항성 관련 기전 연구
① siRNA 전략 및 유전자 과발현 등의 방법으로 galectin-3의 발현을 인위적으로 변화시킨 세포주를 확립함
② 위암세포주 중에서 galectin-3의 발현 정도를 알아내어 발현이 높은 세포주를 선택하여 cell cycle과 관련된 인자 및apoptosis 관련인자의 binding 정도 및 활성화 등이 cell prolieration assay, western blotting, immuno-precipitation, kinase assay 등을 통해 연구
③ galectin-3의 relocation에 의한 역할변화를 관찰
④ 위암세포주의 gaelctin-3 발현과 항암제 내성의 연관성 조사
⑤ galectin-3 과발현 세포주에서 항암제에 의한 DNA 손상 복구 과정에 관여하는 단백질의 활성저하 정도를 조사함
⑥ galectin-3 과발현 세포주에의한 위암에서 anti-apoptosis 기전을 (예, ROS 발현 방해) 연구함
2. galectin-3에 의한 위암의 전이 및 혈관생성 촉진 연구
① migration 및 invasion assay를 통하여 galectin-3를 이상 발현시킨 위암세포주 전이 관찰
② galectin-3 발현 위암세포주의 adhesion 및 움직임을 관찰
③ galectin-3 이상발현 위암세포주에서 배양액을 얻어 전이 관련인자와 혈관생성 관련인자를 Zymograph, elisa assay 및 western blotting 방법으로 연구
④ galectin-3에 의한 위암세포주에서 혈관생성 관련인자 생성유도 기전을 연구
⑤ in vitro에서 hypoxia 유도 후 galectin-3 발현 위암세포주에서 HIF-1a의 발현 유도 및 기전연구 ⑥galectin-3 과발현 위암세포주의 배양액을 이용하여 HUVEC 세포의 in vitro 혈관생성 및 migration 관찰
⑦ 위의 실험결과를 바탕으로 galectin-3 과발현 위암세포주를 xenograph방법으로 쥐에 투여후 종괴형성 및 혈관생성 정도 관찰하고 쥐에 foot-pad방법으로 galectin-3 과발현 위암세포주의 장기특이적 전이성 조사
3. galectin-7의 apoptosis 유도 기전 및 위암에서 galectin-7 promotor methylation 연구
① galectin-7의 발현정도를 위암세포주 및 위암환자군에서 조사
② galectin-7의 과발현 및 siRNA 전략으로 galectin-7 이상발현 세포주 확립
③ 위의 세포주를 이용하여 항암제에 의한 galectin-7와 apoptosis 관련 연구
④ 위암 환자군 및 위암세포주에서 galectin-7의 promotor methylation 연구 및 통계적 방법 시행
⑤ 위암 환자군 및 위암세포주에서 galectin-7의 돌연변이 및 SNP 연구
⑥ 위암 환자군에서 galectin-7와 -3에서 나온 연구 결과와 통계적 연구 병행
Abstract
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Galectin-3, a member of the β-galactoside-binding proteins, is secreted to extracellular space and is also abundantly present in the cytoplasm, the nucleus and the subcellular structures, such as mitochondria and phagosomes. It has been shown to play important roles in some biological responses thro
Galectin-3, a member of the β-galactoside-binding proteins, is secreted to extracellular space and is also abundantly present in the cytoplasm, the nucleus and the subcellular structures, such as mitochondria and phagosomes. It has been shown to play important roles in some biological responses through its intracellular actions, galectin-3 modulates a number of cellular processes such as cell growth, apoptosis, adhesion and invasion. Even though it was reported that galectin-3 is highly expressed in human gastric cancer cells, the mechanism(s) of these functions in cellular galectin-3 are not well elucidated. Gastric cancer is the most commonly incident and the second leading by cancer related death in Northeast Asia including Korea.The mortality of gastric cancer is caused by metastatic spread of gastric cancer cells to peritoneum and liver. However, the mechanism(s) of metastasis in gastric cancer cells are sill unclear.
In this study, to determine the effect on functions of galectin-3 in human gastric cancers, we silenced the expression of galectin-3 by silencing with synthetic double-stranded small interfering RNA (siRNA) in AGS cell lines. After treatment of siRNA of galectin-3, AGS cell numbers decreased and cell shapes also changed in round. To demonstrate mechanism(s), the gene expression in galectin-3 siRNA treated cells was detected by microarray analysis. From these results, it was detected that the expression of genes related to apoptosis mainly increased, whereas expression of genes related to cell cycle tended to decrease. RT-PCR was utilized as a secondary validation for selected genes resulted from the microarray result, and real-time PCR was used to examine to confirm the pattern of genes selected from RT-PCR results. We have got the same pattern of the expression of genes came from microarray result by using RT-PCR and real-time PCR.
Also, we have had the same pattern of the protein expression compare to the mRNA level. Using the siRNA of galectin-3, we show that galectin-3 controls the proliferation and G1 phase progression of AGS cells. From these basic data, we can choose the important regulator of G1-S phase progression like S-phase kinase-associated protein 2 (skp2). At the molecular level, the F-box protein SKP2 functions as a receptor component of the SCF ubiquitin ligase complex, resulting in ubiquitination and degradation of important cell cycle regulator like E2F1. The human skp2 promoter is characterized by three E2F binding sites. To check the contribution of E2F1 toward the transcriptional regulation of skp2 after treatment of siRNA of galectin-3, we did ChIP assay. We could show that E2F1 binding to the skp2 gene promoter is reduced from siRNA treatment. We did western blot analysis to determine whether reduced binding of E2F1 to the skp2 gene is due to impaired E2F1 protein abundance. We show that the expression of E2F1 protein was not changed by treatment of siRNA of galectin-3. These data show that skp2 promoter is indirectly controlled by E2F1 in AGS cells. To detect the modulators related to these mechanisms, we did check several regulators related to the stabilization of E2F1.
Second, we demonstrated the important roles of galectin-3 on the metastasis of gastric cancer cells. To demonstrate the function of galectin-3 on cell migration, we have knock-downed galectin-3 mRNA and protein expression with synthetic double-stranded small interfering RNA (siRNA) in AGS and MKN-28 gastric cancer cells. After treatment of siRNA of galectin-3, two kinds of gastric cancer cells were inhibited the migration by detecting migration assay and cell shapes also changed in round. To clarify the motility related molecules which were modulated by galectin-3, the gene expression in galectin-3 siRNA treated cells was detected by DNA microarray analysis. Several genes were selected by these results, and the changes of mRNA expression by galectin-3 siRNA treatment were confirmed by RT-PCR. Among them, we got a fascin1 is a 55 kDa monomeric which a globular cross-linking and actin bundling protein that provides mechanical support to cellular protrusions and cell motility. The expression of fascin1 in epithelial neoplasms has been recently reported, but its exact mechanism in cancer is unknown. Fascin1 has an influence on migration through modulate F-actin. Expression of mRNA and protein Fascin 1 was reduced by galectin-3 siRNA treatment. Also we demonstrated that changes in F-actin according to fascin 1 reduction through Immunocytochemistry when we treated galectin-3 siRNA in MKN-28. In conclusion, we proposed that galectin-3 regulate in cell migration through modulation with fascin1 into f-actin in human gastric cancer.
Third, we defined the expression of galectin-7 was regulated the methylation in promoter and intron region in galectin-7. In conclusion, we suggest that galectin-3 involves in cancer progression and malignancy by modulating the gene expressions, and further studies are needed.
목차 Contents
- 표지 ...1
- 제출문 ...3
- 목차 ...4
- 요약문 ...5
- 한글 ...5
- 영문 ...7
- 1. 연구사업의 최종목표 ...9
- 2. 연구사업의 내용 및 결과 ...9
- 1) galectin-3에 의한 위암 발암, 진행 및 위암세포의 항암제 저항성 관련 기전 연구 ...9
- 2) galectin-3에 의한 위암의 전이 및 혈관생성 촉진 연구 ...32
- 3) galectin-7의 apoptosis 유도기전 및 위암에서 galectin-7 promoter methylation 연구 ...39
- 3. 연구결과 고찰 및 결론 ...43
- 4. 연구성과 및 목표달성도 ...45
- (1) 연구성과 ...45
- (2) 목표달성도 ...46
- 5. 연구결과의 활용계획 ...47
- (1) 연구종료 2년후 예상 연구성과 ...47
- (2) 연구성과의 활용계획 ...48
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