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암유전자 K-ras에 의한 미토콘드리아의 기능손상 유도기전 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 윤계순 | - |
| dc.contributor.author | 김준형 | - |
| dc.date.accessioned | 2019-10-21T07:25:56Z | - |
| dc.date.available | 2019-10-21T07:25:56Z | - |
| dc.date.issued | 2015-08 | - |
| dc.identifier.other | 20789 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/18751 | - |
| dc.description | 학위논문(박사)--아주대학교 일반대학원 :의생명과학과,2015. 8 | - |
| dc.description.tableofcontents | I. INTRODUCTION 1 II. MATERIALS AND METHODS 20 A. Cell culture and small interfering RNA (siRNA) transfection. 20 B. Human hepatocellular carcinoma samples from patients 20 C. Amplification and production of retrovirus containing activated K-Ras (K-Ras12V) 21 D. Simultaneous measurement of extracellular acidification rate and cellular oxygen consumption rate 21 E. Southern Estimation of lactate dehydrogenase (LDH) activity and cellular ATP levels. 22 F. Glucose uptake assay. 22 G. Fluorescence and live microscopy and electron microscopy. 22 H. Western blot analysis and antibodies. 23 I. Statistical analysis. 23 III. RESULTS 24 A. After infection with K-RasV12 retrovirus, mitochondrial dysfunction and intracellular vesicles appeared. 24 B. Changed cell morphology and induced vesicles during K-RasV12-mediated transformation are reduced by autophagy inhibition. 27 C. During the K-RasV12-mediated transformation, mitochondria are entrapped into the vesicles........ 29 D. Degradation activity of autolysosomes induced by K-RasV12 is not sufficient to completely remove captured organelles. 31 E. Intracellular ATP levels were sustained through accelerated glycolysis in K-RasV12 retrovirus-infected cells. 34 F. Cells transformed by K-RasV12 undergo a metabolic shift from mitochondrial OXPHOS to activated glycolysis. 36 G. Impaired mitochondrial ATP generations were restored by genetic silencing of autophagy-related genes with siRNA. 38 H. K-RasV12 virus-infected cells show unchanged glucose uptake levels. 40 I. Autophagy induction by K-RasV12 is dependent only on low glucose. 42 J. Human hepatocellular carcinoma tissues show induced LC3-II only in low glucose uptake tissues. 44 IV. DISCUSSION 47 V. CONCLUSION 50 VI. REFFERENCE 51 국문요약- 59 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 암유전자 K-ras에 의한 미토콘드리아의 기능손상 유도기전 연구 | - |
| dc.title.alternative | Mechanism of mitochondrial respiratory dysfunction by oncogenic K-Ras | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.department | 일반대학원 의생명과학과 | - |
| dc.date.awarded | 2015. 8 | - |
| dc.description.degree | Doctoral | - |
| dc.identifier.localId | 705719 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000020789 | - |
| dc.subject.keyword | Mitochondrial dysfunction | - |
| dc.subject.keyword | K-RasV12 | - |
| dc.subject.keyword | Mitophagy | - |
| dc.subject.keyword | Tumorigenesis | - |
| dc.subject.keyword | LC3-II | - |
| dc.description.alternativeAbstract | Among the several features of cancer cells, mitochondrial impairment in relation to altered metabolism has been extensively researched. Although the importance of mitochondrial impairment in cancer development has been emphasized, how and why mitochondrial impairment occurs remains unclear. To study the role of mitochondrial impairment in early stages of carcinogenesis, an experimental model for oncogenic transformation without cell death was required. Already known as a strong oncogene driving transformation, oncogenic K-RasV12 was adopted to transform cultured fibroblast. When cultured Rat-2 fibroblast cells were infected with a retrovirus harboring constitutively active K-RasV12, the expressions of both respiratory and non-respiratory proteins were decreased. Such decreased expression of mitochondrial proteins was functionally reflected in the results of significant mitochondrial dysfunction parallel with the acquisition of the transformed characteristics. During the oncogenic transformation by K-RasV12, acidic vesicles appeared simultaneously with mitochondrial respiration defects. Although the role of Ras family proteins in autophagy mediation has gradually emerged through several reports, the importance of the coincidental occurrence of mitochondrial impairment and the autophagy process during the oncogenic transformation process has not been understood. In a previous study, mitochondrial biogenesis, which is another process that maintains whole mitochondrial homeostasis in cells, was not implicated in the mitochondrial impairment of transformed cells. However, the respiration defects of mitochondria in transformed cells were inversely associated with the increased mitophagy accompanying the induction of autophagy-related proteins, such as autophagy-related gene5 (Atg5), Beclin 1, microtubule-associated protein 1 light chain 3-II (LC3-II), and vacuolar adenosine tri-phosphatases (VoATPase). The respiratory protein expression and respiratory activity were recovered by blocking autophagy with conventional inhibitors (bafilomycin A, 3-methyladenin) and siRNA-mediated knockdown of autophagy-related genes. Interestingly, cellular ATP level was not changed during the oncogenic transformation process, and the ATP generation occurred mainly through glycolysis without induction of glucose transporter 1 (GLUT1), which has a low Km value and is a well-known glucose transporter induced by oncogenic Ras signaling. Finally, LC3-II formation, which is indicative of autophagy initiation, was modulated by extracellular glucose levels during the oncogenic transformation; through the study with hepatocellular carcinoma tissues, the modulation of LC3-II formation was also observed only in the tissues exhibiting low glucose uptake and increased K-Ras expression. Taken together, these observations suggest that oncogenic K-RasV12-induced mitophagy leads to mitochondrial functional loss even in the absence of hypoxia during early tumorigenesis, and that this mitophagic process may be an important strategy for overcoming the cellular energy deficit triggered by insufficient glucose supply. | - |
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