초록 ▼

-미토파지를 유도하는 CCCP 처리 - 폐암세포주에서 프로테오믹스를 이용, 변화한 단백질을 동정, S6K1이 미토콘드리아의 기능에 미치는 영향을 확인함. S6K1 KO MEF 세포에서 미토파지의 전단계인 fission을 유도하는 Drp1의 인산화를 확인.

- 미토파지 조절 핵심단백질로 LETM1과 PHF20를 발굴. LETM1과 PHF20 과발현이 미토콘드리아 fission을 유도. 세포주와 동물모델에서 확인. 폐암 모델 마우스인 A/J 마우스를 사용하여 폐암유발 물질인 NKK 를 처리했을 때 PHF20 Tg 마우스에

-미토파지를 유도하는 CCCP 처리 - 폐암세포주에서 프로테오믹스를 이용, 변화한 단백질을 동정, S6K1이 미토콘드리아의 기능에 미치는 영향을 확인함. S6K1 KO MEF 세포에서 미토파지의 전단계인 fission을 유도하는 Drp1의 인산화를 확인.

- 미토파지 조절 핵심단백질로 LETM1과 PHF20를 발굴. LETM1과 PHF20 과발현이 미토콘드리아 fission을 유도. 세포주와 동물모델에서 확인. 폐암 모델 마우스인 A/J 마우스를 사용하여 폐암유발 물질인 NKK 를 처리했을 때 PHF20 Tg 마우스에서 미토콘드리아 특이적 자가포식을 유도함.

- 다양한 폐암세포주에서 과발현되어 있는 AAT 골지체 관련단백질 발굴. 골지체 기능 손상모델인 shGOLGA2/GM130 세포주에서 AAT 의 발현이 현저히 감소. 폐암세포주 A549에 폐암 증식을 감소하는 효과를 보임을 확인.

- 폐암 모델 마우스인 K-rasLA1에 AAT이 폐암의 숫자와 크기에 관련됨. 골지체와 관련이 있는 ERGIC3도 K-rasLA1 마우스에서 폐암형성 조절에 관련이 있음을 확인.

- 다양한 폐암 세포주에 CCCP 처리를 통한 마이토파지 유도 후 발생하는 단백체와 지질 대사체 변화를 확인하여 특이적인 바이오마커를 확인 함.

- K-RAS 변형 모델 마우스의 폐 조직 지질대사체 분자 영상 이미지를 확인하기 위하여 분자 영상 분석법을 최적화 함.

- 각종 지질대사체 그룹의 스탠다드를 이용한 최적화와 약물 이미징 분석을 통한 대사물질의 비표지 분자 영상 분석법을 확립 함.

(출처 : 요약서 3p)

Abstract ▼

Ⅳ. Results

▷ 1st year

○ Establishment of autophagy in the mitochondria damage model
: Established and verification of the autophagy by a chemical substance that inhibits the mitochondria function.
Confirmation the degree of autophagy after the CCCP treatment for mitochondria

Ⅳ. Results

▷ 1st year

○ Establishment of autophagy in the mitochondria damage model
: Established and verification of the autophagy by a chemical substance that inhibits the mitochondria function.
Confirmation the degree of autophagy after the CCCP treatment for mitochondria damage by measuring the change of LC3-II band.

○ Confirmation of Mitochondria-specific autophagy in mitochondria injury model.
: Established and verification of the mitochondria-specific autophagy by a chemical substance that inhibits the mitochondria function.
For confirmation of mitochondria-specific autophagy, the expression of GFP-LC3 and Mito-red and then treatment of CCCP.
We transfected a mitochondrial target red fluorescence plasmid (pDs-Res Mito) to the cell, then observed the mitochondria morphology by confocal imaging.

○ Selection of candidated core protein for Mitophagy using proteomics.
: Selection of candidation core protein for mitophagy using proteomics in cells treated with CCCP and non-treated cells.
Analysis of CCCP-treated cells which are small cell lung cancer, Non-small cell lung cancer and normal lung cells for selection of candidate core protein for mitophagy

○ Comparison of expression of Golgi apparatus-specific autophagy-related proteins in various lung cancer cell-lines.
: RT-PCR was performed for sequence identified 10 genes among about 30 candidate genes which are known as Golgi-related markers in various lung cancer cell-lines [(Non-Small Cell Lung Cancer, NSCLC): A549, NCI-H226, NCI-H322, NCI-H460, NCI-H520; (Small Cell Lung Cancer, SCLC): NCI-H146, NCI-H209); normal lung cell-line 16HBE14o-. RT-PCR was performed using templates which were isolated from 24h cultured lung cancer cell-lines. Golgi-related protein was selected from overexpressed candidates in lung cancer cell compared with normal lung cell.

○ Confirmation of glycosylated/phosphorylated proteins and lipids expression and Golgi apparatus-specific autophagy in Golgi apparatus function damage model.
: In previous study, suppression of protein glycosylation and increase of autophagy were observed in GOLGA2/GM130 shRNA treated A549 cells for the suppression of Golgi apparatus function. Based on this result, we confirmed intra-cellular expression of AAT using Western blot and CLSM for the confirmation of the effect of GOLGA2/GM130 suppression on alpha I-Antitrypsin (AAT) expression. As a result, suppression of AAT expression was observed in shGOLGA2 treated A549 cell.

○ Investigation of protein regulation mechanism through selected Golgi apparatus-specific autophagy core protein overexpression or suppression
: Overexpression of Golgi marker alpha I-Antitrypsin (AAT) was confirmed in A549 lung cancer cell-line and AAT was selected for candidate target for lung cancer therapy. Human AAT overexpression plasmid was purchased from ORIGENE for the finding of mechanism of this protein effect. AAT overexpression cell-line was generated through transfection into normal lung cell-line L-132 and treatment of G418. AAT overexpression cell-line was confirmed through time-dependent Western blot and related protein was investigated.

○ Mitochondrion proteome analysis in the cell line overexpressed LETM1 which is one of the main proteins of autophagy specific to mitochondrion.
: We identified 3542 proteins and 953 proteins are included in mitochondrion database. After investigating relatioinship betwwen differentially expressed proteins, many proteins indicated overexpression in translation, mitochondria transport related proteins are down regulated. UQCRH, exists in complex III in electron transport system, showed the highest upregulation of the all proteins. (5.15 increase in fold change) SIRT3 which interacts with UQCRH also increases 2 fold change. SIRT3 seems like to increase the production of ROS and Respiration causing Mitophagy.

▷ 2nd year

○ Establishment of the cell line of mitophagy regulating core protein
: Establishing the inducible expression lentiviral system that overexpress PHF20 causing the Mitophagy.
Establishing the inducible expression lentiviral system that established cell lines that overexpress PHF20 expression by Doxycycline treatment

○ Establishment of in vivo model of mitophagy regulating core protein
: Establishment of PHF20 Tg mice that was injected with human PHF20 clone.
Establishment of Tg mice that overexpressed PHF20 associated with mitochondria-specific autophagy.

○ Establishment of phosphorylation and complex between S6K1 and core proteins in malnutrition induced mitopahgy
: Selecting a protein showing a difference in expression between the control and LETM1 overexpressing cells using proteomics.
It induces overexpression of S6K1 KO and PHF20 and analyzed using immunological detection of Mitophagy-related proteins, and confirm the phosphorylation of Drp1 induced mitophagy-related fission in S6K1 KO MEF cells. Moreover determine an increase in MFF, related fission, in PHF20-overexpressed cells.

○ Verification by co-expression of interaction between S6K1 and core protein
: Verification by co-expression of interaction between S6K1 and core protein discovered by proteomics.

○ Investigation of intracellular mechanism for Golgi apparatus-specific
autophagy-regulate protein
: We overexpressed Golgi marker AAT in normal lung cell-line L132 for the investigation of intracellular function of alpha I-Antitrypsin(AAT) which is overexpressed in lung cancer. Increases of vesicle-mediated transport, regulation of biosynthetic precess, cellular component movement, and negative regulation of cell death were confirmed in AAT overexpression cell through proteomics analysis compared with normal lung cell-line. This result was confirmed through Western blot, Luciferase assay, immuno-fluorescence staining, angiogenesis array, colony formation, proliferation.

○ Development of overexpression or suppression model for Golgi apparatus-specific autophagy-regulate protein.
: Stable cell-line was generated by inducing shRNA-AAT in AAT overexpressed lung cancer cell-line A549. In AAT suppressed lung cancer cell-line, suppression of cell proliferation, transcription, translation, angiogenesis, migration, and invasion was confirmed which is related to lung cancer development. For in vivo experiment, AAT suppression effect was also confirmed in mouse lung adenocarcinoma cell-line LA4 through lentiviral delivery of shAAT.

○ Aerosol delivery of related gene into lung cancer model mouse
: After aerosol delivery of lentiviral sh-AAT using nose-only inhalation system to K-rasLA1 lung cancer model mouse, suppression of AAT was confirmed by Western blot and fluorescence immuno-staining. Decrease of lung tumor number and size was also observed in this mice.

○ Lipidome analysis of mitophagy induced lung cancer cell (H460 cell) through CCCP treatment
: There are 13 differentially expressed lipids and 11 lipids are down regulated and 2 lipids are up regulated. Especially, there are two unique upregulations of sphingomyeline after mitophagy. Sphingomyeline consists cell organelles cytoskeletons and is quite significant lipid due to its resistant ability to apoptosis. With this fact, down regulation of sphingomyeline may conduct important roles in Mitophagy inducement. PC, SM group look like to decrease breaking down cytoskeleton when mitophagy induces.


▷ 3rd year

○ In-vivo functional verification the overexpression of mitophagy regulating core protein using cell line.
: The overexpression of LETM1 and PHF20, mitophagy-regulating core proteins induce mitochondrial fission and mitochondria specific autophagy.

○ In-vivo functional validation the overexpressing the core protein using an animal model.
: An experiment was carried out using a mouse PHF20 TG, PHF20 overexpression was found to affect the Mitophagy involved in mitochondrial fission.

○ Validation of regulating mitophagy in lung cancer model mouse after overexpression or knockdown of mitophagy regulating core protein.
: An experiment was carried out using the mouse model of lung cancer A / J mouse. When the treatment of NKK, cancer causing substances, induces mitochondria-specific autophagy in PHF20 Tg mice.

○ Investigation of Golgi apparatus-specific autophagy mechanism after core protein expression in lung cancer model mouse
: Gene therapy was performed in lung cancer model mouse by targeting ERGIC3 which is overexpressed in lung tissue and Golgi apparatus and ER-related protein. Investigation of autophagy and ER stress-related mechanism in lung tissues by Western blot. We confirmed that delivery of shERGIC3 stimulated ER stress-induced autophagy.

○ Confirmation of Golgi apparatus-specific autophagy regulation after core protein expression in lung cancer model mouse
: Damages of Golgi apparatus, endoplasmic reticulum (ER), mitochondria and autophagy were confirmed in ERGIC3 suppressed lung cancer cell line. Based on this result, we delivered shERGIC3 in lung cancer mouse model and confirmed ER stress-induced autophagy in lung tissue through Western blot, etc. In special, increase of autophagy in lung tissues was confirmed through LC3-II protein increase. Moreover, cancer cell invasion, proliferation, angiogenesis, and cell cycle proteins were significantly increased in shERGIC3 delivered lungs.

○ Analysis of proteome related with autophagy specific to mitochondrion induced by PHF20, nucleus protein.
: We identified 3202 proteins and 867 proteins are confirmed with mitochondrion database. 203 proteins indicated significant expression, 109 proteins are up-regulated and 94 proteins are down regulated. Anticipated protein because of PHF20 overexpression, Mfn2 showed 0.4 fold change and Mfn1 showed 3.3 foldchange.

○ Preparation of lipid analysis using MALDI IMG in K-RAS mouse model.
: we are carrying out K-RAS mouse model lipid imaging based on optimized ion isolation and mass spectrometer condition. Finally, our researchers will confirm the distribution of lipid exist in K-RAS overexpressed mouse model lung.

○ Molecular imaging analysis of cisplatin in Rat brain.
: We confirmed the quantifying distribution of cisplatin through molecular imaging technique. 281.99m/z, 283m/z, 305.45m/z peaks are identified and validated. (figure 10) We can recognize that cisplatin is distributing in brain broadly rather than concentrated.

(출처 : SUMMARY 15p)

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 보고서 요약서 ... 3
• 요 약 문 ... 5
• SUMMARY ... 13
• CONTENTS ... 21
• 목차 ... 22
• 제1장 연구개발과제의 개요 ... 23
• 제2장 국내외 기술개발 현황 ... 23
• 제3장 연구개발수행 내용 및 결과 ... 25
• 1절. 제1세부 연구개발수행 결과 ... 26
• 2절. 제1세부(위탁과제) 연구개발수행 결과 ... 59
• 3절. 제2세부 연구개발수행 결과 ... 94
• 제4장 목표달성도 및 관련분야에의 기여도 ... 121
• 제5장 연구개발결과의 활용계획 ... 128
• 제6장 연구개발과정에서 수집한 해외과학기술정보 ... 129
• 제7장 연구시설ㆍ장비 현황 ... 130
• 제8장 참고문헌 ... 131
• 끝페이지 ... 132